Percutaneous vertebroplasty (VP) and balloon kyphoplasty (BKP) for treating painful osteoporotic vertebral fractures are controversial.We assessed the regional variation in the use of VP/BKP in Switzerland.We conducted a population-based small area variation analysis using patient discharge data for VP/BKP from all Swiss hospitals and Swiss census data for calendar years 2012/13. We derived hospital service areas (HSAs) by analyzing patient flows, assigning regions from which most residents were discharged to the same VP/BKP specific HSA. We calculated age-/sex-standardized mean VP/BKP-rates and measures of regional variation . We estimated the reduction in variation of VP/BKP rates using negative binomial regression, with adjustment for patient demographic and regional socioeconomic factors . We considered the residual, unexplained variation most likely to be unwarranted.Overall, 4955 VP/BKPs were performed in Switzerland in 2012/13. The age-/sex-standardized mean VP/BKP rate was 4.6/10,000 persons and ranged from 1.0 to 10.1 across 26 HSAs. The EQ was 10.2 and the SCV 57.6, indicating a large variation across VP/BKP specific HSAs. After adjustment for demographic and socioeconomic factors, the total reduction in variance was 32.2% only, with the larger part of the variation remaining unexplained.We found a 10-fold variation in VP/BKP rates across Swiss VP/BKP specific HSAs. As only one third of the variation was explained by differences in patient demographics and regional socioeconomic factors, VP/BKP in the highest-use areas may, at least partially, represent overtreatment. Symptomatic vertebral fractures are common in the elderly and potentially result in pain, deformity, disability, and substantial costs , 2. TreaIn 2009, two placebo-controlled randomized trials demonstrated that VP was not superior in alleviating pain than a sham procedure in up to 12 months old painful osteoporotic fractures , 12. As Although the Swiss Medical Board recommended to use VP only in patients with ongoing pain and after interdisciplinary consensus in 2011 , Swiss hth revision, German Modification (ICD-10-GM), up to 100 procedure codes based on the Swiss Classification of Operations , and the admission type. The area of patient residence and hospital location within one of 705 Swiss MedStat regions are recorded. MedStat regions represent aggregated ZIP-codes regions, with a population size of approximately 10,000 persons per region [We conducted a population-based small area variation analysis based on routinely collected patient discharge data from all Swiss acute care hospitals and Swiss census data for calendar years 2012 and 2013. With the introduction of a diagnosis related group reimbursement system (SwissDRG) in 2012, hospital payments in Swiss acute care hospitals are linked to the provision of accurate diagnoses/interventions, leading to an unprecedented level of data quality. Swiss hospitals are legally obligated to provide the Swiss Federal Statistical Office (SFSO) an anonymized, standardized data set for each hospital discharge. These data are centrally stored in the Swiss Hospital Discharge Masterfile hosted at the SFSO. Recorded variables include patient age, sex, nationality, insurance type, the principal and up to 49 secondary diagnostic codes based on the International Classification of Diseases, 10r region . The SFSr region , 27.Using registry-based 2012 census data from the SFSO, we determined the number of inhabitants, age- and sex distribution, main language , socioeconomic status , and the level of urbanization  for each MedStat region. The SFSO defines the level of urbanization using a multistep approach based on spatial  and functional criteria (commuter travel patterns) . BecauseSwitzerland has compulsory basic health insurance coverage, with voluntary semiprivate and private insurance plans covering additional services. Although Swiss hospital care is primarily organized based on 26 geographic regions, the cantons, patients may utilize hospital services outside their canton of residence and the use of cantons as a unit of observation may skew procedure rates. We therefore used Wennberg and Gittelsohn’s approach to derive Swiss Hospital Service Areas (HSAs) , which tWe initially identified 2,318,595 patient discharges from 155 Swiss acute care hospitals during calendar years 2012 and 2013. Of these, 153,678 patients (6.6%) were discharged for one of the eight predefined preference-sensitive procedures. After the exclusion of 3117 discharges with residence outside Switzerland or missing/invalid MedStat region and 159 discharges aged <18 years, we analyzed the flows of 150,402 discharges (98%). We assigned MedStat regions from which the highest proportion of residents was discharged to the same HSA  , which yWe used the Swiss Hospital Discharge Masterfile to identify patient discharges with a specific Swiss Classification of Operations procedure code for VP  or BKP  from all Swiss acute care hospitals during 2012/2013. Overall, we identified 4946 adult discharges with specific codes for VP/BKP and complete MedStat region information. Because pathologic fractures due to vertebral metastases or multiple myeloma may be appropriate indications for VP/BKP,, 32 we eWe calculated unadjusted and age-/sex-standardized VP/BKP procedure rates per 10,000 persons for each VP/BKP-specific HSA using procedure counts and 2012 census data for the adult Swiss population . We used2, proportion of households led by a person with no/low education, proportion headed by a person in manual/unskilled occupation, and mean crowding, all on the neighborhood level). The SSEP varies between 0 (worst) and 100 (best) and correlates well with mortality [Because differences in illness incidences and socioeconomic factors are possible causes of variation , we explortality .We used progressively adjusted multilevel negative binomial regression to model the procedure rates in each HSA using 5-year age- and sex-strata. Model 1 was an intercept-only model. Model 2 was adjusted for age and sex. Model 3 was additionally adjusted for SSEP, level of urbanization, and language region. We depicted the variation in HSA rates as average predicted VP/BKP rates per 10,000 per HSA derived from the multilevel regression models. To assess the impact of patient demographics and socioeconomic factors on the VP/BKP rates, we used the same progressively adjusted models. We expressed the impact of patient demographics and socioeconomic factors on VP/BKP rates as incidence rate ratios (IRRs), defined as the VP/BKP rate in the defined category  relative to the estimated VP/BKP rate in the reference category . We also determined the percentage reduction in procedure variation across the 26 VP/BKP specific HSAs by examining the variance of the random intercept. We considered the residual, unexplained variance of the fully adjusted model a proxy for unwarranted variation. Statistical analyses were performed using Stata version 14.2  and R statistical software version 3.2.3 .Fig 1 shows the geographic boundaries of the 26 VP/BKP specific HSAs and the population density. The mean number of acute care hospitals per HSA was 12 (range 2–54). The mean population size per HSA was 307,211 persons , with a mean population density of 335 persons per km2 (range 28–2221), and a median SSEP of 62 points (range 51–78). Overall, 19 HSAs were located in the Swiss German-speaking, 6 in the French-speaking, and 1 in the Italian-speaking part of Switzerland.Table 1). Overall, 80% of patient discharges had one or two vertebral bodies treated. Thirty-four patients (1%) died during hospitalization.Of the 4559 adult patients discharged with VP/BKP, 2894 (63.5%) underwent VP, 1278 (28.0%) BKP, and 387 (8.5%) both procedures. The majority of patients were older (mean age 73 years), women (71%), and Swiss nationals (93%)  in HSAs located in the greater Bern area, Uri, and Schwyz, and the lowest rates  in Zurich, Jura, Basel, Glarus, Geneva, and the western Valais. The EQ was 10.2, the CV 0.59, and the SCV 57.6, indicating a very high variation across HSAs.The mean unadjusted VP/BKP rate across VP/BKP specific HSAs was 5.2 procedures (range 1.2–11.9) per 10,000 persons. The mean age-/sex-standardized procedure rate was 4.6 (1.0–10.1) per 10,000 persons. In patients aged ≥65 years, the mean unadjusted and age-/sex-standardized VP/BKP rates across HSAs were 17.9 procedures (range 3.7–37.5) and 17.4 (range 3.6–37.4) per 10,000 persons, respectively. Crude and age-/sex-standardized procedure rates for each HSA are shown in the Supplement  or from >6–8 to 0–2 procedures per 10,000 persons (Schaffhausen and the northern part of Zurich). The decrease was less impressive in other high-volume areas (Bern and Neuchâtel), where predicted average VP/BKP rates decreased from >8 to >4–6 per 10,000 persons. Additional adjustment for socioeconomic/cultural factors did not change the average predicted VP/BKP rates in most regions, except for Schaffhausen and the northern part of Zurich, where adjustment led to increased predicted average rates .Table 2). For each 5-year increase in age, the procedure rate increased by 8% . Women had a 59% higher procedure rate than men . Despite resulting in a reduction in variance amongst HSAs, SSEP, urbanization, and language region were not statistically significantly associated with procedure rates.Age and sex were the main determinants of procedure variation across VP/BKP specific HSAs , are associated with the rate of VP/BKP. However, the large differences in VP/BKP across metropolitan areas , where such specialists tend to concentrate, and the high rates in some non-metropolitan areas  indicate that the regional density of specialists performing VP/BKP might not be the only explanation for variation. Finally, we included inpatient VP/BKP only and could not examine outpatient VP/BKP because of lack of national data. However, according to a major Swiss health insurer, the vast majority of BKP was performed in hospitalized patients and the proportion of outpatient VP procedures was small (<10%).To reduce practice variation, further high-quality evidence from placebo-controlled randomized trials is needed to better define which patients benefit most from vertebral augmentation procedures  , 50. ThiIn conclusion, we found a 10-fold variation in VP/BKP procedure rates across Swiss regions. As the larger portion of the variation remains unexplained by demographic and socioeconomic factors and is unlikely to be explained by differences in patient preferences, regional procedure rates are mostly likely driven by differing practices of local physicians, in line with the professional controversy regarding the benefits of VP/BKP. As VP/BKP is currently not recommended in most instances of osteoporotic vertebral fracture, VP/BKP in the highest-use areas may, at least partially, represent overtreatment.S1 Table§academic university hospital located in the HSA.(DOCX)Click here for additional data file.


