Nymphaea hybrid flower. The extraction conditions were optimized using the response surface method (RSM) coupled with a Box-Behnken design. The crude extract of Nymphaea hybrid (NHE) was further purified using AB-8 macroporous resins, and the purified extract (NHEP) was characterized by FTIR and HPLC. In vitro activity determination by chemical method showed that NHEP displayed strong free radical scavenging abilities against the DPPH and ABTS radicals, good reduction power, and hyaluronidase inhibition. The cell viability by CCK-8 assays showed that NHEP had no significant cytotoxicity for B16 and HaCaT cells when the concentration was below 100 μg/mL and 120 μg/mL, respectively. NHEP with a concentration of 20–160 μg/mL can more effectively reduce the ROS level in H2O2 damaged HaCaT cells compared with 10 μg/mL of VC. The 40 μg/mL of NHEP had similar activity against intracellular melanin production in the B16 melanoma cells compared with 20 μg/mL Kojic acid. Good activities of antioxidation, whitening and protective effect against H2O2-induced oxidative damage promote the potential for NHEP as a functional raw material in the field of cosmetics and medicine.In this study, ultrasonic-assisted cellulase extraction (UCE) was applied to extract flavonoids and polyphenols from the  Nymphaea hybrid is a perennial aquatic herb of Nymphaea in Nymphaeaceae [Nymphaea and found that the extracts of some Nymphaea plants displayed good effects of antioxidation [Nymphaea, which are impressive for the activities of good antioxidation and cell protection [Nymphaea has widespread application prospects in functional food, medicine and cosmetics industries. However, the studies about Nymphaeahybrid have been focused on the aromatic components for fragrance application, while there are only a few studies about flavonoids and polyphenols and other active ingredients.haeaceae . It is fhaeaceae ,3. In thxidation , whitenixidation , antibioxidation , anti-inxidation , liver pxidation , preventxidation ,10. Flavotection ,11. TherNymphaea hybrid, especially few reports on how to extract flavonoids sufficiently. Hence, in this paper, UCE coupled with the response surface method (RSM) was selected to develop the flavonoids and polyphenols extraction process of Nymphaea hybrid.A suitable extraction method is very important for preparing the natural plant active extracts. In recent years, experts have explored new methods of extraction that have a better extraction efficiency, enhanced greenness and shorter extraction time, such as microwave , ultrasoNymphaea extracts and are playing an important role in bioactivities [Natural plant extracts are usually complicated mixtures consisting of a variety of active ingredients. Recent research has shown that flavonoids and polyphenols are the largest groups of secondary metabolites of the tivities . The mactivities . It has tivities ,33,34,352O2-induced oxidative stress in human cells [Oxidative stress is one of the main risk factors in aging and most age-related diseases. It causes ROS levels to exceed the antioxidant capacity of cells, resulting in oxidative damage . There aan cells .Nymphaea hybrid flower by RSM coupled with Box–Behnken design; (2) characterize the purified extract (NHEP) by FTIR and HPLC; (3) evaluate in vitro the bioactivities of extract assays to provide theoretical support for the product development and application of Nymphaea hybrid extract.The focus of this paper aims to (1) develop and optimize the ultrasound-assisted cellulase extraction process of the flavonoids from The ultrasonic-assisted cellulase method was used to prepare NHE. The process included enzymatical hydrolysis with cellulase followed by ultrasonic extraction of flavonoids and polyphenols. Single-factor experiments and RSM were applied to optimize the preparation process. To explore the influence of different conditions, we developed four factors and five levels of an experimental program in the single-factor experiments. The factors including cellulase amount liquid-to-solid ratio, enzymatic hydrolysis time and ethanol concentration were selected to determine the influence on the content of flavonoids. As shown in Based on the results of the single factor experiments, the liquid-to-solid ratio (A), cellulase amount (B) and ethanol concentration (C) were selected as the independent variables for optimizing the extraction process using the flavonoids content as an index. The results of the Box–Behnken experiment were shown in p-value is 0.0001 (p < 0.001), indicating that the model has an extremely significant statistical difference, and the model is meaningful. The high correlation coefficient value (R2 = 0.9886) indicates that more than 98.86% of the response variability is explained by the model. The p-value of lack of fit is 0.2159, the F-value of lack of fit is 2.3293 and the three linear coefficients, three quadratic coefficients and two interaction terms are significant and interact. The results indicate that the quadratic regression model fitted well with the actual situation, and the model could be used to analyze the flavonoid content of NHE.The variance analysis of the Box–Behnken regression model results in p-value, we can determine two interaction coefficients . p < 0.05). The flavonoid content increases with the increasing liquid-to-solid ratio and cellulase amount, while at a higher liquid-to-solid ratio (40:1–60:1 mL/g) and cellulase amount (4–6%), the interaction effect was not obvious. When the amount of cellulase reaches a certain point, the flavonoid content may decrease with the increase in cellulase amount and enlarge the liquid-to-solid ratio. The reason for this may be due to the appropriate cellulase amount and an enlargement of the liquid-to-solid ratio will improve the solubility and increase the mass transfer rate, but the excessive cellulase amount with too high a liquid-to-solid ratio will affect the reaction efficiency and mass transfer efficiency, resulting in a flavonoid content decrease with the liquid-to-solid ratio. A significant interaction effect (p < 0.05) between cellulase amount and ethanol concentration (BC) was also observed in According to the result of regression analysis, the response surface 3D map and the contour plot maps were drawn in w/w), the ethanol concentration was 52% (v/v). Under such conditions, the predicted flavonoid content of NHE was 26.92 mg/g. To verify these optimal conditions, the experiment was repeated five times and the average actual flavonoid content of NHE was 27.26 ± 0.64 mg/g (n = 5), which was close to the predicted value and proved the validity of the models.According to the response surface experiment, the optimal process conditions for extraction were as follows: the liquid-to-solid ratio was 40.45:1 (mL/g), cellulase amount was 5.23% (n = 3). The flavonoids into the resin AB-8 were desorbed, and the desorption ratio increased rapidly first and then slowed down within the first 240 min. After 240 min, the flavonoid content in the solution of desorption did not change significantly when the desorption capacity was 15.61 ± 0.25 mg/g (n = 3).The static adsorption and desorption curves of AB-8 macroporous resin for the flavonoid content of NHE were shown in The effects were determined with pH b, samplev/v) was the optimum ethanol concentration.Ethanol is commonly used as an eluent. It has a good solubility for flavonoids. A small amount of water solution can dissolve the part of water-soluble components and improve the desorption of flavonoids ,46. The The influence of sample volume (a) and adsorption flow velocity (b) were determined on the dynamic adsorption capacity of flavonoids into the resin AB-8 in The dynamic adsorption (c) and desorption (d) curves were drawn in NHEP was prepared by the purification of NHE with AB-8 macroporous resin. As shown in 50 values of the DPPH radical of NHE, NHEP and VC were 11.09 ± 0.15 μg/mL, 3.28 ± 0.03 μg/mL and 5.18 ± 0.06 μg/mL, respectively. The IC50 values of the ABTS radical of NHE, NHEP and VC were 18.21 ± 0.05 μg/mL, 10.50 ± 0.10 μg/mL and 7.77 ± 0.04 μg/mL, respectively. The reduction power of NHE, NHEP, VC for Fe3+ were strong with high absorbances [50 value of NHE and NHEP in hyaluronidase inhibition assay was 0.55 ± 0.01 mg/mL and 0.32 ± 0.01 mg/mL. The inhibition rate of 10 μg/mL dipotassium glycyrrhizinate (Dg) was 94.23 ± 1.72%. This result showed that both NHE and NHEP display an anti-inflammatory effect, and NHEP has a stronger anti-inflammatory effect than NHE. Hence, in vitro bioactivity evaluation by chemical assays showed that both NHE and NHEP displayed strong free radical scavenging abilities against the DPPH and ABTS radicals, good reduction power, and anti-inflammatory effect.To explore in vitro antioxidant activity of NHE and NHEP, the free radical scavenging abilities against ABTS, DPPH, and reduction power, were detected compared with VC. As shown in orbances . The abs−1 is relatively wide and strong, which is attributed to the stretching vibration of O−H. The signal in wavelength of 2930 cm−1 is assigned to the absorption peak of stretching vibration of the C−H bond of aliphatic hydrocarbon [−1 is due to the stretching vibration of C−H of the aromatic ring. The aromatic C−H bond was out-of-plane bending vibration, resulting in absorption peaks at 834 and 760 cm−1 wavelengths [−1 may be assigned to the carboxylic acid of a sharp stretching band of the C=O. The peaks at 1613 and 1449 cm−1 wavelength belong to the C=C bond of the aromatic ring vibrates [−1 may be assigned to the aromatic skeletal vibrations, ring breathing with C−O starching vibration [−1 are assigned to the bending vibrations of −CH3 and −CH2. The peaks at 1210 and 1038 cm−1 wavelength belong to the stretching vibration of C−H and C−O bonds [Infrared spectroscopy analysis can be used as a powerful tool to characterize and identify functional groups present in the compounds. As shown in rocarbon . The banelengths . A charavibrates . The absibration . The ads−O bonds . The cha−O bonds .Nymphaea nouchali using an HPLC system [Nymphaea nouchali and that (6.16 ± 0.019 mg/g) of ethanolic extract of Water Lily (Nymphaea Tetragona Georgi) [The total polyphenol and flavonoid compositions of NHEP were identified by the characterization of HPLC in C system . Among t Georgi) .2O2 on HaCaT cells. As shown in p < 0.001) and 81.14% ± 3.66% (p < 0.001). As shown in p < 0.001), 160 μg/mL  and 200 μg/mL . We can obviously find that while the culture time of the cells extended, the cytotoxicity significantly increased as the cell viability decreased by 10.81%, 8.04%, 8.29%, respectively when the cells were treated with a higher concentration of NHEP (120–200 μg/mL). The CCK-8 assay results were consistent with cell growth density and cellular morphology, as shown in CCK-8 kit assay was used to determine the cytotoxicity of NHEP and H2O2 can be used as an inducer to promote intracellular ROS production in certain concentrations [2O2-induced injury model in vitro, the nominal concentration of H2O2 (25–300 μM) exposed to HaCaT cells was assessed by the CCK-8 kit. As shown in p < 0.001) with a concentration of H2O2 increased to 100 μM. Therefore, 100 μM of H2O2 was selected to induce cellular ROS oxidation damage on HaCaT cells in the subsequent experiments.Htrations . In orde2O2 for 2 h. Compared with the control group treated only with H2O2, HaCaT cells had higher cell viability when pretreated with NHEP in the range of 20–160 μg/mL. The higher concentration of NHEP, the higher the HaCaT cell viability. The cell viabilities of HaCaT cells cultured with 20, 40, 80, 120, 160 μg/mL NHEP were 52.66% ± 2.36% (p < 0.05), 60.19% ± 3.71% (p < 0.01), 66.85% ± 2.00% (p < 0.001), 75.77% ± 3.79% (p < 0.001) and 82.17% ± 4.70% (p < 0.001), respectively. Although 160 μg/mL NHEP had a low cytotoxicity compared with HaCaT cells, it displayed a better inhibition against the stimulation of H2O2. The cell viabilities of 80–160 μg/mL NHEP pretreatment groups were higher than that of the 10 μg/mL VC pretreatment group . It was reported that polyphenols and flavonoids can effectively inhibit the generation of intracellular ROS and protect cells against oxidative damage [2O2.As shown in e damage . In this2O2 is a precursor of many radicals and can increase the intracellular ROS levels by crossing the cell membrane [2O2 and then treated with NHEP of different concentrations.Excessive ROS may destroy the structural integrity of the cell and eventually lead to oxidative damage of organisms . H2O2 ismembrane ,59. In t2O2. The ROS levers of the stimulated injury group were 295.17% ± 9.29% (p < 0.001), which is significantly higher than that of the control group and NHEP post-treatment groups. With the increasing concentration of NHEP, the ROS levels reduced gradually, indicating that NHEP in the concentration range of 10–160 μg/mL can efficiently inhibit the ROS production in HaCaT cells stimulated by H2O2. The ROS levers of 20–160 μg/mL NHEP groups were lower than that of the VC group , and displayed weaker fluorescence intensity. The ROS production of 20–160 μg/mL NHEP were 202.35% ± 10.65% (p < 0.01), 169.74% ± 7.88% (p < 0.01), 132.43% ± 11.25% (p < 0.001), 116.87% ± 4.22% (p < 0.001) and 108.52% ± 5.75% (p < 0.001), respectively. In conclusion, NHEP has a better protection ability antioxidative damage. NHEP can effectively reduce the ROS levels in HaCaT cells induced by H2O2 so that NHEP has good cell protection against oxidative damage in the range of 20–120 μg/mL.The effect of intracellular ROS level in the HaCaT cells was detected by a fluorescent probe DCFH-DA. The percentage of ROS proportion was taken in the control group as the index. As shown in p < 0.001) when the NHEP concentration was 140 μg/mL. As shown in p < 0.001), 72.98% ± 2.96% (p < 0.001) and 63.54% ± 3.73% (p < 0.001), respectively. Comparing the results of different culture times, we can obviously find that the culture time of the cells extended the cytotoxicity increased significantly with cell viability decreased by 10.79%, 18.26% and 21.11%, respectively, when the cells were treated with a higher concentration of NHEP (100–140 μg/mL). It was reported that flavonoids and polyphenols would affect anti-tumor cell proliferation and apoptosis [Nymphaea extract may induce apoptosis of B16 cells [The cytotoxicity of 10–140 μg/mL NHEP on B16 cells was determined by the CCK-8 kits. As shown in poptosis ,61. In c16 cells ,63.p < 0.001) when treated with 40 μg/mL NHEP. A similar melanin content of B16 cells  was produced in contrast with the positive control group (20 μg/mL KA). When the NHEP concentration increased to 100–140 μg/mL, the melanin content obviously decreased to 42.65% ± 2.34% (p < 0.001), 36.53% ± 1.81% (p < 0.001) and 24.69% ± 5.18% (p < 0.001), respectively. This may be due to the fact that NHEP can induce apoptosis of B16 cells or inhibit tyrosinase activity [As shown in activity ,63.Nymphaea hybrid flowers were purchased from the Liandao Agricultural Development Co., Ltd. . Nymphaea hybrid flowers were rinsed with distilled water and frozen. Then, the frozen Nymphaea hybrid flowers were placed into a vacuum freeze-dryer  for freeze-drying to a constant weight. Then Nymphaea hybrid flowers were crushed into a fine powder (50 mesh) by a shredder and stored in air-sealed bags at a low temperature (−20 °C) for use. The AB-8 macroporous resins were purchased from the San Xing Resin Co., Ltd. .3, Na2CO3, HCl, NaOH, CaCl3, NaH2PO4, Na2HPO4, KBr, acetic acid, sodium acetate, acetylacetone, 4-Dimethylaminobenzaldehyde, DPPH, ABTS, Dipotassium glycyrrhizinate, cellulase, potassium persulfate, hyaluronidase , sodium hyaluronate , DMEM, CCK-8 assay, PBS, pancreatin, reactive oxygen species assay kit, H2O2, methanol, formic acid, acetonitrile and other chemicals, rutin standard, gallic acid standard, ellagic acid standard, corilagin standard, myricetin standard, quercetin standard, naringin standard, all reagents is analytical grade. All reagents, solvents and standards were purchased from MACKLIN . HaCaT cells and B16 mouse melanoma cells were purchased from the Shanghai Cell Bank of the Chinese Academic of Sciences.Ultrapure water, anhydrous ethanol, Folin-phenol, AlClNymphaea hybrid flowers powder with petroleum ether and weighs 1.0 g. Then Nymphaea hybrid flowers powder was mixed with PBS buffer solution , and adjusted the pH of the solution to be five. After adding an amount of cellulase , the mixture was enzymatically hydrolyzed for 20–100 min at the thermostatic water bath of 50 °C. Then, absolute ethanol was added to the mixture  and the mixture was associated at the ultrasonic power of 150 W and the temperature of 50 °C for 30 min. Finally, the resulting mixture was centrifuged at 10,000 rpm for 15 min. The liquid NHE was separated from the supernatant.NHE was prepared by a method of UCE . Degreas3 method [2 = 0.9992). The total flavonoids of NHE were detected by the above method.In this study, the total flavonoids were determined using the colorimetric AlCl3 method . The tot2 = 0.9991). The total polyphenols of NHE were detected by the above method.The total polyphenols were determined using the method of Folin–Ciocalteu . It was Based on single-factor results, Design-Expert software 7.1.3 was used for the experimental design, the result analysis and response prediction. According to the Box–Behnken experiment, the extraction process was optimized by three factors and three levels. The flavonoid content was taken as the index.2/g. Macroporous resin can be selectively absorbed flavonoids and polyphenols constituents by electrostatic force, hydrogen bonding interactions, complexation and size sieving [0 and Cr are flavonoids content of NHE at the beginning and at time t (mg/mL), V0 is the volume of sample (mL), m is the weight of the resin (g), Cd is flavonoids content of the desorption solution (mg/mL), V1 is the desorption solution volume (mL).NHE was prepared by the experiment method of the  sieving . EnrichmIn this study, the raw extract NHE and its purified product NHEP were lyophilized into powder. Their DPPH, ABTS radical scavenging activities and reducing power were measured by an ultraviolet spectrophotometer using VC as the positive control according to the method of Kunnaja et al.  and ShadIn this study, the HPLC experiment of the instrument used the Agilent 1260 high-performance liquid chromatography system . Gallic acid, ellagic acid, rutin, corilagin, myricetin, quercetin, naringin standards and NHEP were determined. The powder of NHEP was prepared to the solution of 1 mg/mL. Chromatographic conditions ,54,55: C−1 [The powder of NHEP was mixed with spectroscopic KBr, pressed into a sheet for IR analysis using the FTIR spectrometer  in the range of 4000–500 cm−1 .2. The cells were seeded in sterile 96-well plates according to the density of 10,000 per well for 24 h and 48 h into the carbon dioxide incubator at 37 °C and 5% CO2. The NHEP solution of 1 mg/mL was prepared to remove bacteria and impurities by a 220 nm microporous membrane and the sample was prepared into a mixed medium with a concentration of 10–200 μg/mL. The activity of HaCaT cells was determined by the CCK-8 assay after incubation for 4 h. The optical density (OD) value was measured at the wavelength of 450 nm by the enzyme reader , which was expressed as the percentage of HaCaT cell activity in the control group. Then, the HaCaT cells were seeded in sterile 6-well plates according to the density of 200,000 per well for 24 h and 48 h. The morphology of cells was observed by microscope .The experiment referred to the method of ZHANG J et al. ,68, the 2O2 is one of the ways that induced intracellular production of ROS [2O2 (25–300 μm) for 2 h. Then, the activity of cells was detected by the CCK-8 assay.Hn of ROS ,70. The 2O2 for 2 h. Then, the cells were mixed with the DCFH-DA fluorescent probe for 30 min. The fluorescence intensity was detected at the excitation wavelength of 488 nm and the emission wavelength of 525 nm, which was expressed as the percentage of ROS in the control group of HaCaT cells.In this experiment, the level of reactive oxygen species (ROS) was detected by a fluorescent probe DCFH-DA in the HaCaT cells . The celThe experiment referred to the method of AIMVIJARN P et al. , the actThe melanin content of B16 cells was detected by referring to the method of Teng et al.  The sampp < 0.05, p < 0.01 and p < 0.001 were performed *, ** and ***, respectively. The data shows the mean ± S.D.Design-Expert software 7.1.3 was used for response surface analyses. Other Statistical analyses were performed with OriginPro 2021b. All experiments tested replicate at least three times. The significant represented that sample group compared with the control group, Nymphaea hybrid flowers for the first time. The optimum extraction conditions obtained by RSM were as follows: liquid-to-solid ratio of 40.45:1 (mL/g), cellulase ratio of 4.23%, and ethanol concentration of 52%. After purification by macroporous resins, the flavonoid content increased from 55.60 mg/g (NHE) to 157.37 mg/g (NHEP), and the polyphenols content increased from 414.97 mg/g (NHE) to 888.63 mg/g (NHEP), respectively. Seven major flavonoids or polyphenols in NHEP were identified and quantified by the HPLC method. The chemical evaluation method of the antioxidant and anti-inflammatory effect of NHEP was determined by scavenging free radicals , reduction power and inhibiting hyaluronidase assay. The intracellular antioxidant and whitening effects of NHEP on HaCaT cells and B16 cells were detected. These experiments results indicated that the NHEP displayed significantly antioxidant, whitening, anti-inflammatory effects and the protective effect against H2O2-induced oxidative damage in human cells, which promote the potential as a functional raw material in the field of cosmetics and medicine.In this paper, ultrasound-assisted cellulase extraction method associated with macroporous resin purification step was successfully applied to enrich antioxidant flavonoids and polyphenols from