Structural variations involved the sugar stereochemistry and size as well as the anchoring point of the NHI on the carbohydrate frame (either C-1 or C-6). In the case of the galactose anomeric glycoconjugate (C-1), intriguing solvent-dependent effects were observed in the glycosylation stereochemical outcome. The biological activity of the deprotected glycoconjugates in contrasting lactate formation and cancer cell proliferation are described.Conjugation of known biologically active molecules to carbohydrate frameworks represents a valuable option for the preparation of hybrid, structurally-related families of compounds with the aim of modulating their biological response. Therefore, we present here a study on the preparation of  Deregulation of normal cell metabolism is one of the most important hallmark of cancer cells. In fact, metabolic activities are often reprogrammed in neoplastic masses relative to healthy tissues . The WarNumerous research lines have so far focused on the development of new therapeutic agents that can potentially take advantage of this characteristic metabolic profile present in cancer cells by acting as anti-glycolytic agents ,4. FurthOne of the key enzymes in glycolysis is lactate dehydrogenase (LDH), which catalyzes the reversible transformation of pyruvate into lactate. This enzyme is constituted by four monomeric subunits (LDH-A and LDH-B) and may, therefore, be found in humans as five different isoforms (hLDH1-5). Cancer cells are often found to overexpress subunit LDH-A and, thus, its tetrameric form hLDH5, therefore may represent a promising therapeutic target . In factN-hydroxyindole-based compounds (NHIs), such as 1  position of monosaccharides of the d-galacto, d-manno, d-gluco series and of d-lactose.In order to get a further insight into the potential role played by the carbohydrate structure on the inhibitor activity and on the cellular uptake through facilitative glucose transporters—namely its size, stereochemistry, and anchoring point, herein we describe a structural modulation of this class of glycoconjugate inhibitors, exemplified by 3 and 4 , with a 6α, 6β, and lactose glycoside 8β, known glycosyl donors 5α 12, 16, a D -10.3 (c 0.90 in CHCl3); 1H NMR  δ: 7.99 , 7.86 , 7.80–7.74 , 7.55–7.40 , 7.30 , 5.91 , 5.61 , 5.47 , 5.36 , 5.02 , 4.05 , 4.00 , 3.91 , 2.12, 2.11, 1.99, 1.67 ; 13C NMR  δ: 171.2, 171.1, 170.8, 170.7 (4 × COMe), 160.6 (COOMe), 140.4, 139.7, 138.0, 130.4 (4 × Ar-C), 130.1, 129.2, 128.5 (Ar-CH), 124.8–123.2 (CF3 + Ar-C), 120.4 (Ar-CH), 117.2 (Ar-C), 113.3, 107.1 (2 × Ar-CH), 104.4 (C-1), 70.6 (C-5), 68.9 (C-4), 67.6 (C-2), 66.8 (C-3), 62.5 (C-6), 53.1 (COOMe), 21.8, 20.4 (4 × COMe). Anal. Calcd for C31H30F3NO12: C, 55.94; H, 4.54%; N, 2.10%; Found: C, 55.98; H, 4.56%; N, 2.14%.Compound 6β is a pale yellow solid; Rf 0.35 ; mp 139–142 °C (crom); [α]D + 26.3 (c 1.0 in CHCl3); 1H NMR  δ: 8.12 , 7.84 , 7.75 , 7.71 , 7.54–7.40 , 7.23 , 5.58 , 5.40 , 5.27 , 4.10–3.98 , 3.91 , 2.18, 2.16, 1.98, 1.60 ; 13C NMR  δ: 171.2, 171.1, 170.8, 170.7 (4 × COMe), 160.5 (COOMe), 140.5, 139.8, 139.5, 130.4 (4 × Ar-C), 130.1, 129.1, 128.2 (Ph-CH), 124.1–123.2 (CF3 + Ar-C), 120.3 (Ar-CH), 117.2 (Ar-C), 114.8, 106.8 (2 × Ar-CH), 106.2 (C-1), 72.1 (C-5), 71.3 (C-3), 68.4 (C-4), 68.1 (C-2), 62.4 (C-6), 52.9 (COOMe), 21.2, 20.8, 20.7, 20.4 (4 × COMe). Anal. Calcd for C31H30F3NO12: C, 55.94; H, 4.54%; N, 2.10%; Found: C, 55.97; H, 4.58%; N, 2.13%.Compound 8α D +49.4 (c 1.0 in CHCl3); 1H NMR  δ: 8.08 , 7.80 , 7.71–7.66 , 7.58–7.38 , 7.22 , 5.57 , 5.33 , 5.30 , 5.23 , 5.03 , 4.90 , 4.59 , 4.19 , 4.13–4.02 , 3.99 , 3.91 , 3.75 , 2.13, 2.09, 2.06, 2.02, 1.93, 1.89, 1.58 ; 13C NMR  δ: 171.1–170.2 (7 × COMe), 160.3 (COOMe), 140.6, 139.8, 135.5, 130.4 (4 × Ar-C), 129.9, 128.9, 128.2 (Ph-CH), 123.9–122.9 (CF3 + Ar-C), 120.3 (Ar-CH), 117.3 (Ar-C), 114.9, 106.8 (2 × Ar-CH), 105.6 (C-1), 101.4 (C-1′), 76.9 (C-4), 73.4 (C-5), 72.7 (C-3), 71.5 , 70.5 (C-2), 69.8 (C-2′), 68.0 (C-4′), 62.7 (C-6′), 62.0 (C-6), 52.8 (COOMe), 21.1–20.4 (7 × COMe). Anal. Calcd for C43H46F3NO20: C, 54.15; H, 4.86%; N, 1.47%; Found: C, 54.18; H, 4.89%; N, 1.50%.The glycosylation of 8α ,13  in anhyd THF (20.0 mL) was cooled to 0 °C and treated with dry PPh3  and di-isopropyl azodicarboxylate (DIAD) or di-2-methoxyethyl azodicarboxylate (DMEAD) . The reaction mixture was stirred at room temp until TLC analysis showed the complete disappearance of the starting compound. The reaction mixture was diluted with CH2Cl2 (35.0 mL) and washed with satd aq Na2HCO3 (35 mL). The aqueous phases were extracted with CH2Cl2 (2 × 25 mL) and the combined organic layers were dried over MgSO4, filtered and concentrated under diminished pressure. The crude residue was subjected to flash chromatography on silica gel.A solution of appropriate 6-OH-sugar 11 , 14 [20]D +34.2 (c 0.89 in CHCl3); 1H NMR  δ: 8.13 , 7.81 , 7.77–7.64 , 7.55–7.39 , 7.22 , 5.33 , 5.23 , 4.90 , 4.86 , 4.68 , 4.52 , 4.28 , 3.92 , 3.44 , 2.04, 2.02, 1.95 ; 13C NMR  δ: 171.0–170.8 (3 × COMe), 160.5 (COOMe), 140.5, 139.4, 137.2, 130.1 (4 × Ar-C), 130.1, 129.1, 128.3 (Ph-CH), 124.3–123.3 (CF3 + Ar-C), 120.0 (Ar-CH), 117.6 (Ar-C), 112.7, 105.4 (2 × Ar-CH), 99.5 (C-1), 78.9 (C-6), 72.7 (C-2), 70.1 (C-3), 69.4 (C-5), 66.4 (C-4), 55.9 (OMe), 52.9 (COOMe), 22.1, 21.0, 20.9 (3 × COMe). Anal. Calcd for C30H30F3NO11: C, 56.52; H, 4.74%; N, 2.20%; Found: C, 56.55; H, 4.77%; N, 2.23%.Mitsunobu reaction applied to 14  was performed with DMEAD  in accordance with the general procedure. The reaction was stopped after 4 h and the crude product was purified by flash chromatography on silica gel (65:35 hexane-EtOAc) gave pure 16  as a yellow solid; Rf 0.53 (1:1 hexane-AcOEt); mp 191–194 °C (crom); [α]D –11.5 (c 1.0 in CHCl3); 1H NMR  δ: 8.04 , 7.80 , 7.76 , 7.73 , 7.59–7.40 , 7.18 , 5.27 , 5.10 , 4.93 , 4.63 , 4.56 , 4.49 , 4.10 , 3.91 , 3.33 , 2.02, 2.00, 1.95 ; 13C NMR : δ 170.9–170.4 (3 × COMe), 160.5 (COOMe), 140.5, 139.5, 137.2, 130.4 (4 × Ar-C), 130.0, 129.1, 128.3 (Ph-CH), 124.3–123.3 (CF3 + Ar-C), 120.1 (Ar-CH), 117.5 (Ar-C), 112.5, 105.4 (2 × Ar-CH), 102.1 (C-1), 78.4 (C-6), 73.5 (C-3), 72.5 (C-5), 71.9 (C-2), 69.2 (C-4), 57.3 (OMe), 52.9 (COOMe), 21.0–20.8 (3 × COMe). Anal. Calcd for C30H30F3NO11: C, 56.52; H, 4.74%; N, 2.20%; Found: C, 56.54; H, 4.76%; N, 2.24%.Mitsunobu reaction applied to 15 D + 28.5 (c 0.96 in CHCl3); 1H NMR  δ: 7.99 , 7.78 , 7.77–7.72 , 7.54–7.38 , 7.18 , 5.46 , 5.10 , 5.03 , 4.64 , 4.62 , 4.51 , 4.35 , 3.91 , 3.35 , 2.07, 2.01, 1.92 ; 13C NMR  δ: 171.4, 170.9, 170.7 (3 × COMe), 160.5 (COOMe), 140.5, 139.6, 137.2 130.8 (4 × Ar-C), 130.0, 129.5, 128.3 (Ph-CH), 124.3–123.2 (CF3 + Ar-C), 120.0 (Ar-CH), 117.4 (Ar-C), 112.5, 105.4 (2 × Ar-CH), 102.5 (C-1), 78.9 (C-6), 71.7 (C-3), 71.6 (C-5), 69.7 (C-2), 69.1 (C-4), 57.5 (OMe), 52.9 (COOMe), 20.9, 20.8, 20.7 (3 × COMe). Anal. Calcd for C30H30F3NO11: C, 56.52; H, 4.74%; N, 2.20%; Found: C, 56.57; H, 4.75%; N, 2.25%.Mitsunobu reaction applied to 20 D -14.1 (c 1.0 in CHCl3); 1H NMR  δ: 8.05 , 7.77 , 7.76–7.72 , 7.56–7.40 , 7.25 , 4.92 , 4.79 , 4.73 , 4.68 , 4.39 , 4.30 , 4.29–4.21 , 4.16 , 4.15–3.96 , 3.99 , 3.96 , 3.83 m, , 3.29, 3.24 , 2.06 , 1.49, 1.38 , 1.28 , 1.27, 1.25 ; 13C NMR : δ 170.3 (COMe), 160.3 (COOMe), 140.6, 139.8, 137.6, 131.4 (4 × Ar-C), 129.8, 128.9, 128.4 (Ph-CH), 124.7–123.5 (CF3 + Ar-C), 120.2 (Ar-CH), 117.3 (Ar-C), 112.3 (Ar-CH), 111.1, 110.8, 108.5 (3 × CMe2), 106.1 (C-1), 105.6 (Ar-CH), 101.0 (C-1′), 78.7 (C-6′), 78.4 (C-5), 78.2 (C-3), 77.6 (C-3′), 76.7 (C-2), 76.0 (C-4), 74.7 (C-4′), 73.3 (C-2′), 70.5 (C-5′), 65.2 (C-6), 56.2, 54.5 (2 × OMe), 52.8 (COOMe), 28.1, 27.7, 26.8, 26.5, 26.3, 24.8 (3 × C2Me), 21.1 (COMe). Anal. Calcd for C42H52F3NO15: C, 58.13; H, 6.04%; N, 1.61%; Found: C, 58.16; H, 6.07%; N, 1.64%.Mitsunobu reaction applied to 20   in a 2:3 mixture of CH2Cl2-MeOH (46.0 mL) was cooled at 0 °C and treated with a methanolic solution of MeONa . The reaction mixture was stirred at room temp until TLC analysis showed the complete disappearance of the starting material (2–7 h) and the formation of a lower moving product. The solution was neutralized with Amberlite® IR120 H resin and the suspension was filtered and concentrated under diminished pressure. Purification of crude product by crystallization or trituration with Et2O afforded pure NHI-glycoconjugates 7α, 7β, 10β, 13, 18, or 19.To a solution of appropriate acetylated NHI-glycoconjugates 6α  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . The crude residue  was constituted (NMR) exclusively by a title compound 7α. The trituration of the crude product (52 mg) with Et2O afforded a sample of pure 7α  as a yellow solid; Rf 0.54 (9:1 EtOAc-MeOH), mp 159–162 °C (Et2O); [α]D -15.3 (c 0.63 in MeOH); 1H NMR  δ: 8.41 , 7.86–7.62 , 7.48–7.33 , 7.27 , 5.37 , 4.51 , 4.15 , 4.08 , 4.00 , 3.95 , 3.91 , 3.80–3.65 , 3.36 ; 13C NMR  δ: 161.5 (CO), 140.6, 139.6, 137.5, 131.9 (4 × Ar-C), 129.7, 128.7, 128.0 (Ph-CH), 125.2–123.0 (CF3 + Ar-C), 119.8 (Ar-CH), 118.1 (Ar-C), 113.4 (Ar-CH), 109.4 (C-1), 106.4 (Ar-CH), 75.1 (C-5), 70.4 (C-2), 69.9 (C-3), 69.3 (C-4), 61.9 (C-6), 52.8 (OMe). Anal. Calcd for C23H22F3NO8: C, 55.54; H, 4.46%; N, 2.82%; Found: C, 55.57; H, 4.49%; N, 2.86%.Deacetylation of 6β  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . The crude residue  was constituted (NMR) exclusively by a title compound 7β. The trituration of the crude product with Et2O afforded a sample of pure 7β  as a white solid; Rf 0.83 (9:1 EtOAc-MeOH); mp 207–210 °C (Et2O); [α]D +39.4 (c 0.505 in MeOH); 1H NMR  δ: 8.24 , 7.71 , 7.68–7.73 , 7.48–7.39 , 7.38–7.32 , 7.27 , 5.09 , 3.95 , 3.94 , 3.81 , 3.67 , 3.62 , 3.60 , 3.49 ; 13C NMR  δ: 161.8 (CO), 140.5, 139.5, 137.4, 132.1 (4 × Ar-C), 129.6, 128.9, 128.4 (Ph-CH), 125.4–123.6 (CF3 + Ar-C), 119.7 (Ar-CH), 118.3 (Ar-C), 113.6 (Ar-CH), 110.3 (C-1), 106.8 (Ar-CH), 76.7 (C-5), 74.3 (C-3), 72.3 (C-2), 69.9 (C-4), 62.2 (C-6), 53.4 (COOMe). Anal. Calcd for C23H22F3NO8: C, 55.54; H, 4.46%; N, 2.82%; Found: C, 55.57; H, 4.49%; N, 2.85%.Deacetylation of 9β  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . The crude residue  was constituted (NMR) exclusively by a title compound 10β. The trituration of the crude product (37 mg) with Et2O afforded a sample of pure 10β  as a white solid: Rf 0.10 (9:1 EtOAc-MeOH); mp 199–202 °C. [α]D +63.3 (c 0.59 in MeOH); 1H NMR  δ: 8.27 , 7.83–7.80 , 7.55–7.49 , 7.10 , 5.78 , 5.14–5.01 , 4.87–4.78 , 4.69–4.61 , 4.56–4.43 , 4.31–4.16 , 3.88 , 3.46–3.22  δ: 159.7 (CO), 139.0, 139.0, 137.0, 130.0 (4 × Ar-C), 129.2, 128.2, 127.3, 127.2 (Ph-CH), 124.0–121.8 (CF3 + Ar-C), 118.8 (Ar-CH), 116.7 (Ar-C), 113.5, 113.0 (2 × Ar-CH), 107.7 (C-1), 103.7 (C-1′), 79.4 (C-4), 75.6 (C-5′), 74.8, 74.3 , 73.3, 72.0 , 70.5 (C-2′), 68.1 (C-4′), 60.4, 60.3 , 52.4 (COOMe). Anal. Calcd for C29H32F3NO13: C, 52.81; H, 4.89%; N, 2.12%; Found: C, 52.84; H, 4.91%; N, 2.15%.Deacetylation of 12  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . The crude residue  was constituted (NMR) exclusively by a title compound 13. The crystallization (Et2O-hexane) of crude product (42 mg) afforded a sample of pure 13  as a pale yellow solid; Rf 0.69 (9:1 EtOAc-MeOH); mp 59–62 °C (Et2O-hexane); [α]D +21.6(c 0.92 in MeOH); 1H NMR  δ: 7.95 , 7.67–7.61 , 7.49–7.32 , 7.16 , 4.81 , 4.64 , 4.53 , 4.19 , 4.02 , 3.93–3.75 , 3.89 , 3.35 ; 13C NMR  δ: 161.2 (CO), 140.6, 139.5, 136.8, 131.9 (4 × Ar-C), 129.7, 128.6, 128.1 (3 × Ph-CH), 124.8–122.7 (CF3 + Ar-C), 120.1 (Ar-CH), 117.2 (Ar-C), 112.0, 106.4 (2 × Ar-CH), 101.9 (C-1), 78.1 (C-6), 72.1 (C-3), 71.2 (C-2), 71.0 (C-5), 67.5 (C-4), 55.9 (OMe), 51.3 (COOMe). Anal. Calcd for C24H24F3NO8: C, 56.36; H, 4.73%; N, 2.74%; Found: C, 56.39; H, 4.75%; N, 2.77%.Deacetylation of 16  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . The crude residue  was constituted (NMR) exclusively by a title compound 18. The trituration of the crude product (40 mg) with Et2O afforded a sample pure 18  as a white solid; Rf 0.53 (9:1 EtOAc-MeOH); mp 211–214 °C (Et2O); [α]D -73.7 (c 0.66 in MeOH); 1H NMR  δ: 8.10 , 7.70–7.62 , 7.50–7.38 , 7.15 , 4.79 , 4.58 , 4.18 , 3.95 , 3.70 , 3.41–3.30 , 3.36 , 3.24 ; 13C NMR  δ: 161.2 (CO), 141.0, 140.1, 137.8, 131.8 (4 × Ar-C), 130.1, 129.1, 128.4 (Ph-CH), 124.9–123.2 (CF3 + Ar-C), 119.9 (Ar-CH), 117.8 (Ar-C), 112.7 (Ar-CH), 105.8 (C-1), 105.4 (Ar-CH), 80.1 (C-6), 77.8, 71.3 , 75.5 (C-5), 74.9 (C-2), 57.5 (OMe), 52.7 (COOMe). Anal. Calcd for C24H24F3NO8: C, 56.36; H, 4.73%; N, 2.74%; Found: C, 56.40; H, 4.76%; N, 2.78%.Deacetylation of 17  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . The crude residue  was constituted (NMR) exclusively by a title compound 19. The trituration of the crude product (52 mg) with Et2O afforded a sample pure 19  as a white solid; Rf 0.60 (9:1 EtOAc-MeOH); mp 191–194 °C (Et2O); [α]D –21.6 (c 0.70 in MeOH); 1H NMR  δ: 7.99 , 7.68–7.62 , 7.47–7.32 , 7.24 , 4.74 , 4.59 , 4.15 , 4.01 , 3.96 , 3.93 , 3.53 , 3.44 ; 13C NMR : δ 160.6 (CO), 140.3, 139.4, 139.3, 131.7 (4 × Ar-C), 129.2, 128.3, 127.7 (Ph-CH), 124.7–123.3 (CF3 + Ar-C), 119.6 (Ar-CH), 117.2 (Ar-C), 111.6, 105.8 (2 × Ar-CH), 104.5 (C-1), 79.0 (C-6), 73.5 (C-2), 72.5 (C-5), 71.3 (C-4), 69.5 (C-3), 57.2 (OMe), 52.4 (COOMe). Anal. Calcd for C24H24F3NO8: C, 56.36; H, 4.73%; N, 2.74%; Found: C, 56.38; H, 4.76%; N, 2.78%.Deacetylation of 21  was performed in accordance with the general procedure. The reaction was stirred until the starting compound was completely reacted . Purification of the crude product by flash chromatography on silica gel (1:1 hexane-EtOAc) gave pure 22  as a white foam; Rf 0.43 (1:1 hexane-EtOAc); mp 70–73 °C (chrom); [α]D -23.2 (c 1.1 in CHCl3); 1H NMR  δ: 8.03 , 7.79 , 7.76–7.71 , 7.54–7.39 , 7.23 , 4.70 , 4.63 , 4.51 , 4.46 , 4.30 , 4.29 , 4.23 , 4.15 , 4.14–4.00 , 4.03 , 3.94 , 3.88 , 3.55 , 3.43 , 3.27, 3.20 , 1.46, 1.37, 1.33, 1.29, 1.27, 1.26 ; 13C NMR  δ: 160.4 (CO), 140.7, 139.8, 137.5, 131.4 (4 × Ar-C), 130.0, 129.1, 128.6 (Ph-CH), 124.7–123.3 (CF3 + Ar-C), 120.3 (Ar-CH), 117.5 (Ar-C), 112.6 (Ar-CH), 110.7, 110.5, 109.0 (3 × CMe2), 106.2 (C-1), 105.5 (Ar-CH), 104.4 (C-1′), 80.0 (C-3), 78.6 (C-6′), 78.5 (C-3′), 78.3 (C-4), 77.8 (C-5), 74.5 (C-4′), 74.4 (C-2′), 72.6 (C-2), 71.0 (C-5′), 65.9 (C-6), 56.3, 54.2 (2 × OMe), 52.9 (COOMe), 28.4, 27.5, 27.0, 26.5, 26.3, 25.0 (3 × CMe2). Anal. Calcd for C40H50F3NO14: C, 58.18; H, 6.10%; N, 1.70%; Found: C, 58.21; H, 6.13%; N, 1.74%.Deacetylation of 22  in 80% aq AcOH (1.3 mL) was stirred at 80 °C. After 4 h, the TLC analysis (9:1 EtOAc-MeOH) showed the complete disappearance of the starting material and the formation of a lower moving product (Rf 0.10). The reaction mixture was concentrated under diminished pressure and repeatedly coevaporated with toluene (4 × 20 mL). Trituration of the residue (45 mg) with Et2O afforded an amorphous white solid  composed  by a α-anomer 23 (>95%). 1H NMR  δ: 8.15 , 7.92–7.83 , 7.57–7.41 , 7.15 , 6.40 , 5.23 , 4.92 , 4.83 , 4.72–4.67 , 4.58–4.47 , 4.34 , 4.12 , 3.91 , 3.78–3.48 , 3.40–3.20 ; 13C NMR  δ: 159.0 (CO), 138.8, 138.2, 135.8, 129.2 (4 × Ar-C), 129.3, 128.1, 127.6 (Ph-CH), 123.5–122.3 (CF3 + Ar-C), 118.8 (Ar-CH), 115.9 (Ar-C), 111.8, 104.1 (2 × Ar-CH), 104.0 (C-1′), 92.1 (C-1), 82.2 (C-4), 79.4 (C-6′), 72.7 (C-2′), 72.3 (C-5′), 72.0 (C-5), 71.7 (C-3), 70.3 (C-4′), 69.8 (C-2), 68.9 (C-3′), 60.7 (C-6), 52.4 (COOMe). Anal. Calcd for C29H32F3NO13: C, 52.81; H, 4.89%; N, 2.12%; Found: C, 52.84; H, 4.92%; N, 2.15%.A solution of 50 measurements were performed by fluorescence  to monitor the amount of consumed NADH. Assays were carried out in wells containing 200 μL of a reagents solution, dissolved in 100 mM phosphate buffer (pH = 7.4), in the presence of 200 μM pyruvate and 40 μM NADH. For the IC50 calculations of the compounds, seven different concentrations (in duplicate for each concentration) were used to produce the concentration–response curve. As well as the sample test wells, maximum and minimum controls were also included in each plate. After 15 min of incubation, the final measurements were carried out by using a Victor X3 Microplates Reader (PerkinElmer®). IC50 values were produced using GraphPad Prism software .The LDH inhibition activities of the compounds were evaluated against purified human lactate dehydrogenase isoform 5 . The ‘forward’ direction (pyruvate→lactate) of the lactate dehydrogenase reaction was conducted, and the IC−1 was reached. The minimization was carried out in a water environment model  using the MMFF force field and a distance-dependent dielectric constant of 1.0. The hLDH5 chain was extracted from the minimized average structure previously obtained by us [The seven compounds were built using Maestro 9.0 and were minimized using the conjugate gradient method until a convergence value of 0.05 kcal (mol Å)ed by us . AUTODOCed by us  was empled by us . The num50 values was performed under normoxic conditions as described previously [2 atmosphere for 72 h. Medium was removed and cells were fixed by the addition of 50 µL 10% trichloroacetic acid in water, 4 °C, to each well. Plates were incubated at 4 °C for at least one hour, and the sulforhodamine B colorimetric assay was performed to assess remaining biomass in each well. Cells treated with 1% DMSO were used as the 100% live control for biomass, and wells incubated with medium alone were used as the baseline zero biomass control. IC50 values were calculated using SoftMax Pro software .Determination of cellular ICeviously . Briefly2 atmosphere. Duplicate wells were prepared for each treatment. Following treatment, medium was collected, and 100 µL were added to 2 µL 50 mM chlorophenylalanine . Samples were concentrated, derivatized by a four-hour incubation with MTBSTFA + 1% TBDMCS  in acetonitrile at 85 °C, and immediately analyzed using GC-MS  and an electron impact ionization source. One microliter of each sample was injected using an automated injector, and a solvent delay of 8.20 min was implemented. The initial oven temperature was 120 °C, held for 5 min; then the temperature was increased at a rate of 10 °C min−1 until a temperature of 250 °C was reached. Temperature was then increased by 40 °C min−1 until a final temperature of 310 °C was reached. Total run time per sample was 22.5 min. Compounds were identified using AMDIS Chromatogram software  and programmed WIST and Niley commercial libraries. The integration area of lactate in each sample was divided by the integration area of CPA in the same sample to achieve a lactate/internal standard ratio. The ratios were averaged for duplicates, and percent lactate production over the vehicle was calculated for each independent experiment. The mean lactate production/vehicle was then averaged between three or more independent experiments.This assay was performed under normoxic conditions as previously described . BrieflyhLDH5, whereas conjugates at C(6) were completely devoid of any enzyme inhibitory activity. Among the anomerically-linked conjugates, monosaccharide derivatives 7α and 7β displayed potent antiproliferative activities against lung and cervical cancer cells, which are comparable to those observed with reference gluco-conjugate 2, whereas disaccharide-linked 10β proved to be much less potent in the same assays. Finally, the configuration of the anomeric carbon atom seems to strongly affect the ability of these compounds to reduce the cellular production of lactate. In fact, an α-anomeric configuration (7α) proved to be positively correlated to a significant and dose-dependent inhibition of lactate production in HeLa cancer cells, when compared to its β- configured anomer (7β). Overall, these results shed further light about the structural motifs of glycoconjugates that can be further developed with the aim of exploiting the Warburg effect and selectively counteract the peculiar metabolism displayed by cancer cells. The studies reported here, therefore, have the potential to help develop therapeutic agents characterized by fewer side effects.Seven NHI glycoconjugates, which differ for the kind of sugar and for the conjugation branching point, were prepared and studied as potential LDH inhibitors. The chemical strategy was aimed at successfully accessing a wide structural variation while keeping the synthetic plan simple. Therefore, C-6 and C-1 were selected as the two anchoring options, as it is quite straightforward to functionalize these positions in a large variety of carbohydrates. For the C-6 glycoconjugation, the Mistunobu protocol was employed, while classical trichloroacetimidate precursors were employed in the C-1 glycosylation reactions. For the galactose donor, it was possible to switch the glycosylation stereochemical outcome by changing the nature of the solvent used. Indeed, despite the presence of an acetate participating group at C-2, the alpha anomer was obtained with high stereoselectivity by exploiting the nitrile solvent effect. After cleavage of the protecting groups, the biological results of the resulting glycoconjugates revealed that only those linked at the anomeric carbon inhibited


Furthermore, experiments with aqueous disodium succinate and solutions of sodium sulfate in sodium hydroxide were carried out. The influence of concentration ratios and temperature was studied. The Spiegler and Kedem model as well as the Pusch model were applied to fit the experimental data. Additionally, scanning electron microscopy (SEM) and infrared (ATR–IR) measurements were performed to validate the chemical and thermomechanical stability of the membrane. The succinic acid retention varies with its degree of dissociation. In a fully dissociated form, the NaOH concentration shows no impact on the retention. In contrast, the retention of sulfate decreases with increasing NaOH concentration.Nanofiltration is considered to be an appropriate separation technique in the production of bio-based materials. For the utilization of process streams from the viscose-fiber production, understanding the separation behavior of organic compounds in highly alkaline solutions is necessary. Experiments with succinic acid in sodium hydroxide (NaOH) solutions with varying concentrations up to 5 mol L Furthermore, it can be used as a downstream process for the purification of process chemicals. A possible application is the removal of hemi celluloses and other wood degradation products from the highly caustic steeping lye. Hence, the lye could be recycled to the process and the demand of the make-up caustic solution can be significantly reduced. The total organic carbon (TOC) content can basically be divided into two fractions. The xylose oligo- and polymers with a molecular weight from ~600 to ~50,000 g mol−1) from steeping lye containing 18% sodium hydroxide was tested by Saurabh  which is declared as the main membrane polymer by the supplier.