The comparative effects of different types of cardiac resynchronization therapy (CRT) delivered by biventricular pacing (BVP), His bundle pacing (HBP), and left bundle branch area pacing (LBBAP) remain inconclusive.HBP and LBBAP may be advantageous over BVP for CRT.PubMed, Embase, Web of Science, and the Cochrane Library were systematically searched for studies that reported the effects after BVP, HBP, and LBBAP for CRT. The effects between groups were compared by a frequentist random‐effects network meta‐analysis (NMA), by which the mean differences (MDs) and 95% confidence intervals (CIs) were calculated.Six articles involving 389 patients remained for the final meta‐analysis. The mean follow‐up of these studies was 8.03 ± 3.15 months. LBBAP resulted in a greater improvement in LVEF% , followed by HBP  compared with BVP. HBP resulted in a narrower QRS duration , followed by LBBAP  compared with BVP. No significant differences of changes in LVEF improvement and QRS narrowing were observed between LBBAP and HBP. The pacing threshold of LBBAP was significantly lower than those of BVP and HBP.The NMA first found that LBBAP and HBP resulted in a greater LVEF improvement and a narrower QRS duration compared with BVP. Additionally, LBBAP resulted in similar clinical outcomes but with lower pacing thresholds, and may therefore offer advantages than does HBP for CRT. Review articles, case reports, editorials/letters, abstracts, and studies with patients <10 were excluded. The full texts of all potentially relevant articles were assessed for compliance with the inclusion and exclusion criteria. Two reviewers (Qiling Kong and Yichu Zhang) screened the selected articles independently based on the title and abstract. Any discordance was settled through discussion between the reviewers. The pacing threshold was the His lead for HBP and LBBAP and LV lead for BVP at implantation or a week after implantation at 0.4, 0.5, or 1.0 ms.Articles reporting HBP or LBBAP in patients undergoing CRT were included in the English language. The 2.3Data from each enrolled article were independently extracted by two reviewers (Qijun Wang and Ziyi Xiong). Background information such as authors, years, the region of trial, indication, intervention, duration of follow‐up, and outcomes were extracted from each article. All conflicts were resolved through discussion between the reviewers. The Cochrane Handbook for Systematic Reviews of Interventions (version 5.4.0) was used to evaluate the quality of the selected RCTs.2.4Network meta‐analysis using a network analysis tool that combined direct and indirect evidence in a mixed‐intervention model was performed.33.1The literature search yielded 1471 articles , which were considered as potential studies. After the removal of duplicates, 679 publications remained. Afterward, 650 articles were excluded by preliminary screening of titles and abstracts, and a total of 29 were further evaluated comprehensively. Then, 22 records were excluded because of various reasons. Seven articles were found to be eligible for the present meta‐analysis after a full‐text review.3.2All articles selected involving a total of 375 subjects who reported changes in LVEF, including 203 patients for BVP, 84 for HBP, and 88 for LBBAP. When compared to BVP, LBBAP resulted in the greatest LVEF% improvement with a MD of 7.17 (95% CI = 4.31 to 10.04), followed by HBP with a MD of 4.06 (95% CI = 1.09 to 7.03) Figure . In addi3.3All articles selected involving a total of 388 subjects who reported changes in QRSd, including 208 patients for BVP, 90 for HBP, and 90 for LBBAP. When compared to BVP, HBP resulted in narrower QRS duration with a MD of 31.58 ms (95% CI = 12.75 to 50.40), followed by LBBAP with a MD of 27.40 ms (95% CI = 10.81 to 43.99) Figure . In addi3.4All articles selected involving 388 subjects and reported pacing thresholds, including 208 patients for BVP, 90 for HBP, and 90 for LBBAP. When compared to BVP, LBBAP patients had a lower pacing threshold with a MD of −0.46 V (95% CI = −0.56 to −0.36). Conversely, HBP patients had a higher pacing threshold with a MD of 0.48 V (95% CI = 0.24 to 0.71) compared with BVP Figure . In addi3.5No inconsistency was found to be in both global Figure  and loca4Several case reports and single‐center studies that explored the use of the HBP and LBBAP for CRT have been published. The sample sizes in the published studies were relatively small, and the comparisons across them were limited, especially between HBP and LBBAP. Therefore, we aimed to systematically review the literature on HBP or LBBAP for CRT and conduct a network meta‐analysis (NMA) of the available data. Notably, this is the first NMA on the topic of CRT delivered by BVP, HBP, and LBBAP. One advantage of this NMA was to indirectly compare the head‐to‐head of the three types of pacing interventions simultaneously. Another advantage of this NMA was the calculation of the ranking and hierarchy of these treatments for CRT. In this study, we found that HBP and LBBAP delivered greater LVEF improvement and narrower QRS duration than BVP. Additionally, the pacing threshold of LBBAP was significantly lower than those of BVP and HBP.CRT is recommended for symptomatic patients with HF in sinus rhythm with a QRS duration ≥150 ms and in those with LBBB QRS morphology with an LVEF ≤ 35% despite optimized medical treatment to improve symptoms and reduce morbidity and mortality, with Class I recommendation of the 2019 American College of Cardiology/American Heart Association (ACC/AHA) and European Society of Cardiology (ESC) heart failure guidelines.The His‐Purkinje system pacing is currently considered to be the optimal physiologic pacing technique, with the pacing lead directly implanted in the conduction system to narrow the QRS complex and improve cardiac function by selective or nonselective HBP.The QRS duration has been identified as a powerful prognostic marker, and its significance is well known in patients with heart failure.additional evidence of the His‐Purkinje pacing for CRT response. With more methodological and clinical research and a better understanding of the features of the His‐Purkinje pacing, HBP/LBBAP would be more applicable as a supplement to BVP.In theory, HBP and LBBAP confer physiologic pacing with a highly ventricular resynchronization; thus, there should be more LVEF improvement than BVP. We observed that all these resynchronization approaches had a significant improvement in LVEF in this meta‐analysis. Furthermore, the improvements of HBP and LBBAP are greater than those of BVP. Nevertheless, HBP and LBBAP do not have a large impact, as hypothesized. Previous studies showed that the absolute LVEF improvement of HBP ranges from 6% to 23%.also important in pacing treatments, such as pacing threshold and impedance. The early studies found that the pacing threshold of LBBAP was significantly lower than that of HBP,implantation.In addition to the clinical benefits and electrical synchrony, pacing parameters were 4.1because the study with positive results were easier to be reported. Last, the present study should use more indicators of clinical outcomes, such as left ventricular end‐diastolic diameter, blood B‐type natriuretic peptide, New York Heart Association classification, to evaluate the results. Unfortunately, very little data were reported for these outcomes, and network meta‐analyses were not possible. Despite the above limitations, this is the first NMA to provide the latest evidence of changes in LVEF, changes in QRS duration, and pacing threshold of BVP, HBP, and LBBAP delivered for CRT.This meta‐analysis has several potential limitations. First, the small sample size may therefore possibly contribute to an underestimation of the accuracy of this study. Second, the lack of uniform criteria for LBBAP may influence its actual effects. The characteristics of the ECG and the EGM in the LBBAP procedure, such as stim‐LVAT, paced QRS morphology, and discrete component in the EGM, as the indirect criteria for LBB capture, were mainly used to distinguish LBBP from LVSP in previous study.5This is the first NMA that has analyzed the types of CRT and has demonstrated that LBBAP and HBP result in a greater LVEF improvement and a narrower QRS duration compared with BVP. Additionally, LBBAP was associated with a similar electromechanical resynchronization but lower pacing thresholds compared with HBP; therefore, it may offer advantages over HBP for CRT.The authors declare no conflict of interest.Juan Hua and Chenxi Wang performed the meta‐analysis; Qiling Kong, Yichu Zhang, Qijun Wang, Ziyi Xiong were responsible for the statistical analysis; Jinzhu Hu, Juxiang Li, and Kui Hong provided editing assistance; and Qi Chen prepared the manuscript. All the authors have reviewed and agreed to this information before submissionSupporting information.Click here for additional data file.