−1 represents the O–H stretching band. In contact with NaOH solution, a change in the ATR-IR diagram can be observed but is not related to any changes in the membrane polymer composition. These changes appear within the first hour and do not alter significantly over time.The band at a wavelength from 3200–3500 cm−1 represent aliphatic C-H stretching bonds which decrease in contact with NaOH solution. The bands at 1040 and 925 cm−1 refer to glycerol, which was used for membrane preparation or storage and is washed out immediately by the solutions. The band at 1400 cm−1 indicates decompositions of carbonates on the membrane. Carbonates were formed from carbon dioxide, which is dissolved in the solution. By longer exposure to the NaOH solution, the band increases slightly. None of the characteristic polymer bands changes significantly with the treatment time. Consequently, NP030 can be considered as chemically stable in alkaline solutions with concentrations up to 5 mol L−1 NaOH. This is in good agreement with the data published by Schlesinger [The bands at 2934 and 2879 cmBased on the retention results of aqueous solutions, it can be concluded that the organic salt (disodium succinate) behaves similarly to the inorganic sodium sulfate. A different retention behavior is observed in NaOH solutions. Sulfate ions are more affected by the presence of co-ions than succinate ions. Degree of dissociation is the factor with the highest impact on the retention of succinic acid. Consequently, the charge selectivity of the NP030 nanofiltration membrane could be verified with an organic salt by changing its degree of dissociation. A higher NaOH concentration in the feed solution leads to a significant decrease of permeate flux.The retention of completely dissociated succinate, is unaffected by the NaOH concentration. Hence, a dilution during the nanofiltration process may improve the process efficiency, as the flux increases. Furthermore, the selectivity between sulfate and succinate can be controlled by the NaOH concentration.The results of the ATR–IR measurements and the thermomechanical stress tests indicate an applicability of the NP030 membrane in highly alkaline conditions. Further performance tests with model solutions are necessary to prove this.Furthermore, it is shown, that the Spiegler and Kedem model appropriately describes the retention behavior of all investigated species, even in five molar NaOH.


Atrial fibrillation (AF) is the most common cardiac arrhythmia, with its incidence making up nearly one‐third of all hospital admissions. Atrioventricular block (AVB) is a conduction abnormality along the atrioventricular node or the His‐Purkinje system. The relationship between atrioventricular conduction block and AF is controversial.This study is designed to observe whether there is a correlation between AVB and AF, and which type of AVB has the most obvious correlation with AF.This study retrospectively reviewed 1345 patients. We classified the AVB according to the AVB classification criteria. One hundred and two patients were excluded, and the final total sample size was 1243 patients, including 679 patients in the AF group  and 564 patients in the non‐AF group . AF group and non‐AF group were compared to observe the relationship between AVB and AF.I AVB have a relative statistical risk of 1.927  with the occurrence of AF. II AVB occupied the largest proportion, accounting for 67 cases (9.87%), and the statistical risk of II AVB in AF is 16.845 . III AVB has a comparative statistical risk of 17.599 .The The three types of AVB in the AF group were significantly higher than that in the non‐AF group. II AVB has the highest incidence rate compared with other types of AVB in the AF group. AVB can be used as a risk factor for AF occurrence. Second‐degree AVB remains poorly understood despite major advances in cardiac electrophysiology in the past three decades.At present, there are not too much controversies about third‐degree AVB. Third‐degree AVB is generally accompanied by the pathological state of the disease and is closely related to the occurrence of the disease. Therefore, the current controversy over third‐degree AVB is not too much exalted. The purpose of this study is to analyze the occurrence of atrioventricular conduction block in patients with AF and non‐AF, and to explore the relationship between the AF and the three types of atrioventricular conduction block, and find out which one is the most common type of AVB occurred in AF patients.2The clinical data process of AF and non‐AF groups Figure :Figure The inclusion and exclusion criteria of AF and non‐AF:More than two episodes of AF occurred and recorded by ECG before.Aged from 18 to 80 years old.The inclusion criteria of AF:Valvular heart disease and rheumatic heart disease excluded.Dilated cardiomyopathy, hypertrophic cardiomyopathy, and ischemic cardiomyopathy excluded.Congenital heart disease excluded.Anemia excluded.Hyperthyroidism excluded.Cardiac insufficiency (EF < 35%) excluded.The exclusion criteria of AF:Diagnosis of AVBI AVB:The PR interval is greater than 0.2 seconds, and each atrial excitement is transmitted to the ventricle.II AVB of Type I:ECG shows a gradual extension of time from the atrium to the ventricle, and an atrial excitement cannot be transmitted to the ventricle.II AVB of Type II:It means that the atrial impulse cannot be transmitted to the ventricle, and the ECG is represented as the leakage of QRS interphase.III AVB:It means that all atrial excitations cannot be transmitted to ventricles, and the activities of atria and ventricles are independent, respectively, and unrelated to each other.Case selectionThis experiment is designed to study the relationship between AVB and AF. The study retrospectively reviewed 1345 consecutive patients from August 2017 to August 2018, who were diagnosed with AF and non‐AF. All patients were enrolled from the Atrial Fibrillation Center, Department of Cardiology, the First Affiliated Hospital of Xi'an Jiaotong university. The clinical data were obtained from medical record review and analyzed in AF center. Patients were divided into the AF group and non‐AF group. Among the total of 1345 patients, 102 patients who did not meet the inclusion criteria were excluded:26 cases were diagnosed with Valvular heart disease; 12 cases had dilated heart disease; 6 cases had hypertrophic heart disease; 4 cases had ischemic cardiomyopathy; 17 cases had Congenital heart disease; 18 cases had anemia; 17 cases had hyperthyroidism; and 4 presented with severe left ventricle dysfunction (EF < 35%). Meanwhile, patients in the AF group must comply with the following conditions: (a) more than two episodes of AF occurred and recorded by ECG before and (b) aged from 18 to 80. The final total sample size was 1243 patients, including 679 patients in the AF group  and 564 patients in the non‐AF control group .3t test and Pearson χ2 test were used to compare the continuous variables and the classified variables. Single factor analysis method was used to preliminarily analyze related risk factors. Statistically significant factors were taken into multiple factors analysis model by using negative binomial regression for the analysis of the related factors. The aim is to evaluate whether there is statistical significance between AF and corresponding covariates in the negative binomial regression model. Finally, the correlation between AVB interval and AF was found.Independent sample 44.1From Table P < 0.05) with the occurrence of AF. II AVB occupies the largest proportion, accounting for 67 cases (9.87%), and the statistical risk of II AVB of AF is 16.845  compared with the non‐AF group. III AVB has a relative statistical risk of 17.599  in the AF group compared with the non‐AF group.The I AVB has a relative statistical risk of 1.927 .According to the analysis results of this study, second atrioventricular conduction block occupies the largest percentage, accounting for 67 cases (9.87%) among the three types of atrioventricular conduction block in the AF group. The correlation between second atrioventricular conduction block and AF is the most obvious (4.2P < 0.000), SBP , DBP , RBC , PLT , CHOL , TG , FDP , HGB , K , QRS , left atrial diameter , left ventricular diameter , EF , AVB , CO , hypertension , coronary heart disease , and cerebral infarction . The above statistical factors were then included in the multivariate analysis model for further correlation analysis.The statistical results are shown in Table 4.3P < 0.000), age , left ventricular diameter , cerebral infarction , hypertension , coronary heart diseases , diabetes , and gender . We set AF as a dependent variable, related factors are included into the multifactor analysis model. Finally, after adjusting age, gender, left atrial diameter, hypertension, diabetes, coronary heart diseases, and cerebral infarction, the no AVB/AVB still have a relative statistical risk of 0.811  with the occurrence of AF.We select variables to be included in the multivariate regression model by consulting relevant literature. Variables to be included are selected by DAG diagram and results of univariate analysis. As it is shown in Table 5AF is the most common sustained cardiac arrhythmia in clinical practice, and its incidence is increasing rapidly worldwide accounting for nearly one‐third of all hospital admissions.5.1The relationship between I AVB and arrhythmia has been controversial. The reported results of studies on the relationship between I AVB and AF are inconsistent. Some studies suggest that individuals with first‐degree AVB are at a substantially increased risk of future AF (~2‐fold)P < 0.05) with the occurrence of AF. I AVB is a risk factor for AF.Through the statistical analysis of the AF data in the Atrial Fibrillation Center of The First Affiliated Hospital of Xi'an Jiaotong University during 2017‐2018, it is found that: I AVB is increased in the AF group  compared with the non‐AF group . The I AVB has a relative statistical risk of 1.927  compared with the non‐AF group. II AVB is an obvious risk factor for AF.The relationship between II AVB and arrhythmia is not as controversial as I AVB is. By data analysis, among the three types of AVB in the AF group, II AVB occupied the largest proportion, accounting for 67 cases (9.87%); II AVB holds the statistical risk of 16.845  in the AF group compared with the non‐AF group. III AVB is also an obvious risk factor for AF.At present, there are not too much reports and controversies over III AVB and AF. III AVB is usually accompanied with the pathological state of the disease and is closely related to the occurrence of the disease. Therefore, the current controversy over III AVB is not too much great. Through the statistical analysis of the data, we draw the conclusion that: III AVB has a relative statistical risk of 17.599  with the occurrence of AF. II AVB takes the largest percentage, accounting for 67 cases (9.87%), and the statistical risk of II AVB in AF is 16.845  compared with the non‐AF group. III AVB has a relative statistical risk of 17.599  in the AF group compared with the non‐AF group. Although I AVB has been controversial, this study agrees that I AVB is a risk factor for AF. The comparatively relevant and highest incidence of AVB is II AVB. Second‐degree AVB is the most correlated type with AF among the three types of AVB. At the same time, third‐degree atrioventricular conduction block is also strongly related to the occurrence of AF. The results point out that II and III degree AVB are also risk factors for AF.To sum up, the three types of AVB are risk factors for AF. The proportion of three types of AVB in the AF group is, respectively, increased compared with the non‐AF group, and all of them have statistical significance. The I AVB has a relative statistical risk of 1.927 (95% CI: 1.160‐3.203, The three types of AVB are risk factors for AF occurrence, and II AVB has the highest proportion and high correlation with the occurrence of AF.5.4This study is an observational research conducted in a single center. The research center is relatively single, and the sample representation is not enough.The authors declare no potential conflict of interests.Figure S1Click here for additional data file.


ZFHX3 (16q22), a major regulator of inflammation, has been reported linking to atrial fibrillation (AF) by genome-wide association studies. Inflammation is known to be a strong predictor of atrial fibrillation recurrence after ablation, so we examined the association of the ZFHX3 SNP rs2106261 to inflammation marker expression and recurrence after AF ablation.The single nucleotide polymorphism (SNP) rs2106261 in the transcription factor gene ZFHX3 SNP rs2106261 and compared the minor (T) allele frequency between 362 paroxysmal AF (PAF) patients underwent pulmonary vein isolation (PVI) and 627 non-AF controls. We also analyzed associations between ZFHX3 SNP rs2106261 genotype and recurrence rate after pulmonary vein isolation and the inflammation markers.We genotyped ZFHX3 SNP rs2106261 was significantly higher in AF patients than non-AF controls . Multivariable analysis revealed that the minor allele (T) decreased AF recurrence rate after pulmonary vein isolation . Further, neutrophil/lymphocyte (N/L) ratio, C-reactive protein (CRP), and interleukin-6 (IL-6) expression levels were lower in PAF patients with the ZFHX3 SNP rs2106261 minor allele (TT+TC) than in CC patients .The minor (T) allele frequency of the ZFHX3 SNP rs2106261 minor allele is associated with lower AF recurrence rate after pulmonary vein isolation. Low baseline inflammation conferred by this allele may reduce AF recurrence risk.The  ZFHX3  has been associated with AF by genome-wide association studies (GWASs) . Longer duration of AF and longer CSRT also tended to increase AF recurrence risk but these trends did not reach statistical significance. Multivariable analysis revealed that presence of the ZFHX3 SNP rs2106261 minor allele (TT or TC) was independently associated with lower AF recurrence rate after RFCA (p = 0.04).Univariate analysis revealed that presence of the ZFHX3 SNP rs2106261 genotypes and the inflammation markers N/L ratio, CRP level, and plasma IL-6 level. All three were lower in patients with the ZFHX3 SNP rs2106261 minor allele than in those without .p = 0.018; CRP: CC 0.103 ± 0.009 mg/dl, TT + TC 0.076 ± 0.007 mg/dl, p = 0.016; IL-6: CC 60.3 ± 3.0 pg/ml, TT + TC 52.8 ± 2.3 pg/ml, p = 0.04).The N/L ratio, CRP level and plasma IL-6 level were lower in the TT + TC group than the CC group  were more likely to maintain sinus rhythm after RFCA.In our study, we confirmed the previously reported association between AF and  by GWAS  in JapanZFHX3 SNP rs2106261 minor allele (TT or TC genotype) than in those without (CC genotype).We also found that inflammation markers  were lower in PAF patients with the ZFHX3 gene SNP rs12373097 was associated with AF recurrence in both PAF and persistent AF patients [ZFHX3 gene SNP rs2106261 was an independent predictor of good responders to RFCA among long-standing persistent AF patients [ZFHX3 polymorphisms did not predict clinical recurrence of AF after catheter ablation among PAF and persistent AF patients [ZFHX3 SNPs to AF recurrence after RFCA remain controversial. Although different SNPs (rs12373097 vs. rs2106261) may account for the discrepancy, cohort and treatment heterogeneity may also contribute. First, these previous studies analyzed recurrence rate in a combined cohort of persistent AF and PAF patients. Second, strategies for RFCA are multifarious, especially in persistent AF patients. In the present study, we enrolled only PAF patients schedule for first AF RFCA and the treatment strategy was PVI exclusively. In this restricted cohort, we found that the ZFHX3 SNP rs2106261 minor allele (T) was independently associated with low AF recurrence rate.Husser et al. reported that the patients . Howeverpatients . Thus, tA systematic review of predictors for atrial fibrillation recurrence after ablation  reportedZFHX3 SNP rs2106261 minor allele (T) is associated with high AF occurrence but low recurrence after AF ablation. The ZFHX3 SNP rs2106261 was linked to AF onset by a GWAS, but the precise mechanism of association with AF pathogenesis has not been elucidated. Similarly, the mechanisms underlying this discrepancy between onset risk and recurrence risk remain to be revealed. The minor allele of ZFHX3 SNP rs2106261 was also associated with high BMI [ZFHX3 SNP rs2106261 genotypes. ZFHX3 knockdown in atrial myocytes was reported to dysregulate calcium homeostasis and increase atrial arrhythmogenesis, ultimately contributing to AF occurrence [ZFHX3 SNP and PITX2 SNP. They also reported that ZFHX3 positively regulated PITX2c expression [PITX2 regulates process that pulmonary mesenchyme differentiates in myocardium, initiates a phase of rapid proliferation and expands to form the myocardial sheet around the pulmonary vein branches. Overexpression of PITX2 may promote the proliferation of myocardial sleeve around the pulmonary vein branches and increase automaticity from there [ZFHX3 SNP rs2106261 may modulate PITX2c expression levels and associate with high occurrence of AF. We performed PV isolation and paroxysmal AF patients with ZFHX3 SNP minor allele have lower AF recurrence rate than those without. Given that ZFHX3 SNP acts through PITX2c expression levels, AF trigger in paroxysmal AF patients with ZFHX3 SNP minor allele was related to PV and they easily cured by PVI.In this study, we revealed that the high BMI , but in currence . Huang eom there . ZFHX3 SZFHX3 SNP rs2106261 minor allele is associated with low baseline inflammation levels. There are many reports that inflammatory factors are involved in AF occurrence and recurrence [ZFHX3 SNP rs2106261 minor allele may reduce the risk of recurrence. If the paroxysmal AF patients possess the ZFHX3 SNP minor allele, we can preprocedurely select only PVI as AF ablation strategy.We also reported in this paper that the currence , 24. Howcurrence . Lin et ZFHX3 SNP rs2106261 was associated with PAF among Japanese patients in our replication study. The ZFHX3 SNP rs2106261 minor allele (T) was associated with lower AF recurrence rate after PVI, and low baseline inflammation may contribute to this reduced recurrence risk. The ZFHX3 SNP rs2106261 may be an important genetic marker for prediction of AF non-recurrence after AF RFCA and a useful guide for selecting therapeutic interventions.The S1 Table(DOCX)Click here for additional data file.


This study was undertaken to detect if free fatty acids (FFA) induce hepatocyte senescence in L-02 cells and if huperzine A has an anti-aging effect in fatty liver cells.L-02 cells were treated with a FFA mixture  at different concentrations. Cell viability and fat accumulation rate were assessed by a Cell Counting Kit 8 and Nile Red staining, respectively. The mixture with the highest cell viability and fat accumulation rate was selected to continue with the following experiment. The L-02 cells were divided into five groups, including the control group, FFA group, FFA + 0.1 μmol/L huperzine A (LH) group, FFA + 1.0 μmol/L huperzine A (MH) group and FFA + 10 μmol/L huperzine A (HH) group, and were cultured for 24 h. The expression of senescence-associated β-galactosidase  was detected by an SA-β-gal staining kit. The expression levels of aging genes were measured by qRT-PCR. The expression levels of apoptosis proteins were detected by a Western blot. ELISA kits were used to detect inflammatory factors and oxidative stress products. The expression of nuclear factor (NF-κB) and IκBα were detected by immunofluorescence.The FFA mixture  of 0.5 mmol/L had the highest cell viability and fat accumulation rate, which was preferable for establishing an in vitro fatty liver model. The expression of inflammatory factors (TNF-α and IL-6) and oxidants Malonaldehyde (MDA), 4-hydroxynonenal (HNE) and reactive oxygen species (ROS) also increased in the L-02 fatty liver cells. The expression levels of aging markers and aging genes, such as SA-β-gal, p16, p21, p53 and pRb, increased more in the L-02 fatty liver cells than in the L-02 cells. The total levels of the apoptosis-associated proteins Bcl2, Bax, Bax/Bcl-2, CyCt and cleaved caspase 9 were also upregulated in the L-02 fatty liver cells. All of the above genes and proteins were downregulated in the huperzine A and FFA co-treatment group. In the L-02 fatty liver cells, the expression of IκBα decreased, while the expression of NF-κB increased. After the huperzine A and FFA co-treatment, the expression of IκBα increased, while the expression of NF-κB decreased.Fatty liver cells showed an obvious senescence and apoptosis phenomenon. Huperzine A suppressed hepatocyte senescence, and it might exert its anti-aging effect via the NF-κB pathway. Currently, nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease, and it is a major public health hazard. The prevalence of NAFLD in western countries is variable, ranging from 24–46% . The estThe two-hit hypothesis is a wildly accepted mechanism of NAFLD. Fat deposition in the cytoplasm of the hepatocytes is claimed to be the first hit. Oxidative stress, lipid peroxidation and inflammation serve as the second hit, which contributes to the progression of steatosis to steatohepatitis. Increasingly more evidence indicates that age is an independent predictor of NAFLD . The preRecent studies have reported that there is a close association between oxidative stress and cellular senescence . To studHuperzine A, a selective AChE inhibitor, is widely used to treat Alzheimer’s disease . However2  supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 U/mL streptomycin  for 24 h. All the cell lines were maintained at 37 °C in a humidified incubator with an atmosphere of 5% CO2 .2O2 and NaOH solutions, respectively, and then, they were added to a 10% solution of free fatty acids (FFA) bovine serum albumin (BSA) , which was dissolved overnight at 55 °C with gentle shaking; then, the mixture was cooled to room temperature, filtered and stored at 4 °C in a refrigerator  were added to the Higerator .Stock solutions of 50 mmol/L sodium oleate and 50 mmol/L sodium palmitate were mixed at five different ratios  and were then conveniently diluted in culture medium containing 1% BSA to obtain the desired final concentrations  . To indu4 cells/well. Then, the cells were maintained at 37 °C for 24 h. Cell viability was determined with Cell Counting Kit 8 (CCK8)  following the manufacturer’s instructions. After treatment with FFA mixture for the times and concentrations indicated, cell viability was assessed by incubation with 10 μl of tetrazolium substrate for 1 h at 37 °C, followed by measurement of absorbance at 450 nm  and were then stained with the Nile Red solution at a concentration of 1 mg/L in PBS for 15 min at 37 °C. Then, the cell monolayers were washed with PBS. The images were recorded at 543-nm excitation and 598 nm emission wavelengths using fluorescence microscopy  to analyze the accumulation rate of the lipids in the L-02 cells  group, FFA + 1.0 μmol/L huperzine A (MH) group and FFA + 10 μmol/L huperzine A (HH) group. In the huperzine A treatment group, the L-02 cells were pretreated with RPMI 1640 containing different concentrations of huperzine A for 12 h, after which the FFA mixture was added to the culture system, with exception of the control group; the groups were then incubated for a further 24 h.A mixture of sodium oleate and sodium palmitate, mixed at a concentration of 0.5 mmol/L with a ratio of 2:1 , was selected as the optimum FFA mixture. The L-02 cells were seeded in six-well plates at a density of 2.0 × 10Apoptotic cells were assessed using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I  following the manufacturer’s instructions. L-02 cells in each group (mentioned above) were digested with trypsinization after treated for 24 h. Detached cells were collected by centrifugation at 1,500 rpm for 6 min. Then the harvested cells were washed by PBS for twice, resuspended in the binding buffer, and incubated with Annexin V-FITC and propidium iodide staining solution. Samples of 10,000 stained cells were analyzed using a flow cytometer  .A senescence-associated β-galactosidase staining  kit  was used for SA-β-gal staining. The cells in each group were washed three times using PBS and were then fixed in the SA-β-gal fixing solution for 15 min after removing the cell-culture medium. Then, the cells were again washed three times in PBS and were incubated overnight at 37.0 °C with the SA-β-gal working solution. The cells were stained with DAPI for 1 h and were then dehydrated and stored at 4 °C. The images were captured at 200× magnification using a regular microscope and a fluorescence microscope . The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments for each experiment. Each sample was repeated three times.4 cells/well and were maintained at 37 °C for 24 h. A total of 10 µm Edu was added to the 24-well plate, and it was then