Rehmannia glutinosa is an important medicinal plant that has long been used in Chinese traditional medicine. Acteoside, one of the bioactive components from R. glutinosa, possessed various pharmacological activities for human health; however, the molecular mechanism of acteoside formation is not fully understood. In the current study, a novel tyrosine decarboxylase (designated as RgTyDC2) was identified from the R. glutinosa transcriptome. Biochemical analysis of RgTyDC2 showed RgTyDC2 uses tyrosine and dopa as the substrate to produce tyramine and dopamine, respectively, and it displays higher catalytic efficiency toward tyrosine than dopa. Moreover, the transcript level of RgTyDC2 was consistent with the accumulation pattern of acteoside in R. glutinosa, supporting its possible role in the biosynthesis of acteoside in vivo. Rehmannia glutinosa Libosch. (Dihuang), a medicinal herb from the Scrophulariaceae family was widely used in traditional Chinese medicine for thousands of years. R. glutinosa showed positive effects on the blood system, immune system, cardiovascular system, nervous system and of being anti-tumor, etc [R. glutinosa, including iridoids, saccharides, and phenylethanoid glycosides (PhGs) have been discovered by pharmacological research [mor, etc ,5,6,7,8.research .R. glutinosa. Acteoside drew attention due to its various biological activities, such as anti-inflammatory, antioxidant, anti-tumor, and neuroprotective properties [l-phenylalanine is converted to the caffeoyl group via several intermediates, including cinnamic acid, coumaric acid, caffeic acid, and caffeoyl CoA under series enzymes [Syringa vulgaris revealed tyrosine and tyramine can be efficiently converted into acteoside [Olea europaea showed that dopa produced from tyrosine is the main precursor for generating acteoside; dopa is converted to dopamine by dopa decarboxylase and then oxidated to aldehyde, reduced to hydroxytyrosol, and glycosylated to form acteoside in order [Rhodiola revealed that tyrosine can be converted to form 4-hydroxy-phenylacetaldehyde directly without the intermediate tyramine [Acteoside, an important bioactive compound of PhGs, accumulated in the leaves and tuberous roots of operties . Pharmacoperties . Wang’s operties . Acteosioperties . In addioperties ,14. Althoperties . The pro enzymes ; whereascteoside . On the in order . In addityramine  and furttyramine ,17,18,19R. glutinosa, the TyDC (denoted RgTyDC2) was isolated and characterized by biochemical assays. The role of RgTyDC2 in acteoside biosynthesis was explored by analyzing the RgTyDC2 expression level and acteoside abundance in R. glutinosa. The findings here will aid understanding of the acteoside production in R. glutinosa.Tyrosine decarboxylase (TyDC) from plants supported the biosynthesis of the PhGs and alkaloids by catalyzing the decarboxylation of tyrosine and dopa . As showR. glutinosa transcriptome based on functional annotation, including 6 for TyDC, 13 for MAO, 138 for ADH, 12 for TRY, and 12 for PPO; the information of these sequences is shown in Based on the proposed biosynthesis pathway of acteoside in R. glutinosa transcriptome. Cluster-11149.26764 and Cluster-11149.28833 were assembled into one sequence. Considering the other tyrosine decarboxylase  was isolated from R. glutinosa Wen 85-5 cultivar in a previous study [RgTyDC2 and selected for further analysis. Other sequences were discarded due to the uncompleted open reading frame (ORF) and low expression level (RgTyDC2 was amplified from leaves of the R. glutinosa Beijing No.3 cultivar by polymerase chain reaction (PCR) with gene-specific primers, and the sequence of RgTyDC2 was deposited in the NCBI database (GenBank accession number OL744234). The ORF of RgTyDC2 was 1524 bp and encoded a 56.05 kDa protein (507 amino acids). As literature reported, TyDCs together with tryptophan decarboxylases (TDCs) and aromatic acetaldehyde synthases (AASs) encompassed the aromatic amino acid decarboxylase (AAAD) family which was responsible for discrete decarboxylation or decarboxylation-deamination reactions of different aromatic amino acids. TyDC, TDC, and AAS shared high sequence similarity, which made it difficult to predict their function just based on the primary sequence [347  was highly conserved in all identified TyDCs and TDCs, while Phe346  was present in AASs [372  residue was strongly conserved in TyDCs but substituted with glycine in TDCs [Thalictrum fiavum (76% amino acid identity).Based on functional annotation, 6 sequences  were identified as tyrosine/dopa decarboxylase candidate genes from the us study , the TyDon level . RgTyDC2 in AASs . Ser372  in TDCs . The phyE. coli BL21(DE3) cells and purified using His tag. The recombined protein was assayed to verify its activity with the substrate tyrosine, dopa, tryptophan, and phenylalanine. HPLC detection of the enzymatic products showed that compared with the control reaction, the new compounds tyramine and dopamine are produced by RgTyDC2 protein when using tyrosine and dopa as substrate  of RgTyDC2 was about 4.4-fold higher for tyrosine than for dopa.To examine the biochemical activity of RgTyDC2, it was expressed in ubstrate , respectR. glutinosa, the correlation between acteoside content and RgTyDC2 gene expression was evaluated. The production of acteoside in leaves and tuberous roots of R. glutinosa was detected by HPLC. Among three independent biological experiments, one plant sample showed higher acteoside abundance in leaves than in tuberous roots, while the others showed similar concentrations between the two organs . The catalytic efficiency (kcat/Km) of RgTyDC2 was about 4.4 times higher for tyrosine than for dopa, and the Vmax of RgTyDC2 for tyrosine was about 3.4-fold higher than those for dopa, suggesting that tyrosine was the better substrate for RgTyDC2. Similarly, a higher affinity toward tyrosine was observed in TyDCs from Rhodiola sachalinensis and Pertoselinum crispum, whereas TyDCs from Hordeum vulgare, Sanguinaria canadensis, and Cytisus scoparius showed a preference for dopa [R. glutinosa; in addition, the pathway from tyrosine to dopa was also available in the R. glutinosa.TyDC catalyzed the decarboxylation of aromatic amino acids with phenol side chains, such as tyrosine. In this study, one sequence was identified as TyDC from the ’s study . The amiof TyDCs  and was m flavum , suggest crispum , RgTyDC2 rugosum ,27, whillinensis . RgTyDC2for dopa . Based oR. glutinosa Beijing No.3 cultivar was selected as our plant material. From three biological replicates, only one R. glutinosa plant sample showed significantly higher acteoside content in leaves than that in tuberous roots, revealing the accumulation pattern of acteoside in leaves and tuberous roots of R. glutinosa Beijing No.3 cultivar was not regular among different plant samples. This result was different from Wang’s studies showing the acteoside content in leaves is often ten times more than that in roots of R. glutinosa Wen 85-5 and QH cultivars [R. glutinosa plant which produced more acteoside in leaves was chosen for further gene expression analysis and RgTyDC2 expressed higher in leaves compared with tuberous roots  at 23 ℃. The leaves and tuberous roots were harvested 180 days after sprouting. Acteoside, l-tyrosine, tyramine, l-tryptophan, tryptamine, l-dopa, dopamine, l-phenylalanine, phenethylamine, and pyridoxal 1-phosphate were purchased from Shanghai Source Leaf Biological Technology Company . Methanol was from Thermo Fisher Scientific Inc. . HCl was from the Shanghai Hushi laboratory equipment company. Unless specified otherwise, all enzymes were purchased from Takara Company .R. glutinosa transcriptome database derived from the leaves and tuberous roots was recently constructed by our group . R. glutinosa tyrosine/dopa decarboxylase genes were screened based on functional annotation in the transcriptome database. Multiple sequence alignment was conducted using the CLC Sequence Viewer 6.8 program and the phylogenetic tree was generated by the neighbor-joining method of the MEGA 7.0, using 1000 bootstrap replications.RgTyDC2 by TransStart FastPfu DNA polymerase . RgTyDC2 were subcloned into the plasmid pET-28a at the EcoR I/Not I site, giving the pET-28a-RgTyDC2. The construct pET-28a-RgTyDC2 was transferred into E. coli BL21 (DE3) cells. The expression of RgTyDC2 was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), which was cultivated at 16 ℃, 180 rpm for 14 h. After induction, transgenic E. coli cells were harvested by centrifugation at 8000× g for 5 min, resuspended in the chilled lysis buffer  and disrupted by ultrasonication. The crude protein extracts were collected by centrifugation at 12,000× g for 10 min at 4 ℃ and then loaded onto a column packed with HisPur Ni-NTA resin to purify RgTyDC2 according to the manufacturer’s instructions. The purified recombinant proteins were desalted into 50 mM Tris-HCl (pH 7.2) buffer with 5% glycerol. The purity of recombinant RgTyDC2 was assessed by SDS-PAGE and its concentration was determined by the Bio-Rad protein assay.For the functional characterization, primers 1 and 2  were desl-tyrosine, l-tryptophan, l-dopa, l-phenylalanine), 1.0 mM EDTA, and 10 μg of isolated protein. After incubation at 30 ℃ for 2 h, the reaction mixture was collected for high-performance liquid chromatography (HPLC) analysis.The standard in vitro enzymatic activity assays were performed in 250 μL of 50 mM Tris-HCl buffer (pH 7.2) containing 25 μM pyridoxal 1-phosphate (PLP), 1 mM aromatic amino acid substrate , 25 μM PLP, 1.0 mM EDTA and 10 μM to 2.5 mM of substrates including tyrosine and dopa, were performed at 30 ℃ for 50 min before HPLC analysis. The kinetic values were determined by hyperbolic regression analysis using the GraphPad Prism 5 program.R. glutinosa, three unfolded leaves were mixed and ground to powder in liquid nitrogen, which was used as the leaves material of this plant. Tuberous roots were also ground to powder. For the acteoside extraction, 100 mg of fresh plant materials (leaves and tuberous roots) were extracted twice with 500 μL of methanol for 30 min by ultrasonic extraction at room temperature. The supernatants were combined, then dried by vacuum and re-dissolved in 800 μL of 50% methanol, which was filtered through the 0.22 μm microfilter prior to HPLC detection.To explore the organ specificity of acteoside accumulation in −1. The column temperature was 25 ℃ and absorbance was 330 nm. Quantification of acteoside was determined from three biological replicates using the standard curves method.HPLC detection was conducted using an LC-16AT system equipped with an Inertsil ODS-SP reverse phase column   and mobile phase containing solvent A (water) and solvent B (methanol). To analyze acteoside from plant materials, the samples were eluted with 40% methanol at a flow rate of 0.6 mL min−1. The detection wavelength was set at 280 nm and the column temperature was 30 ℃. The compound was confirmed by comparing their retention times with authentic standards.For detecting the products from RgTyDC2 in vitro assay, 20% methanol was used as the mobile phase at a flow rate of 0.6 mL minTIP41 gene, which was stably expressed in different organs of R. glutinosa, was used as a reference gene [RgTyDC2 for the QRT-PCR  was used to analyze gene expression levels. Total RNA was isolated from the plant sample which produced more acteoside in leaves than in tuberous roots using the EASYspin plus Plant RNA isolation kit . The RNAwas treated with DNase I and then reverse transcribed to first-strand cDNA using Revert Aid reverse transcriptase . QRT-PCR was performed with the LightCycler 480 system  using TransStart Top Green qPCR SuperMix  in three biological replicates with three technical replicates. nce gene  and ampl QRT-PCR . The theRgTyDC2) was isolated from R. glutinosa. RgTyDC2 was responsible for the decarboxylation of tyrosine and dopa and exhibited a preference for tyrosine. The gene expression of RgTyDC2 matched the abundance of acteoside in R. glutinosa.In this study, the cDNA encoding a novel tyrosine decarboxylase (