co-incubated with the cells for 4 h. Then, the cells were fixed with cold 4% paraformaldehyde in 0.1 M PBS for 30 min and were rinsed with PBS three times. Next, the cells were incubated with 0.2% Triton X-100 for 10 min and were then rinsed with PBS three times. The Edu antibody was diluted with cell-culture medium at a ratio of 1:1,000, and 300 µL of the solution was added to each well. The cells were incubated at 4 °C overnight and rinsed with PBS three times, and the nuclei were stained using 0.5 µg/mL DAPI. The images were captured using a regular microscope and a fluorescence microscope . Each sample was repeated three times.For the Edu  immunofluorescence, the L-02 cells were seeded in 24-well plates with cover slips at a concentration of 2.0 × 10The L-02 fatty cells were treated by huperzine A for 24 h, and then, the cell culture supernatants were collected. TNF-α and IL-6 ELISA kits  were used to measure the expression of the inflammatory factors TNF-α and IL-6. Malonaldehyde (MDA), 4-hydroxynonenal (HNE) and reactive oxygen species (ROS) kits  were used to measure the expression of MDA, HNE and ROS to analyze oxidative stress. The content of the inflammatory factors and oxidative stress products above were measured according to the kit instructions. Each sample was measured in triplicate.The cells were lysed with RIPA lysis buffer  with protease inhibitor cocktails  on ice for 5 min. Then, the lysates were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected in a new fresh EP tube. Protein was extracted with chilled lysis buffer containing 1 mmol/L PASF. The protein concentration was detected using a BCA protein assay kit . The protein samples were separated on SDS–PAGE gels and were then transferred to PVDF membranes. The membranes were first probed with primary antibodies overnight at 4 °C and were then incubated with an HRP-conjugated goat anti-rabbit/mouse secondary antibody at room temperature for 1 h; then, the blots were detected using the ECL system. The primary antibodies were β-actin , 8-oxoG , Bcl-2 , Bax , cytochrome C , cleaved caspase 9  and procaspase 9 . The secondary antibodies were goat anti-rabbit and goat anti-mouse that were HRP-conjugated . The GAPDH, 8-oxoG, CytC, cleaved caspase 9 and procaspase 9 antibodies were purchased from Proteintech , and Bcl-2 and Bax were from Cell Signaling Technology . The secondary antibodies were from Jackson .® MMLV Reverse Transcriptase kit . For the RT-PCR, the cDNA and primers were prepared with a SYBR Green qPCR SuperMix Kit , according to the instruction manual. GAPDH was used as a reference gene. The following primers were used: p16, forward 5′-CCTGGAGGCGGCGAGAACAT-3′ and reverse 5′-CGCGGGATGTGAACCACGAAA-3′; p53, forward 5′-CCTCCTCAGCATCTTATCCG-3′ and reverse 5′-CAACCTCAGGCGGCTCATAG -3′; p21, forward 5′-ACCGAGACACCACTGGAGGG-3′ and reverse 5′-CCTGCCTCCTCCCAACTCATC-3′; pRb, forward 5′-CCAGGCGAGGTCAGAACAGG-3′ and reverse 5′-CCTCTGGAAGTCCATTAGATGTTAC-3′; and GAPDH, forward 5′-AATCCCATCACCATCTTCCAG-3′ and reverse 5′-ATCAGCAGAGGGGGCAGAGA-3′. The primers above were designed using Primer Express software 5.0 . They were synthesized by Suzhou Genewiz Biology Company . One times cDNA template, two times cDNA template and 0.5 times cDNA template were used to detect dose-effect relationship of p16, p53, p21, pRb and GAPDH respectively on ABI 7500 fluorescence quantitative PCR apparatus. The results showed that the quantitative relationship between the fluorescence of PCR products and the cDNA template was R2 > 0.99, and their line slope were comparable, which proved that these primers were appropriate and the experimental results based on this method were accurate and credible. Thus we calculated relative fold changes in gene expression normalized to GAPDH by the ΔΔCT method using the equation 2−ΔΔCT. The amplification conditions were as follows: 95 °C for 5 min for initial denaturation, followed by denaturation at 95 °C for 10 s, and a combined annealing and extension at 60 °C for 30 s through 40 amplification cycles. Each sample was measured in triplicate.The cells were rinsed with PBS three times after removing the culture medium. Total RNA was extracted from the experimental cells with Trizol  for cDNA synthesis. A total of 2 µg of RNA was used to generate cDNA with the SMART4 cells/well and were maintained at 37 °C for 24 h; then, the cells were rinsed with PBS three times, 3 min each time. Then, the cells were fixed with 4% paraformaldehyde for 15 min and rinsed with PBS three times, 3 min each time. Next, the cells were incubated with 0.5% Triton X-100 for 20 min at room temperature and were then rinsed with PBS three times, 3 min each time. The PBS on the cover slips was dried with wipes, and normal goat serum was added on the cover slips. They were incubated for 30 min at room temperature. The goat serum on cover slips was dried with wipes, and then, the cover slips were first probed with primary antibodies overnight at 4 °C in a wet box. Then, the cover slips were rinsed with Phosphate Buffered Solution with Tween-20 (PBST) three times. The cover slips were dried with wipes and were incubated with secondary antibodies for 1 h at room temperature. Then, the cover slips were rinsed with PBST three times (3 min each time), and the nuclei were stained with DAPI. Then, the cover slips were rinsed with PBST 4 times (5 min each time), and the cover slips were dried with wipes. The cover slips were treated by antifade mounting medium. The images were captured using a fluorescence microscope . The primary antibodies were NF-κB p65 (diluted 1:100) , IκBα (diluted 1:100) , and NF-κB p-p65 (diluted 1:100) , and the secondary antibodies were goat anti-mouse with HRP-conjugated .The L-02 cells were seeded in 24-well plates with cover slips at a concentration of 2.0 × 10P < 0.05 was considered a statistically significant difference.Data are presented as the means ± SD for at least three independent experiments for every group. Differences among groups were all compared using a one-way ANOVA, Fisher’s LSD test and fixed effects model. All the data were analyzed using GraphPad Prism  and SPSS 18.0 . P < 0.05). The viability of the L-02 cells incubated with FFA mixed at a ratio of 2:1  was higher than that for the other ratios (P < 0.05). The viability of the L-02 cells incubated with 0.25, 0.5, 0.75 and 1.0 mmol/L of the FFA mixture  were all higher than those incubated with 1.5 mmol/L of the FFA mixture (P < 0.05)  and concentrations  after 24 h of treatment. The results showed that the cell viabilities were different with different ratios of the FFA mixture. The FFA mixture  produced a significant toxicity at those ratios with a higher palmitic acid content  ( < 0.05) .P < 0.05)  had a higher lipid accumulation rate than did the other concentrations of FFA ( < 0.05) –1C. ThesP < 0.01). The expression levels of the SA-β-gal-positive cells in the FFA + LH, FFA + MH and FFA + HH groups were all lower than those in the FFA group (P < 0.01). The expression levels of the SA-β-gal-positive cells in FFA + HA group were the lowest (P < 0.01) , was selected to establish the L-02 fatty liver model in the following experiments. To determine the cell senescence status, immunofluorescence labeling for SA-β-gal was selected to analyze the number of aging cells. The results showed that the expression of the SA-β-gal-positive cells was nearly threefold in the FFA group compared with in the control group under fluorescence microscopy ( < 0.01) –2F. ThesP < 0.01). The expression levels were all higher in FFA + LH, FFA + MH and FFA + HH groups than in the FFA group (P < 0.01), but with no significant difference among FFA + LH, FFA + MH and FFA + HH groups (P > 0.05)  –2L.P < 0.01). The percentage of apoptotic cells were 5.690% ± 0.405%, 5.263% ± 0.276% and 3.640% ± 0.907% in LH, MH and HH groups, respectively, which were all lower than in the FFA group (P < 0.05) (Flow cytometry was used to detect cell apoptosis. The results showed that the percentage of apoptotic cells was increased from 3.127% ± 0.308% in control cells to 8.800% ± 0.089% in FFA-treated cells ( < 0.05) –3B. ThesP < 0.01), while the protein levels of Bcl2 were downregulated in the FFA group compared with the levels in the control group (P < 0.01). The expression levels of 8-oxoG, Bax, Bax/Bcl2, CytC and cleaved caspase 9 decreased in the FFA + LH, FFA + MH and FFA + HH groups compared with those of the FFA group, but with no significant difference among FFA + LH, FFA + MH and FFA + HH groups (P > 0.05)  –3I. ThesP < 0.01) (P < 0.01) (P < 0.01) (P < 0.01), but with no significant difference among FFA + LH, FFA + MH and FFA + HH groups (P > 0.05) (P < 0.01), and they decreased in the FFA + LH, FFA + MH and FFA + HH groups compared with those in the FFA group (P < 0.01) (P < 0.05)  –4D. All  < 0.01) –4D. To a < 0.01) –4F. The  > 0.05) –4F. To f < 0.01) –4I. In a < 0.05) –4I. ThesP < 0.01) (P < 0.01) (P < 0.01) (P > 0.05). These results suggested that the NF-κB-p-p65 pathway was activated after FFA treatment. Huperzine A might suppress cell senescence induced by FFA via the NF-κB pathway.To further confirm whether the NF-κB pathway plays an important role in cell senescence, we measured the levels of NF-κB-p-p65 and IκBα by an immunofluorescence assay. The mean optical density (MOD) value was used to analyze the quantitative of NF-κB-p-p65 and IκBα. The results showed that the MOD value of IκBα was decreased in the FFA group compared with that of the control group ( < 0.01) –5B. The  < 0.01) –5B. The  < 0.01) –5D. TherThe pathogenesis of NAFLD is widely interpreted by the “two-hit” hypothesis. Lipid metabolism disorder, such as an increased concentration of FFA and a disturbed fatty acid oxidation process, serve as the first hit in the liver. The treatment of HepG2 and L-02 cell lines with monounsaturated and saturated fatty acids is commonly used to make an in vitro NAFLD model and to study the mechanism of hepatic steatosis . In our The second hit is correlated with increasing oxidative stress in hepatocytes. In normal circumstances, oxidation and antioxidation are kept in a dynamic balance. Oxidation can exceed antioxidation if it is influenced by infection disease or the environment. ROS accumulate and damage the organ, a process called oxidative stress. Oxidative stress induces the upregulation of inflammatory factors. This study found that the levels of oxidants, such as MDA, 4-HNE and ROS, were higher in the L-02 fatty liver cells. The inflammatory factors, such as TNF-α and IL-6, were increased in the L-02 fatty liver cells as well. The above results revealed that the in vitro NAFLD model also represents some aspects of hepatic steatosis.Recent studies reveal that hepatocyte senescence predicts the progression of NAFLD . HepatocStudies report that oxidative stress plays an important role in cellular senescence and aging . Aging iHuperzine A is proved to have anti-inflammation and antioxidative effects in the immune and central nervous systems of mammals . Our preIt has been reported that oxidative stress induces the expression of p53 . The tumIn our study, we also found that the cell density was significantly decreased after FFA treatment compared with that of the control. The levels of apoptosis proteins, such as Bcl2, Bax, CytC and cleaved caspase 9, were higher in the fatty liver cells than in the control. As is well known, the acceleration of apoptosis is one feature of aging. It is reported that the expression levels of p53, p21 and Bax are increased in HepG2 cells during serum withdrawal-induced apoptosis . p53 traNuclear factor-κB is a nuclear transcription factor with a very wide function, regulating a series of genes, such as those of cytokines, inflammatory factors, immune related receptors and adhesion molecules . VariousIn summary, the results of the present study proved that fatty liver cells might be liable to senescence. Oxidative stress may play an important role in promoting cell senescence in NAFLD. Oxidation resistance may promote cell apoptosis and inhibit cell senescence in NAFLD.Huperzine A may inhibit the progression of NAFLD via NF-κB signaling. Further studies are needed to test hepatic cell senescence in animal models of NAFLD and in patients with NAFLD. Further studies about the critical genes involved in regulating hepatic cell senescence in NAFLD are also needed.10.7717/peerj.5145/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5145/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.5145/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.5145/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.5145/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5145/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.5145/supp-7Supplemental Information 7Click here for additional data file.


While androgen receptor (AR) and stress may influence the development of the major depressive disorder (MDD), the detailed relationship, however, remains unclear. Here we found loss of AR accelerated development of depressive-like behaviors in mice under chronic mild stress (CMS). Mechanism dissection indicated that AR might function via altering the expression of miR-204-5p to modulate the brain-derived neurotrophic factor (BDNF) expression to influence the depressive-like behaviors in the mice under the CMS. Adding the antiandrogen flutamide with the stress hormone corticosterone can additively decrease BDNF mRNA in mouse hippocampus mHippoE-14 cells, which can then be reversed via down-regulating the miR-204-5p expression. Importantly, targeting this newly identified AR-mediated miR-204-5p/BDNF/AKT/MAPK signaling with small molecules including 7,8-DHF and fluoxetine, all led to alter the depressive-like behavior in AR knockout mice under CMS exposure. Together, results from these preclinical studies conclude that decreased AR may accelerate the stress-induced MDD via altering miR-204-5p/BDNF/AKT/MAPK signaling, and targeting this newly identified signaling may help in the development of better therapeutic approaches to reduce the development of MDD.  Major depressive disorder (MDD) is highly associated with marked personal, social and economic impact, and the susceptibility of MDD is affected by the interactions of multiple functional genetic variants and environmental factors. MDD has the greatest impact of all diseases on disability in the U.S, and it is the third leading cause of disability in Europe . While tThe biological actions of androgens are mediated by a ligand-dependent nuclear transcription factor, the androgen receptor (AR). An inverse relationship between AR transcriptional activity and the number of polyglutamine repeats in the AR transcriptional activation domain is well documented ,8. A preAccumulating evidence suggests that brain-derived neurotrophic factor (BDNF) is associated with the pathophysiology of MDD. The plasma/serum levels of BDNF were decreased in MDD patients compared with healthy controls ,17 and tThe microRNAs  are non-coding RNA molecules with 20–22 nucleotides, which modulate gene expression at the post-transcriptional level and brain specific miRNAs were found to play important roles in neurotrophin expression . BDNF waHere we found that decreased AR may accelerate the stress-induced MDD via altering the miRNA-204-5p/BDNF/AKT/MAPK signaling, and targeting this newly identified signaling with small molecules may help in the development of better therapeutic approaches to reduce the development of MDD.All animal studies were handled in accordance with the “Guide for the Care and Use of Laboratory Animals” . All procedures for testing and handling were approved by the Committee on Animal Resources of Kaohsiung Chang Gung Memorial Hospital. The strains of the mosaic founder mice were C57BL/6 and 129Sv background and Floxed AR/AR female mice were established and the procedures used for genotype determination as described in our previous publication . The ACTIn the CMS paradigm, the animals were exposed to several stressors for 6 weeks A and 2 wThe protocol of sucrose preference test with 1% sucrose solution and forced swimming test was adapted from a previous study . Mice weGlass beakers (13 cm diameter × 24 cm high) were filled with fresh water (23–25 °C) to a depth of 12 cm. Mice were placed into the test beaker and were unable to escape or rest by touching the bottom of the beaker. Sessions were video recorded for 5 min and analyzed offline with ethovision XT . Mice were scored by visual inspection for immobility defined as motionless floating in the water.Fluoxetine  was dissolved in sterile phosphate-buffered saline (PBS) and administrated i.p. daily at 10 mg/kg of body weight  for 14 dBDNF, pAKT and p-p38 staining were performed on 5 μm paraffin brain sections . The sections were deparaffinized in xylene and then hydrated in 100–70% alcohol. Sections were incubated with the anti-BDNF , anti-pAKT(10 μg/mL Bioss #bs-5209R), or p38 MAPK (Thr180 + Tyr182) (10 μg/mL Bioss #bs-2210R) antibodies for 24 h at 4 °C. The sections were then washed three times with 0.2% Triton X-100 in PBS and then incubated with Dako REAL EnVision/HRP, Rabbit/Mouse (ENV) detection kit for 30 min at room temperature, followed by a color reaction using Dako REAL DAB+ chromogen for 1 min for BDNF, pAKT, and p-p38.In situ probe generation: The probe sequence was as follows: CAG TTG GCC TTT GGA TAC CGG GAC TTT CTC TAG GAC TGT GAC CGT CCC.Coronal sections (5 μm) were mounted on poly-l-lysine-coated slides and allowed to air-dry for 16 h at 45 °C and were then de-paraffinized and rehydrated. The BioTnA Biospot Kit protocol was applied to the sections. The ISH was completed after applying DAB Solution to the slides for 1 min and the slides washed for 2 min under running tap water. We then counted the cells with positive staining and calculated the density.LCM was performed with Veritas Automated Laser Capture Microdissection (LCM) System . A total of 10–15 series of sections were dissected for each sample. The RNA of microdissected cells was extracted using the PicoPure RNA isolation kit . The isolated RNAs were reversely transcribed using QuantiTect Reverse Transcription Kit  following the manufacturer’s instructions. The cDNA was then pre-amplified using the TaqManPreAmp Master Mix (Life Technologies) according to the manufacturer’s instructions.5 cells/mL in RPMI 1640 media supplemented with 2 mM l-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% fetal bovine serum (FBS). All media contained 1.5 μg/mL penicillin/streptomycin/neomycin, and cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. In corticosterone and flutamide experiments, mHippoE-14 cells were seeded in 6-well plate treated with 1 μM flutamide for 48 h, and then treated with 100 ng/mL corticosterone. After 48 h, cells were harvested for further analysis. RNAs from harvested cells were extracted using Quick-RNA MiniPrep .The embryonic mouse hippocampus, mHippoE-14, cell line was obtained from Cedarlane and maintained at 2 × 10ON-TARGET plus SMART pool small interfering RNA (siRNA) against mouse miR-204-5p and ON-TARGET plus non-targeting siRNA pool were purchased from Dharmacon. The TransIT-X2 Dynamic Delivery System  was used for siRNA transfection, as described by the manufacturer. In corticosterone treatment and miR-204-5p knockdown experiments, the mHippoE-14 cells in 6-well plates were transfected with 30 nM miR-204-5p siRNA or control siRNA. After 2 h transfection, cells were treated with 100 ng/mL corticosterone  or ethanol for 72 h. In flutamide treatment and miR-204-5p knockdown experiments, the mHippoE-14 cells in 6-well plates were treated with 1 μM flutamide for 48 h and then were transfected with 30 nM miR-204-5p siRNA or control siRNA for 24 h.Mouse RNAs were reversely transcribed using QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s instructions. cDNA for microRNA were synthesized using the TaqMan Micro RNA Transcription Kit (Life Technologies) according to the manufacturer’s instructions. The qRT-PCR was performed using the following sets of primers: Human AR 5′-TCA CCG CAC CTG ATG TGT G-3′ (sense) and 5′-ACA TGG TCC CTG GCA GTC TC-3′ (antisense); human GAPDH, 5′-TGC ACC ACC AAC TGC TTA GC-3′ (sense) and 5′-GGC ATG GAC TGT GGT CAT GAG-3′ (antisense); mouse BDNF, 5′-TGA AGG GGC ATA GAC AAA AGG-3′ (sense) and 5′-CTT ATG AAT CGC CAG CCA ATT CTC-3′ (antisense); and mouse GAPDH, 5′-GCA CAG TCA AGG CCG AGA AT-3′ (sense) and 5′-GCC TTC TCC ATG GTG GTG AA-3′ (antisense), as well as miR-204-5p , and U6 . Relative expression levels of target genes in each sample were calculated based on the threshold cycle (CT), where the difference in CT (−ΔCT) used to represent relative expression of clinical samples was defined as CTGAPDH – CT sample. The 2−ΔΔCT method was used to calculate relative changes in expression of target genes for cell assays, where ΔΔCT = ΔCT treatment group − ΔCT control group.p < 0.05 or p < 0.01 was used as a criterion for statistical significance.All sections used for IHC staining and ISH were microphotographed together in order to diminish the difference as much as possible. Data related to the IHC and ISH were analyzed with optical intensity staining using Image-Pro Plus software. The cell density of BDNF protein immunoreactive cells and BDNF RNA positive cells were counted by using 3-point scale . The celp < 0.05; To link the function of AR to the CMS-mediated depressive-like behavior in male mice, we first set up the protocol of CMS model of depression in wild type (WT) mice under CMS in different periods of time, and compared to the control group of non-stressed WT mice by Sucrose preference test A. It is The expression of AR has been reported to localize mainly in pyramidal neurons from hippocampal CA1 area and scarcely in some neurons from CA3 area and dentate gyrus . MoreoveWe also applied the ISH assay to examine the BDNF mRNA expression and localization in the hippocampus and found that the cell density of BDNF mRNA positive cells in the CA1 area was significantly decreased after the CMS procedure S,T,W. HoTogether, results from Recent studies demonstrated BDNF signaling, via its TrkB receptor. It cooperatively interacts with androgens to maintain somal and dendritic morphology of bulbocavernosus (SNB) motoneurons  and the Recent studies indicated that decreasing the miR-204 expressing resulted in increased the BDNF/TRKB expression , and theImportantly, we also applied the interruption approach using siRNA of mir-204 (si-mir-204 5p) to examine its impact on androgens/AR effects on BDNF expression in the mHippoE-14 cells. The results revealed that corticosterone- C and fluTogether, results from BDNF/TrkB signaling has been shown to activate AKT and MAPK kinase cascades . To targTogether, these results from Previous studies indicated the positive linkage between androgen and depression symptom severity ,44, and The effects of androgen/AR signals on behavior and psychopathology have been proposed to be of value in preventing anhedonia in middle-aged males ,47. MalePrevious studies have demonstrated the consistent relationship between loss of androgen and depression symptom severity ,44. SeveImmunoreactivity of AR has been reported to localize mainly in hippocampal CA1 pyramidal neurons and scarcely in CA3 and dentate gyrus neurons , brain rWhile impacts of androgens on promoting neonatal neurogenesis in the developing rat hippocampus had been studied , studiesCastration of adult male rats caused the dendrites of androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) to retract and the frequency of motoneurons intensely immunolabeled for TrkB receptors was regulated by the presence of testosterone . Here wePrevious studies have shown that the overexpression of BDNF/TrkB might not only provide an autocrine survival pathway for neurons, but also has been implicated in poor prognosis in several tumors ,72. InteIn conclusion, the present study describes a depression model in response to stress-based gene-hormone/environment interactions. Our work identifying the miR-204 that is directly affected by AR during CMS has revealed novel protective mechanisms of AR actions on depression. Loss of AR accentuates the noxious effects of CMS on the development of depressive-like phenotypes and 7,8-DHF, a TrkB receptor agonist, could ameliorate the depressive-like behavior. Future development of novel antidepressant agents targeting AR, which, together with other testosterone interventions, could allow the AR/BDNF/TrkB axis to function properly for optimizing the management of depressive-related diseases.