This study aimed to determine whether hesperetin (HPT) has chondroprotective effects against the TNF-α-induced inflammatory response of chondrocytes and related mechanisms and clarify the impact of HPT on osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). Under tumor necrosis factor-α (TNF-α) stimulation, rat chondrocytes were treated with or without HPT. The CCK-8 assay was used to detect viability and cytotoxicity. RT-qPCR and Western blot were used to examine the expression of aggrecan, collagen type II, and inflammatory and proliferative genes/proteins in chondrocytes. Flow cytometry was used to check the cell cycle to determine whether HPT protects chondrocytes against the inhibitory effect of TNF-α on chondrocyte proliferation. In addition, RNA sequencing was used to discover possible molecular targets and pathways and then validate these pathways with specific protein phosphorylation levels. Finally, immunofluorescence staining was used to examine the phosphorylation of the AMP-activated protein kinase (AMPK) pathway. The results showed that HPT restored the upregulation of interleukin 1β (IL-1β), PTGS2, and MMP-13 induced by TNF-α. In addition, HPT reversed the degradation of the extracellular matrix of chondrocytes induced by TNF-α. HPT also reversed the inhibitory effect of TNF-α on chondrocyte proliferation. RNA sequencing revealed 549 differentially expressed genes (DEGs), of which 105 were upregulated and 444 were downregulated, suggesting the potential importance of the AMPK pathway. Progressive analysis showed that HPT mediated the repair of TNF-α-induced chondrocyte damage through the AMPK signaling pathway. Thus, local treatment of HPT can improve OA induced by ACLT. These findings indicated that HPT has significant protective and anti-inflammatory effects on chondrocytes through the AMPK signaling pathway, effectively preventing cartilage degradation. Given the various beneficial effects of HPT, it can be used as a potential natural drug to treat OA. Osteoarthritis (OA) is a connective tissue degenerative disease and frequently associated with joint inflammation and substantial pain, possibly debilitating the affected patient . It is cTNF-α is a key inflammatory factor, with a leading role in the inflammatory process. Several studies have demonstrated that TNF-α downregulates the synthesis of major extracellular matrix (ECM) components by inhibiting the anabolic activities of chondrocytes . It has Citrus L. plant in the Rutaceae family, has diverse pharmacological capacities, including anti-inflammatory and antioxidative properties , a natural flavonoid from the operties . HPT hasoperties . Althougoperties , it remain vitro OA model of chondrocytes (with TNF-α induction) was prepared to assess the inhibitory effects of HPT on the inflammation caused by abnormal gene/protein phenotypic changes. To further unravel the underlying molecular mechanisms, high throughput RNA sequencing quantification was used to analyze mRNA-level changes induced by TNF-α with or without HPT treatment in global chondrocytes. Finally, the AMPK signaling pathway was demonstrated to relieve OA, which could be inhibited by HPT treatment.This study investigated whether HPT has chondroprotective effects against the inflammatory responses of chondrocytes induced by TNF-α and the related mechanisms. First, an Rat chondrocytes were isolated and cultured as described in a previous study . SpragueHesperetin (HPT) and dexamethasone (DEX) were purchased from Selleck  and then dissolved in DMSO as a 10 mM stock solution. Rat chondrocytes were treated with HPT at different concentrations ; DMSO was used as a control. Meanwhile, exposure to 10 μM HPT was implemented at different time intervals . Morphology caused by HPT at different concentrations  was detected by microscopy . In addition, 10 ng/ml of TNF-α alone (the TNF-α-treated group) or TNF-α plus HPT at 10 μM (TNF-α + HPT-treated group) or TNF-α plus DEX at 10 μM (TNF-α + DEX-treated group) was used for treating chondrocytes for 48 h (TNF-α and HPT or TNF-α and DEX were added in the same order). All the experiments were performed in triplicate independently.Cell Counting Kit 8 (CCK-8)  was used to investigate cell viability and cytotoxicity. Chondrocytes were seeded at a density of 1,000 cells/well in a 96-well plate and tested for toxicity for 1–5 days with different concentrations of HPT. DMSO was used as a control. At designated time intervals, the cells were incubated with 10% CCK-8 reagent at 37°C for 2–4 h. After incubation, the corresponding absorbance was measured with a microplate reader at a wavelength of 490 nm. The data were analyzed with GraphPad 8.0. All the experiments were performed in triplicate independently.Chondrocytes were treated with TNF-α alone or TNF-α plus HPT and then harvested for the qRT-PCR assay. DMSO was used as control. Each sample was harvested using TRIzol reagent, and RNA was isolated. Total RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Then, the cDNA was used as template to perform real-time polymerase chain reaction (PCR) on a StepOne Plus thermal cycler (Applied Biosystems). Primers were designed by NCBI primer blast and synthesized using Invitrogen. Data were analyzed according to the 2−ΔΔct method.Rat chondrocytes were lysed by RIPA lysis buffer to extract the total protein which was separated by SDS-PAGE gel electrophoresis and transferred to PVDF (polyvinylidene fluoride) membrane . After 1 h blocking with 5% skimmed milk, the membrane was incubated with primary antibody ACAN , COL2A , IL-1β , IL-6 , MMP-13 , PTGS2 , Bcl-2 , CDK1 , Cyclin D1 , p-AMPKα1 , AMPKα , and GAPDH  antibodies overnight at 4°C. After washing 3 times with 1×TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody  at room temperature for 1 h. Finally, target protein was visualized by the ECL Western Detection Kit  and quantified by ImageJ software.TM flow cytometer .Flow cytometry was used to determine the effect of HPT on the chondrocyte cycle. After being treated with TNF-α alone or TNF-α plus HPT, the chondrocytes were harvested and washed, followed by fixation using 70% alcohol at 4°C overnight. After washing the cells with PBS, they were treated with ribonuclease RNase A and incubated with propidium iodide (PI) for the cell cycle analysis on a BD FACSVerseRNA sequencing was performed on chondrocytes in different treatments to determine the changes in their mRNA expression profiles. RNA extraction kit  was used to extract total RNA from each group of cells. Send the obtained RNA sample to BGI Co., Ltd., Shenzhen, China, and perform RNA-seq operation on BGISEQ-500. The R language program was used to further analyze the sequencing results to find differential genes in the chip expression profile. Then, use the R program to perform the gene ontology (GO) biological function enrichment analysis and genome encyclopedia (KEGG) signal pathway enrichment analysis. All samples were repeated three times.The chondrocytes were seeded on coverslips coated with 0.01% poly L-lysine. After treatment, the cells were fixed in 4% paraformaldehyde for 30 min, followed by rinsing with PBS and permeating with 0.1% Triton X-100 on ice for 5 min. Then, the cells were blocked with a blocking solution at 37°C for 1 h and then incubated with the primary antibody p-AMPKα1  at 4°C overnight and with a fluorescently labeled secondary antibody (concentration 1:200) at 37°C for 1 h, respectively. After counterstaining with DAPI, the samples were imaged and captured under a fluorescence microscope .The animal experiments were carried out following the protocol approved by the Animal Care and Use Committee of Chongqing University. The OA model was achieved by anterior cruciate ligament transection (ACLT) in SD rats, as described in our previous study . There ain vivo on regulating the AMPK pathway. In the microscopic examination, the histological score was scored according to the histological evaluation performed in the previous study  and safranin-O fast green. H&E staining was used to observe changes in the surface of articular cartilage, cartilage levels, and the number and morphology of cartilage cells. In addition, the histological immunofluorescent staining for p-AMPKα1  was performed to verify HPT exhibits the effect us study . The , and p < 0.05; TNF-α vs. control), while HPT recovered these gene levels   . Like ge. TNF-α) . Moreove. TNF-α) . Finally. TNF-α) . These fp < 0.05; TNF-α vs. control), while HPT recovered these gene levels   , while the percentages of chondrocytes in the S phase were restored to the control group level in the group with TNF-α + HPT   . Similar. TNF-α) . We furt. TNF-α) .The RNA-seq experiments were performed in cells affected by TNF-α induction with or without HPT treatment to further investigate the protective and anti-inflammatory mechanism of HPT in TNF-α-induced chondrocytes. The results demonstrated that compared with the TNF-α group, there were many differentially expressed genes (DEGs) in chondrocytes treated with TNF-α and 10 μM HPT, of which 105 genes were upregulated and 444 genes were downregulated . Furtherp < 0.05). Furthermore, immunofluorescence staining with p-AMPKα1 was performed, which further supported the Western blot results (ap < 0.05). In addition, it also diminished the upregulation of p-AMPKα1 and COL2A expression by HPT treatment (ap < 0.05). These data suggested that HPT relieves TNF-α-induced inflammation of chondrocytes through the AMPK signaling pathway.Based on the results of highly enriched signaling pathways, we selected AMPK signaling pathways for further analyses. Western blot analysis showed that TNF-α induction suppressed the phosphorylated-/total-AMPKα1 (p/t-AMPKα1) ratios; however, the phosphorylation of AMPKα1 increased after HPT supplementation  . HPT treatment significantly changed this situation (#p < 0.05). In addition, compared with the healthy group, the histological score of the OA group was significantly different (*p < 0.05); however, DEX and HPT treatment significantly improved cartilage degeneration, and the histological score was significantly different from that of the OA group (#p < 0.05), indicating that HPT treatment successfully inhibited cartilage degeneration. In addition, the expression of p-AMPKα1 was measured in vivo by immunofluorescent staining (α1 was recovered after DEX and HPT supplementation.ACLT was used to establish the rat OA model. Healthy rats served as a control group. HPT was locally injected into the joint cavity 4 weeks after ACLT surgery. As shown in staining . The resOA is a chronic disease with low-grade inflammation . Thus, iGrowing evidence indicates that HPT positively affects anti-inflammatory reactions . InflammHPT can inhibit inflammatory responses in lipopolysaccharide-induced RAW 264.7 cells via the inhibition of the NF-κB pathway and activation of the Nrf2/HO-1 pathway . ResearcWe examined the effect of HPT on TNF-α-induced inflammation with or without compound C, a selective AMPK inhibitor, to determine the role of the AMPK signaling pathway in HPT-induced alleviation of inflammation. Further verification experiments showed that HPT relieves TNF-α-induced inflammation of chondrocytes through the AMPK signaling pathway. It has also been demonstrated that AMPKα1 deficiency in chondrocytes accelerates the development of both injury-induced and age-related spontaneous OA in mice . Furtherin vivo study provided evidence that HPT prevented the occurrence and development of OA by reducing cartilage degradation, delaying cartilage surface erosion, and maintaining chondrocyte formation. In conclusion, this study suggests that HPT might be used as a potential therapeutic agent for OA.This study established an ACLT rat model and locally administered HPT treatment to further determine the therapeutic effect of HPT on OA. The ACLT-induced OA model we established is very similar to human osteoarthritis, and our previous studies have been reported . It has


In bone-marrow-derived macrophages harvested from these mice, various anti-TNF biologics induced the expression of genes characteristic of alternative macrophages and also inhibited the expression of pro-inflammatory cytokines mainly through the upregulation of arginase-1. Injections of TNF inhibitors during arthritis increased pro-resolutive markers in bone marrow precursors and joint cells leading to a decrease in arthritis score. These results demonstrate that the binding of anti-TNF biologics to tmTNF results in decreased arthritis severity. Collectively, our data provide evidence for the significance of tmTNF reverse signaling in the modulation of arthritis. They suggest a complementary interpretation of anti-TNF biologics effects in the treatment of inflammatory diseases and pave the way to studies focused on new arginase-1-dependent therapeutic targets.In order to ascertain the significance of transmembrane tumor necrosis factor (tmTNF) reverse signaling   •In vivo demonstration of tmTNF reverses signaling existence•tmTNF reverse signaling induces anti-oxidative stress response•tmTNF reverse signaling induces an arginase-1-mediated anti-inflammatory response•Reverse signaling is a complementary mechanism to TNF neutralization by anti-TNF Immunology; Cell Biology Tumor necrosis factor (TNF) is a homotrimeric pro-inflammatory and immunomodulatory cytokine. Like most members of the TNF superfamily, it exists either in a transmembrane form (transmembrane tumor necrosis factor [tmTNF]) or in a soluble form (soluble tumor necrosis factor [sTNF]) after cleavage of its precursor—(tmTNF)—by the protease TACE . Both forms are bioactive . Once seRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects 0.5% of the adult population. It is characterized by the infiltration of synovial compartment of joints by immune cells . MacrophThe development of TNF inhibitors has permitted to control the activity of RA in a substantial proportion of patients . There ain vitro as a signaling receptor through a mechanism known as “reverse signaling”  resistance, via the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway . Furtherin vitro neuronal growth  animals is not the aim of this study and will be discussed later. In primary bone-marrow-derived macrophages (BMDMs) from these mice, we evaluated the effects of anti-TNF stimulation on macrophage polarization in vitro. We observed that tmTNF reverse signaling induced a decrease of the pro-inflammatory transcription factor Fra-1 and consequently an upregulation in pro-resolutive transcription factors and effectors such as arginase-1 (Arg-1). Furthermore, we observed that tmTNF reverse signaling induced an early peak of IL-10 and inhibited pro-inflammatory cytokines, such as IL-6 and IL-12. Finally, using the K/BxN serum injection arthritis model in WT and 3TG mice, we showed that anti-TNF injections induced an increase in pro-resolutive transcription factor and enzyme (Arg-1) in bone marrow precursors and inhibited arthritis. Furthermore, pro-inflammatory IL-1β was inhibited, and neutrophils were less numerous in the joints of treated mice, thus demonstrating for the first time the significance of reverse signaling in vivo and implicating a novel interpretation of the effects of anti-TNF therapy in the treatment of inflammatory diseases, such as RA.To this end, we generated a triple transgenic mouse model (3TG) lacking TNFR1 and TNFR2 expression (TNFR1/R2 KO) and expressing TNF at a physiological level exclusively in its transmembrane form (tmTNF KI) due to knock-in mutations . These 3We first validated our mouse model specifically developed to study reverse signaling. By flow cytometric analysis of immune cell populations in the spleen and inguinal lymph nodes, we did not observe significant differences in these cell populations between WT and 3TG mice A–S1C. AsWe then investigated the interaction of soluble TNF receptor 2 (ETA) with tmTNF in BMDMs from 3TG mice. To this end, we performed imaging cytometry on BMDMs after 30 min of stimulation with LPS (50 ng/mL) as a mean to increase tmTNF cell surface expression. Cells were then incubated with ETA conjugated to DyeLight488 (ETA-488), and an internalization assay was performed. As a control, we used anti-H-2 (I-A/I-E) phycoerythrin-conjugated antibody which did not induce any internalization A. HigherTNF inhibitors are known to modulate macrophage polarization . We thusThen, we analyzed the expression of the scavenger receptor CD36 which was previously shown to be upregulated by anti-TNF treatment in human monocytes . In our In vitro, we observed a strong decrease in Fra-1 expression in WT and 3TG M1 macrophages in the presence of anti-TNF but not in the IgG control-treated cells   C. AlthouA] test) D (p < 0.A] test) D. There A] test) A and S7BA] test) C. NevertA] test) E. IL-1β A] test) E. This lA] test) A. By floin vitro B. No effin vitro C and B lin vitro D was notin vitro D, suggesin vitro , were dein vitro E. These in vitro F, althouin vitro E and S7Fin vivo anti-TNF-induced tmTNF reverse signaling and demonstrate for the first time its impact on macrophage polarization and its functional role in the clinical response to TNF blocking therapy during arthritis in vivo.In this work, we present evidence for Contrary to what was previously published , we obseIn vitro, we saw that TNF blockade during WT macrophage differentiation, by antibodies but not ETA, inhibited the expression of pro-inflammatory surface markers, such as CD40 and Ly6c. Such inhibition can be attributed to the neutralization of sTNF as it was not observed in our 3TG model. This difference between antibody and ETA may be due to a different interaction with sTNF and tmTNF  with normal cell-surface expression and function which avoid an interaction with other receptors . Neverthin vivo and prompt us to consider new interpretations to the mechanisms underlying the effects of TNF inhibitors in the treatment of inflammatory diseases. Our work also paves the way to studies focused on new arginase-1-dependent therapeutic target, such as tmTNF agonists.In conclusion, our data provide evidence for the involvement of tmTNF reverse signaling in the anti-TNF-mediated modulation of arthritis Although we did not see any significant differences between WT and 3TG mice in immune cell populations in the spleen and inguinal lymph nodes and we observe an equivalent capacity of anti-TNF to interact with its ligands, we are aware that 3TG mice are just a model to study tmTNF reverse signaling and are quite different from WT mice. Indeed, WT mice express soluble TNF that participate to inflammation, and the effect of anti-TNF administration is mediated not only by tmTNF reverse signaling but also by sTNF neutralization. This lack of sTNF limits also the arthritis model that we could use. Nevertheless, we could demonstrate that tmTNF reverse signaling is a complementary mechanism of anti-inflammatory effect of anti-TNF therapies.benjamin.rauwel@inserm.fr) or Dr. Degboé Yannick (yannick.degboe@inserm.fr)Information and requests for resources should be directed to and will be fulfilled by the lead contact, Dr. Rauwel Benjamin (This study did not generate new unique reagents.All relevant data are available from the lead contact upon request.All methods can be found in the accompanying


The intracellular delivery of emerging biomacromolecular therapeutics, such as genes, peptides, and proteins, remains a great challenge. Unlike small hydrophobic drugs, these biotherapeutics are impermeable to the cell membrane, thus relying on the endocytic pathways for cell entry. After endocytosis, they are entrapped in the endosomes and finally degraded in lysosomes. To overcome these barriers, many carriers have been developed to facilitate the endosomal escape of these biomacromolecules. This mini-review focuses on the development of anionic pH-responsive amphiphilic carboxylate polymers for endosomal escape applications, including the design and synthesis of these polymers, the mechanistic insights of their endosomal escape capability, the challenges in the field, and future opportunities. Most successfully developed biotherapeutics up to date only target extracellular receptors, because the intracellular delivery of biomacromolecules remains a key challenge , poly(amino ester), poly(2-(dimethylamino)ethyl methacrylate), and polyamidoamine] can buffer the endosomal acidification by protonation, and cause osmotic swelling of endosomes, finally leading to endosomal rupture. The polycation induced “proton sponge” effect has long been studied, and reviewed recently , without disrupting the membrane at physiological pH  lipids in the 1980s  has been developed in the late 1990s  methacrylamide) (HPMA), polyethylene glycol , or poly (Jeffamine). The PEG block could enhance polymer solubility by forming micelles and increase the resistance to serum proteins  in the polymerization with PAA monomer generated hyperbranched PPAA, which showed lower hemolytic activity than linear PPAA at endosomal pH conditions  with hydrophobic methacrylates have the coil-to-globule conformational transition, when the pH decreases from neutral to acidic ranges  (PLP), also confirm that pH-dependent membrane-permeabilizing capability could be adjusted by grafting amino acids with different hydrophobicity or alkyl chains  polymerization (Fairbanks et al., Meanwhile, the mechanism of polymer-mediated endosomal escape has been intensively explored, along with the advances in the basic understanding of the endocytosis process itself (Skowyra et al., Notwithstanding the great achievements in both polymer synthesis tools and endosomal escape mechanism investigation, many fundamental issues remain to be addressed in this field. For example, the fate of the amphiphilic carboxylate polymers within the cells after the endosomal escape is rarely covered in the previous publications. It is not known whether the polymer carrier itself undergoes degradation in the cell, or gets expelled from the cell somehow. This issue is critical for biomedical applications, because of the long-term biosafety concerns. Further investigations are expected to result in an improved understanding of the degradative pathways of these polymers within the cells.in vivo environment.In summary, carboxylated amphiphilic polymers with pH-responsive endosomolytic activities demonstrate promising potentials for the intracellular delivery of macromolecules. From a retrospective view, this field has continuously progressed with the application of new synthetic techniques, mechanistic understanding of endocytic trafficking, and better methods for endosomal escape characterization. Up to date, various designs have been made to adapt these polymers for biomedical applications, i.e., the delivery of antigens, genes, and therapeutic peptides. Nevertheless, it is still early to expect clinical translations, due to the lack of biodegradability and long-term biosafety concerns. It would require joint efforts from polymer chemists, biologists, and pharmaceutical scientists to understand how polymers interact with endosomal membranes at the molecular level; how the endosomal escape happens at the cellular level; and finally the delivery in the complicated SW constructed the idea, designed the article, drafted and revised the manuscript. The author has agreed to publish the content of the work.