Astragali Radix (AR) is widely-used for improving liver fibrosis, but, the mechanism of action has not been systematically explained. This study aims to investigate the mechanism of AR intervention in liver fibrosis based on comprehensive metabolomics combined with network pharmacology approach.UPLC–Q-TOF/MS based metabolomics technique was used to explore the specific metabolites and possible pathways of AR affecting the pathological process of liver fibrosis. Network pharmacology analysis was introduced to explore the key targets of AR regarding the mechanisms on liver fibrosis.AR significantly reduced the levels of ALT, AST and AKP in serum, and improved pathological characteristics. Metabolomics analysis showed that the therapeutic effect of AR was mainly related to the regulation of nine metabolites, including sphingosine, 6-keto-prostaglandin F1a, LysoPC (O-18:0), 3-dehydrosphinganine, 5,6-epoxy-8,11,14-eicosatrienoic acid, leukotriene C4, taurochenodesoxycholic acid, LysoPC (18:1 (9Z)) and 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine. Pathway analysis indicated that the treatment of AR on liver fibrosis was related to arachidonic acid metabolism, ether lipid metabolism, sphingolipid metabolism, glycerophospholipid metabolism and primary bile acid biosynthesis. Validation of the key targets by network pharmacology analysis of potential metabolic markers showed that AR significantly down-regulated the expression of CYP1B1 and up-regulated the expression of CYP1A2 and PCYT1A.Metabolomics combined with network pharmacology was used for the first time to clarify that the treatment of AR on liver fibrosis, which is related to the regulation of arachidonic acid metabolism and ether lipid metabolism by modulating the expression of CYP1A2, CYP1B1 and PCYT1A. And the integrated approach can provide new strategies and ideas for the study of molecular mechanisms of traditional Chinese medicines in the treatment of liver fibrosis. As a worldwide clinical problem, liver fibrosis is a wound healing process with recurrent chronic liver damage . It can Astragali Radix (AR), is a worldwide used traditional Chinese medicine (TCM) is the dried root of Astragali radix membranaceus (Fisch.) Bge. or Astragali radix membranaceus (Fisch.) Bge. var. mongholicus (Bge) Hsiao . The sections were washed and incubated with an anti-rabbit Alexa Fluor 488-conjugated IgG secondary antibody. 4,6-diamidino-2-phenylindole counterstaining was used to stain the nuclei. The sections were cover-slipped with fluorescent mounting medium.2O2 for 25 min at room temperature and then blocked with 5% rabbit serum for 30 min. Then, the primary antibody [α-SMA (1:100)] was added for incubation at 4 °C overnight. After washing with PBS, the liver sections were incubated with horseradish peroxidase-conjugated secondary antibody for 50 min at room temperature. Liver sections were immersed in diaminobenzidine for 3 min and then stained with ethanol dehydrated hematoxylin. The stained areas of the sections were observed under an optical microscope of 200 nm.To measure immunohistochemical, liver tissues were immersed in 2% Hg and 4 °C for a 10 min. The supernatant was western blotted with 10% SDS-PAGE and the blot was transferred to the polyvinylidene fluoride membrane. Subsequently, it was incubated with 5% skim milk powder in blocking buffer for 2 h. And polyvinylidene fluoride membrane incubated with α-SMA (1:1000) and GAPDH (1:2000) primary antibodies at 4 °C with gentle shaking overnight. Finally, after incubation with the secondary antibody for 1 h, the membrane was washed 3 times with TBST for 5 min each time and the immunoreactivity bands were detected by chemiluminescence detection kit.Liver tissue (80 mg) was homogenized and lysed in ice-Cold lysis buffer with PMSF and protein phosphatase inhibitor mixture, and then centrifuged at 12,000×600 μL of methanol was added to 200 μL of serum and was mixed. The sample was allowed to stand at 4 °C for 20 min and then centrifuged at 12,000 rpm for 10 min. Finally, the supernatant was absorbed and filtered through 0.22 μm micropore filter. The filtrate was collected for analysis. For the quality control (QC) samples, taken 20 μL from each prepare sample extract and mix, used the rest of the samples for Q-TOF LC/MS test.An Agilent 6550 iFunnel Q-TOF LC/MS  system was used for serum metabolic spectrum analysis. A 4 μL aliquot of each sample was injected into the system on a ZORBOX RRHD C18 analytical column  for sample separation at 30 °C. Solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) were used as the mobile phase for a linear gradient separation at a flow rate of 0.30 mL/min for 25 min a linear gradient of 100% A over 0–1.0 min, 100–60% A over 1.0–9.0 min, 60–10% A over 9.0–19.0 min, 10–0% A over 19.0–21.0 min, 100% B over 21.0–25.0 min.m/z 50 to 1200, gas temperature is 225 °C, gas flow rate is 13 L/min, nebulizer is set to 20 psi, sheath gas temperature is 275 °C, sheath gas flow is 12 L/min, and nozzle voltage is 2000 V in both negative and positive modes.Both positive and negative mode electrospray ionization sources (ESI) were used. The electrospray source parameters are set as follows: electrospray capillary voltage is 3.5 kV in negative ionization mode and 4 kV in positive ionization mode, mass range is from m/z 80–1000 and sets the initial and final retention times for data collection. Data was normalized using MetaboAnalyst 4.0 and then analyzed by principal component analysis (PCA) and orthogonal-partial least squares-discriminant analysis (OPLS-DA) using SIMCA-P 14.1 software .MassHunter Profinder software  was used to extract sample data and perform peak detection and alignment. The full scan mode is applied to the mass range of P<0.05 in ANOVA) were used as potential biomarkers in the OPLS-DA analysis [Biomarkers were discovered by screening for metabolic differences. Differential metabolite satisfying the conditions | ≥ 0.58 and analysis . MetabolThe chemical components of AR  ≥ 30% and a drug-likeness (DL) > 0.18) were collected in the TCMSP database. And the MBROLE 2.0 database was used to collect the protein targets for potential metabolites. UniProt ID was used to replace different types of protein IDs, and then a metabolic–target–component interaction network was established through protein interaction (PPI) information. Finally, the network was visualized and analyzed using Cytoscape 3.6.1 software.−ΔΔCT method. The primers are listed in Table Total RNA was extracted from liver tissue by trizol reagent and RNA (2 μg) was transcribed into cDNA by PrimerScript RT regent kit. The cDNA was subsequently subjected to PCR amplification by ABI Step One Plus. Data analysis was performed by 2P < 0.05 was considered statistically significant.Data were analyzed by one-way ANOVA and Duncan’s multi-range test. The SPSS computer program was used to analyze the mean of Windows. Results are expressed as mean ± standard deviation (SD). P < 0.01) compared with that in the control group, respectively. And serum ALT, AST and AKP concentrations were attenuated in a dose-dependent manner by AR. Compared with the model group, ALT, AKP and AST levels were most significantly reduced after administration of 10.8 g/kg AR alone (P < 0.01). Histologic analysis was performed using HE staining and MASSON staining. As shown in Fig. 4-administration group included pericentral necrosis and fibrosis, vacuolar steatosis, inflammatory cell infiltration, and cytoplasmic degeneration. AR treatment of animals exposed to CCl4 can effectively improve liver necrosis, fibrosis, and reduce inflammatory infiltration, especially in 10.8 g/kg AR group. In the liver section of MASSON staining, the blue area represented the deposited collagen. The results showed that a large amount of collagen was deposited around the hepatic sinus of the model group. And compared with model group, 10.8 g/kg AR could effectively reduce collagen deposition and improve liver fibrosis  in the positive and negative modes were 1 and 1, and the variance for modeling in cross-validations (Q2) were 0.774 and 0.777, respectively  were performed to validate the predictive ability of modes  absolute values over 0.58 means can be considered as potential biomarkers | ≥ 0.58 in the S-plots as candidates for ANOVA analysis. Candidate variables with significant differences were then screened using ANOVA analysis. Candidates with significant changes were identified as biomarkers based on the METLIN and Metaboanalyst databases. A total of 9 metabolic differences were identified, 7 in ESI+ mode and 2 in ESI− mode, which were marked with red in the S-plots diagram Fig. : S3E, F.4-induced liver fibrosis , cytochrome P450 1A2 (P05177), choline-phosphate cytidylyltransferase A (P49585), cytochrome P450 3A4 (P08684), cytochrome P450 1B1 (Q16678), and cytochrome P450 2A6 (P11509), directly regulate the 3 potential metabolites . And the levels of CYP1A2 and PCYT1A were significantly up-regulated after AR treatment in a dose-dependent manner (P < 0.01). Furthermore, the expression of the CYP1B1 in CCl4-induced hepatic fibrosis rats was significantly inhibited after AR treatment  [Serum biochemical indicators showed that AR significantly reduced serum ALT, AST and AKP levels, restored liver function and effectively alleviated liver injury. HE staining and Masson staining also confirmed that AR could effectively reduce liver damage and collagen deposition in liver tissue, as well as improved liver fibrosis. In addition, this study used a variety of methods to detect the expression of two specific makers of liver fibrosis, including α-SMA (a specific marker of hepatic stellate cell activation) and TGF-β1 (an important factor of hepatic fibrosis induced by CCl4) . The res4. At the same time, significant differences were observed between the AR group and the model group, which indicated the regulatory role of AR. Finally, nine differential metabolites were screened to reveal the regulatory mechanism of AR in ameliorating liver fibrosis. These metabolites can interact in different ways. The results suggest that the development and progression of liver fibrosis were caused by the changes in many physiological and pathologically related molecules, and most of the changes in the body are interrelated. AR modulated these nine biomarkers to normalize their expression levels, indicating that AR can treat liver fibrosis through multiple pathways and multiple targets.Subsequently, the metabolomic profiles of AR in the treatment of liver fibrosis were described. AR protected liver fibrosis by reversing potential metabolites to normal levels. Biomarkers can be an effective method for disease diagnosis, treatment and prognosis . MetabolThose metabolites involved five main metabolic pathways including arachidonic acid metabolism, ether lipid metabolism, sphingolipid metabolism, glycerophospholipid metabolism and primary bile acid biosynthesis. The decomposition and metabolism of arachidonic acid plays a major role in triggering and eliminating inflammation . Sustain−/− mice exhibited severe fatty liver, dyslipidemia and other phenotypic characteristics [mogat2−/− mice are protected from hepatic steatosis induced by high-fat diet [4-induced hepatic steatosis and fibrosis by regulating PCYT1A and MOGAT2.In order to better understand the mechanism of AR in the treatment of liver fibrosis and the correlation between the AR chemical constituents and metabolites, a metabolite–target–component interaction network in combination with network pharmacology was further constructed. The results showed that six chemical components directly acted on a variety of targets, including CYP1A2, PCYT1A, CYP3A4, CYP2A6, MOGAT2 and CYP1B1, and were directly related to a variety of metabolites. CYP1A2 is a member of the CYP450s family and plays a crucial role in liver metabolism . It has eristics . MOGAT2 fat diet . NetworkThe results of RT-PCR in liver tissue of rats with liver fibrosis showed that the expression of CYP1A2 and PCYT1A was up-regulated and the expression of CYP1B1 was down-regulated in AR treatment groups, which was consistent with the previously reported results. The mRNA expression of CYP3A4, CYP2A6 and MOGAT2 was not detected, probably because the results of network pharmacology came from big data, and not all results could be verified. It might also be due to improper detection methods. Mass spectrometry data indicated that AR extract contained quercetin and nicotinic acid, which might play an important role in the treatment of liver fibrosis. The other four components were not detected, which might be due to the change of compounds during AR extraction. It might also be that the content of those components in AR extract was too low to be detected. These results not only indicated that AR could ameliorate liver fibrosis by regulating the expression of CYP1A2, PCYT1A and CYP1B1, but also confirmed that the predicted results of network pharmacology were credible.Asiatic acid can ameliorate CCl4-induced liver fibrosis in rats by regulating PI3K/AKT/mTOR, Bcl-2/Bax, Nrf2/ARE, NF-κB/IκBα and JAK1/STAT3 signaling pathways [Salvia miltiorrhiza could inhibit liver fibrosis by regulating TGF-β/Smad, Nrf2/HO-1 and NF-κB/IκBα signaling pathways [Taken together, our study found that AR could be involved in the regulation of arachidonic acid metabolism and ether lipid metabolism by regulating the expression of CYP1A2, PCYT1A and CYP1B1, thereby effectively improving liver fibrosis. This study confirmed the advantages of AR in the treatment of liver fibrosis with multiple targets and multiple pathways. In addition, it has been reported that pathways , 48. Salpathways , 50. The4-induced liver fibrosis, possibly by regulating arachidonic acid metabolism and ether lipid metabolism, which may provide new insights into the mechanism of AR in the treatment of liver fibrosis. In addition, this study also confirmed that TCM has potential advantages in the treatment of liver fibrosis, which is worth further exploring.This study confirmed that AR had a therapeutic effect on CClAdditional file 1: Figure S1. Q -TOF LC/MS total ion chromatogram in ESI+. Table S1. The major chemical compounds of AR extract.Additional file 2: Figure S2. The corresponding loading scores plots from PCA.Additional file 3: Table S2. Differential identified metabolites for discrimination among control and model groups. Table S3. Differential identified metabolites for discrimination among model and 10.8 g/kg AR groups.Additional file 4: Figure S3. The OPLS-DA score plots, S-plots and 100-permutation test generated in ESI− mode.Additional file 5: Figure S5. The interrelation between metabolic differences.