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on  Virus-like particles (VLPs) or chimeric virus particles (CVPs) have been utilized extensively as effective scaffolds for the presentation of epitopes or antigens in the development of vaccine candidates –4. The aBamboo mosaic virus (BaMV), a member of the genus Potexvirus, and validated its applicability in stimulating protective immunity against foot-and-mouth disease virus (FMDV)  , 12, infs (FMDV) , or Japas (FMDV)  in diffeOne of the major limitations of the epitope-presenting VLP or CVP systems is that the fusion of target proteins on the viral coat proteins may impair the self-assembly efficiency of the VLPs or CVPs, which are required for highly ordered presentation of repetitive epitopes to stimulate strong immune responses. In addition, the fusion with the viral CP may interfere with the proper folding of the antigen proteins, leading to inactive conformation. Thus, the sizes of the target antigens to be displayed on VLPs or CVPs are usually limited to short peptide fragments. However, some epitopes are structurally-dependent, which require the formation of specific conformation from different domains of an antigen, not just linear amino acid sequences. The peptide epitopes might not stimulate proper immune responses. Several strategies have been developed to circumvent the above challenges. For example, we have adopted the FMDV 2A peptide sequence, which induces co-translational “cleavage”  between Staphylococcus aureus and found to mediate the ligation of proteins on the surface of bacterial cells. It has been shown that different SrtA may recognize and cleave at specific amino acid sequences in the donor protein and mediate the ligation of the acceptor protein at the exposed N-terminus  residues as the acceptor for SrtA-mediated ligation (hereafter referred to as SrtA-ready CVPs). The second part includes vectors for the expression of target antigen, with calmodulin (CaM) fusion tag to enhance solubility, and SrtA protein, respectively, in bacterial cells. The applicability of this new system was demonstrated by using the recombinant envelope protein domain III (rEDIII) of JEV (111 amino acids) as the target epitope, and the efficacy of the vaccine candidate in the induction of functionalized antibodies was validated in BALB/c mice. The current BaMV-based system provides an efficient alternative for the rapid development of bioactive vaccine candidates.To address these issues, we have devised a binary system in this study. The first part contains a BaMV-based vector which facilitates the efficient Agrobacterium-mediated infiltration. The coding sequence for TEV protease cleavage motif  and tetra-glycine (4G) were amplified by polymerase chain reaction (PCR) using the primer pair, TM-4G-F and TM-4G-R, and inserted into pBS-d35CP at the AgeI/NotI sites, which was further digested with ApaLI and SacI and inserted into the corresponding restriction sites of pKBNd35 to generate pKBTM4G. The coding region for BaMV triple-gene-block proteins (TGBps) was replaced by that of Histidine-tagged TEV protease (7×His-TEV protease), which was amplified by PCR using the primer pair, 7×His-TEV protease-F and TEV protease-stop-R, followed by digestion with DraIII and cloned into the corresponding site of pKBTM4G, generating pKB5G  CaM were digested with NdeI and EcoRI and ligated into the cognate sites of pET29a  to generate pET29a-6×His-CaM-6×His, abbreviated as pET29a-CaM. The coding sequence of JEV rEDIII was amplified from pBJ2A .To improve the solubility of the recombinant proteins, we have employed CaM fusion tag, which has been used for proteins highly expressed in E. coli . The genom pBJ2A  with theom pBVP1  with theE. coli BL21 Star (DE3) cells harboring the desired constructs, the induction and purification of target proteins were performed as described previously . The fractions containing rEDIII-CaM and VP1 peptide-TrxA proteins were confirmed by 12% polyacrylamide gel containing 1% sodium dodecyl sulfate (12% SDS-PAGE). The rEDIII protein, which was expressed from pET21d-rEDIII , and BaMV CVPs were purified using the standard protocol described previously , and the reaction mixture was incubated at 28°C for different time period as indicated. SrtA activity was terminated with the addition of 0.25 reaction volume of 5× sample buffer  glycerol, 2 mM beta-mercaptoethanol, 0.1% (w/v) bromophenol blue). The protein products were analyzed by electrophoresis through 12% SDS-PAGE, followed by Coomassie Blue Staining (CBS) or western blotting with specific antibodies as described below. Large-scale coupling reactions were performed in a 100-ml beaker, and the mixture was incubated for 15 min at 28°C with gentle stirring. Another epitope candidate, the VP1 peptide-presenting BaMV CVPs (designated BVP1LPET5G) were also produced as described above.The CP concentration of BaMV CVP was adjusted to 1 mg/mL and subject to SrtA-mediated ligation reaction. The ratio between the volumes of BaMV CVP and the total reaction mixture was adjusted to 1:10. To determine the optimal reaction condition of the rEDIII coupling to BaMV CVPs, the ratio of B5G CP: rEDIII-CaM: SrtA was varied as indicated in a 1.5-ml Eppendorf tube containing 1× SrtA reaction buffer  were gently added to inhibit SrtA activity; (2) the calcium complex was removed by centrifugation at 7,600 ×g at 4°C for 10 min; (3) the supernatant was purified with the Ni2+-NTA column to remove the unreacted His-tagged proteins; (4) the flow-through fraction was collected and subjected to ultracentrifugation through a 20% sucrose cushion; and (5) the virion pellet was resuspended with Borate–EDTA buffer  and stored at 4°C. The BVP1LPET5G CVPs were inhibited SrtA activity as described above and purified using the PEG 6000 precipitation protocol as described previously (The rEDIII-presenting BaMV CVPs (designated BJLPET5G) were purified as described above with the following modifications: (1) the chelator (10 mM EGTA) and disodium phosphonate  twice, and the sera were collected. The titers of sera were tested through indirect enzyme-linked immunosorbent assay (ELISA).Three groups of five 3-week-old female BALB/c mice were obtained from the National Laboratory Animal Center, Taiwan. Freund’s complete (priming) or incomplete adjuvants (boosting)  were mixed with B5G (BaMV virion scaffold), rEDIII (5 μg), and BJLPET5G (≈5 μg of rEDIII-coupled BaMV virion scaffold) for priming on day 1 and boosting on day 12, respectively, for intraperitoneal inoculation. Whole blood from the orbital sinus was collected on days 0, 11, and 26; the blood cells were removed through centrifugation .C6/36 cells were infected with JEV (RP-9)  at a mulBHK-21 cells were infected with the reporter virus, JEV (RP-9) expressed enhanced green fluorescent protein (JEV-eGFP) , at an M70) data for each antibody were used to calculate relative reduction in fluorescence of JEV-eGFP, determined as follows: % reduction = 100 × [1 − (average number of fluorescent cells for each dilution/average number of fluorescent cells in B5G control group)]. The RVNT70 is evaluated by the concentration that gave 70% fluorescent cells reduction, determined by nonlinear, dose-response regression analysis with GraphPad Prism version 6.0 .The titers of the neutralizing antibodies in the sera from mice vaccinated with various CVPs were determined using the neutralization assay with BHK-21 cells and the reporter virus JEV-eGFP (at an MOI of 0.5) in triplicates following the standard protocol . The JEV2+-NTA resin in batch mode, and bound on resin for the coupling reaction with SrtA-ready B5G CVPs. Mass-produced SrtA-ready B5G CVPs were added to the resin with rEDIII-CaM and SrtA, incubated for 15 min in the SrtA reaction buffer containing 300 mM imidazole. The SrtA-mediated ligation reaction was terminated with the addition of EGTA (10 mM), and the imidazole was removed by using spin column . Supernatant was incubated with Ni2+-NTA resin again to remove his-tagged unreacted proteins. The BJLPET5G CVPs were then harvested from the supernatant after low-speed centrifugation.His-tagged rEDIII-CaM and SrtA proteins were purified from bacterial cell lysates (following sonication) with Nit test was used to compare between the means of two groups; p < 0.05 indicates statistical significance.Statistical analysis and graphical visualizations were performed in Microsoft Excel 2010  and GraphPad Prism version 6.0. A two-tailed Student’s Nicotiana benthamiana via Agrobacterium-mediated infiltration. Total proteins were extracted from the inoculated leaves 7 DPI and analyzed by electrophoresis through 12% SDS-PAGE, followed by CBS , but not pKB5G (lane 4). We hypothesized that the low expression level might be due to the replacement of the original BaMV RNA silencing suppressor, TGBp1, by the TEV protease in pKB5G construct, leading to the silencing of the expressions of B5G CP. To address this problem, we resorted to the usage of the well-known potyvirus silencing suppressor, HC-Pro  with a size of approximately 35 kDa, as observed by SDS-PAGE analysis , 38. WitTo demonstrate the applicability of the B5G CVP platform with SrtA-mediated ligation for other antigens, we used the VP1 peptide derived from FMDV  as the t2+-NTA resin. It was found that the affinity purification method prevented the precipitation problem (Following the addition of 10 mM ethylene glycol tetraacetic acid (EGTA) to terminate SrtA reaction, the virions were extracted from the mixture as described . Unexpec problem . Based oTo verify whether the CVPs of B5G and BJLPET5G were properly assembled, the purified samples were examined by electron microscopy, with the BNd35 virion as a control. The result showed that the CVPs of B5G and BJLPET5G exhibited similar flexible filamentous virion conformations as those of BNd35 ], it was50) values could not be accurately determined since the reaction curves had not reached a plateau at 1/100 dilution in our experiments. The observation indicated that immune responses elicited by the treatments were not especially strong. However, the data indicated that BJLPET5G stimulated higher or comparable titers than rEDIII did at the priming and boosting stages, respectively. In addition, the activities of antibodies were evaluated by using the JEV-eGFP reporter system  with B5G (52.25 µg), free rEDIII (5 µg), or BJLPET5G (corresponding to 5 µg of rEDIII conjugated on 52.25 µg of B5G CP) following the scheme illustrated in r system , in whicr system , 35. The70  and SrtA proteins were lysed by ultrasonication, and the lysates were directly mixed with Ni2+-NTA resin in the Eppendorf tube followed by the addition of previously prepared SrtA-ready B5G CVPs. The supernatant containing BJLPET5G CVPs was then recovered. However, the result of SDS-PAGE analysis of the products , tested for quality control, and stored or applied immediately by SrtA-mediated ligation with desired antigens for the development of other vaccine candidates. In contrast, in the previous CP-epitope fusion approaches, such as , the type species of the genus Potexvirus, is one of the viruses with adjuvant potential  could reach up to 83% by increasing the incubation time and the concentration of M2e peptide . Howeverotential . BaMV isN. benthamiana for the production of BaMV-based vaccine candidates  in the B5G construct and ensure the production of B5G virions in each cell of the transgenic plants. Secondly, our design facilitated auto-processing of CP and self-assembly CVP scaffold. In comparison, the scheme for PapMV-based platform recently published  to enter the lymphatic vessels . In addiN. benthamiana, the other for the generation of target antigens and SrtA in E. coli. Upon mixing the B5G CVPs with the antigen proteins and SrtA, antigen-presenting CVPs (BJLPET5G) could be generated in 1 min and readily purified by removing the unreacted antigen proteins and SrtA through Ni2+-NTA column. Injection of mice with the BJLPET5G CVPs resulted in the stimulation of immune responses against JEV, demonstrating the prospective applicability of our approach. This study thus provides an efficient system for the development of vaccine candidates, adding to the arsenal against the ever-emerging pandemics.An alternative approach for the production of BaMV-based CVPs was provided, facilitating the presentation of antigens (rEDIII and VP1 peptide) through SrtA-mediated coupling. Our approach consists of two subsystems: one for the production of SrtA-ready B5G CVPs in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/This animal protocol was approved by the Academia Sinica Institutional Animal Care and Use Committee (Protocol no. 17-11-1123) and was performed in accordance with the guidelines.M-HY performed the SrtA-ready BaMV construction, the coupling experiments on the B5G BaMV platform, batch production of coupling CVPs, and was involved with immunoelectron microscopy and animal experiments. M-HY also drafted the manuscript. C-CH, C-HW, N-SL, Y-HH, and Y-LL helped draft the article structure of the manuscript. J-JL helped perform mouse experiments. H-YK provided technical assistance for animal experiments and useful advice and discussion. M-HH helped perform immunoelectron microscopy experiments. Y-LL and N-SL guided and provided advice and discussion of the animal experiences and the immunoelectron microscopy. Y-HH supervised the entire study, organized the participation of each lab participant. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Ministry of Science and Technology, Taiwan , and by the Advanced Plant Biotechnology Center from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We prepared bi- or trifunctional nanoparticles bearing tumor necrosis factor-α (TNF) and/or interleukin-12 (IL12) plus a peptide containing isoDGR, and we tested their anti-cancer effects, alone or in combination with doxorubicin, in tumor-bearing mice.Gold nanospheres tagged with peptides containing In vitro biochemical studies showed that both nanodrugs were monodispersed and functional in terms of binding to TNF and IL12 receptors and to αvβ3. In vivo studies performed in a murine model of fibrosarcoma showed that low doses of bifunctional nanoparticles bearing isoDGR and TNF (corresponding to few nanoparticles per cell) delayed tumor growth and increased the efficacy of doxorubicin without worsening its toxicity. Similar effects were obtained using trifunctional nanoparticles loaded with isoDGR, TNF and IL12. Mechanistic studies showed that nanoparticles bearing isoDGR and TNF could increase doxorubicin penetration in tumors a few hours after injection and caused vascular damage at later time points.IsoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multi-cytokine delivery to cells of the tumor vasculature. Extremely low doses of isoDGR-coated nanodrugs functionalized with TNF or TNF plus IL12 can enhance doxorubicin anti-tumor activity.The online version contains supplementary material available at 10.1186/s12951-021-00871-y. The rationale for exploring the TNF/IL12 combination, in addition to TNF alone, relies on the notion that IL12 can induce IFNγ in the tumor microenvironment [isoDGRG) (iso1), a head-to-tail cyclic peptide that recognizes αvβ3 integrin overexpressed in the tumor vasculature [iso1 and TNF  or with iso1, TNF and IL12  can increase the efficacy of doxorubicin without increasing its toxicity. Furthermore, we show that nanogold functionalized with iso1 and TNF increases doxorubicin penetration in tumors a few hours after injection and causes vascular damage at later time points.The major limitation in using cytokines for cancer therapy is often related to their prohibitive toxicity and rapid induction of counter-regulatory mechanisms –3. A gro-α (TNF) , interfe-α (TNF) , or IFNγ-α (TNF)  to tumor-α (TNF) , 10. A p-α (TNF) . Low dos-α (TNF) , 11–14. -α (TNF) –19. We ho tumors –23. Indeo tumors –23. Howeironment , 25, a cironment . Thus, wculature , 28. We isoDGRG) peptide  were prepared as described previously [Iso1-HSA conjugate, comprising iso1 chemically coupled to HSA via 4-cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) and SMCC-HSA (HSA activated with sulfo-SMCC and quenched with β-mercaptoethanol in place of iso1), were prepared as described previously [The following reagents were purchased: human serum albumin (HSA) 20% solution ; bovine serum albumin (BSA) (Sigma); human integrin αvβ3 ; methoxy-PEG-SH  ; soluble TNF receptor Type-II (sTNF-R2) fused to the Fc fragment of an IgG1 antibody (Enbrel) ; monoclonal antibody (mAb) anti-IL12/IL23, p40 subunit, clone C15.6 and C17.8 ; recombinant murine interleukin-12 (IL12) (PeproTech); and 25 nm gold (Au) nanospheres (nanogold Aurion). Recombinant murine TNF and synthetic cyclic head-to-tail c or iso1 and TNF (iso1Au/TNF) were prepared, as described previously, by incubating 1 ml of 25-nm nanogold  with 0.1 ml of solution containing 120 µg of iso1-HSA and 2.7 µg of IL12 or 160 µg of iso1-HSA and 16 µg of TNF, respectively [iso1 (Au/TNF and Au/IL12) were prepared in the same manner, except that SMCC-HSA was used instead of iso1-HSA. Other monofunctional Nps lacking cytokines (iso1Au) were prepared in the same way by omitting the cytokine in the incubation mixture.Bifunctional gold Nps loaded with ectively , 23  were prepared as follows: a solution containing 132 µg of iso1-HSA, 1 µg of TNF (19 pmol), 6 µg of IL12 , and 5 µg of PEG in 100 µl of 5 mM sodium citrate buffer, pH 6.0, was added to 1 ml of 25-nm nanogold (Aurion), with the pH adjusted to 4.3–4.4 with orthophosphoric acid , and left to incubate for 2 h at room temperature under shaking. The product was mixed with 100 µl of 10% HSA in water  and incubated for an additional 5 min to saturate gold Nps. The final product was centrifuged , resuspended in 1 ml of 1% HSA in 5 mM sodium citrate buffer, pH 6.0 (three times), filtered  and stored at − 80 °C  were prepared in the same manner, except that SMCC-HSA was used instead of iso1-HSA.Trifunctional gold Nps loaded with 530 nm values on a calibration curve obtained using uncoated nanogold in 5 mM sodium citrate buffer, pH 6.0 .Visible spectra of the nanodrugs were recorded using an UltroSpec 2100 spectrophotometer (Amersham Biosciences) and a 1-cm-path-length quartz cuvette. The concentration of Nps was calculated by interpolating the A10 Nps/ml) and analyzed using 25–30 independent measurements of 10-s duration at 20 °C. The calculation of the hydrodynamic radius of the Nps was performed using DLS regularization analysis.Dynamic light scattering (DLS) measurements were performed using a DLS DYNAPRO 99 instrument (Wyatt) operating with the laser intensity set to 20% power. The nanodrugs were diluted 1:10 in 5 mM sodium citrate buffer, pH 6.0 (1–3 × 10Transmission electron microscopy (TEM) analysis were performed using a TALOS L120C microscope (ThermoScientific) as described previously . MorphomThe binding assay of nanodrugs to αvβ3, anti-IL12 antibodies or sTNF-R2 (Enbrel) spotted onto nitrocellulose filters (0.45 µm) was performed using a Bio-Dot apparatus (Bio-Rad) as described previously , 21, 23.The amount of IL12 and PEG bound to gold Nps was quantified using the MAX Deluxe Set Mouse IL12 (p70) ELISA kit (Biolegend) according to the manufacturer’s instructions and the Polyethylene Glycol Backbone ELISA Kit  as described previously .The amount of bioactive IL12 bound to gold Nps was determined using an in vitro bioassay based on IL12-induced release of IFNγ from murine splenocytes . IFNγ wa6 WEHI-164 cells. Five days later, the mice were injected (i.v.) with nanodrugs in 0.9% sodium chloride containing 100 µg/ml of HSA, alone or in combination with 150 µg of doxorubicin  in 0.9% sodium chloride. Tumor growth was monitored by measuring the tumor size with a caliper. The tumor volumes were estimated by calculating r1 × r2 × r3 × 4/3π, where r1 and r2 are the longitudinal and lateral radii, respectively, and r3 is the thickness of the tumor protruding from the surface of normal skin. Animals were sacrificed before the tumors reached a diameter of 1–1.5 cm. The tumor sizes are shown as the mean ± SE.Murine WEHI-164 fibrosarcoma cells  were cultured in DMEM with standard supplements and tested for mycoplasma contamination before their use in vivo using the MycoAlert assay Control Set (Lonza). BALB/c mice , weighing 18 to 20 g, were challenged by s.c. injection into the left flank with 10Iso1-coated gold Nps (iso1Au) bearing different amounts of TNF and IL12  were prepared by incubating 25-nm Au nanospheres with solutions containing TNF and IL12 in different molar ratios (1:4.5 or 4:1 molar ratio) and fixed amounts of iso1-albumin conjugate (iso1-HSA) and PEG  and monofunctional (Au/TNF and Au/IL12) nanodrugs were also prepared by omitting one or two components, respectively, in the incubation mixtures. UV–visible spectrophotometric analysis of the mono-, bi-, and trifunctional products showed similar absorption spectra, as judged from the λmax absorption values, peak width at 75% height (PW75), and 650/530 nm ratio  or anti-IL12 mAb C15.6. Mono-, bi- and trifunctional nanoparticles recognized spots on nitrocellulose with patterns consistent with their expected functional properties  and to promote IFNγ release from cultured murine splenocytes (bioassay). Both assays were calibrated with IL12. The immunoreactivity of each bi- or trifunctional nanoparticle was equivalent to that of 5–19 molecules of IL12, depending on the preparation protocol  in the IL12 bioassay corresponded to 4.8 × 106 and 4.2–4.8 × 106 Nps/ml, respectively, despite the different number of immunoreactive IL12 molecules/Np . The administration of doses of iso1Au/TNF, iso1Au/IL12, or iso1Au/TNF + IL12 equivalent to approximately 10 pg of TNF or 70 pg of IL12 induced similar anti-tumor effects  was investigated using the same tumor model. The administration of 150 µg of Doxo alone induced a significant delay in tumor growth Fig. b. This eL12 Fig. b. None oiso1Au/TNF and its synergism with Doxo was then investigated. Considering that TNF can increase tumor vascular permeability and reduce drug penetration barriers, we tested the hypothesis that this nanodrug increased Doxo penetration in tumors. To this aim, we exploited the fact that Doxo is a fluorescent compound and that the fluorescence intensity of the tumor cells recovered from animals after treatment indicates the amount of Doxo that penetrated tumors [iso1Au/TNF increased the fluorescence intensity and the percentage of positive cells recovered from tumors 2 h after treatment . A marked reduction in the signal/time area under the curve (AUC) and peak enhancement of microbubbles was observed 2–3 days after treatment  trigger biological effects more efficiently than the corresponding “free” cytokines. For example, 35- to 47-fold lower amounts of nanoformulated IL12, compared to IL12, were sufficient to evoke the secretion of IFNγ from murine splenocytes. This phenomenon was not related to iso1-mediated targeting mechanisms of nanoformulated cytokines, because iso1Au/IL12 and Au/IL12 showed similar potencies in this assay (data not shown). More likely, this phenomenon was related to high-avidity interactions engaged by multivalent nanoparticles with IL12 membrane receptors. Accordingly, enhanced effects have also been observed with TNF upon nanogold formulation.This work shows that gold nanospheres coated with the iso1Au/TNF or iso1Au/TNF + IL12, corresponding to 3–6 × 107 nanoparticles , were sufficient to exert synergistic effects with doxorubicin in the WEHI-164 fibrosarcoma model. If we assume that approximately 1% of the injected dose accumulated in tumors, as typically observed with nanoparticles [5 nanoparticles reached their target in tumors. Considering that 0.1- to 0.2-cm3 tumors may contain 1–2 × 107 cells [iso1 [iso1Au/TNF, showed that this nanodrug enhanced the amount of doxorubicin that reached cancer cells, likely by increasing endothelial permeability and reducing drug penetration barriers. However, analysis of tumor perfusion by contrast-enhanced ultrasound 2–3 days after treatment showed that iso1Au/TNF, despite its low dose, could also cause vascular shutdown in some tumor areas. Thus, while iso1Au/TNF can reduce doxorubicin penetration barriers at early time points, at later times this nanodrug may also further entrap doxorubicin in tumor tissues by impairing tumor perfusion. The combination of these time-dependent effects on the tumor vasculature may have contributed to the synergism observed with chemotherapy, resulting in a prolonged survival of mice.The enhanced cytokine activity, together with peptide- and gold-mediated active and passive targeting mechanisms (which may occur in vivo), may contribute to reducing the dose of cytokines necessary for inducing pharmacological effects. According to this view, we found that extremely low, nontoxic doses of articles , we esti07 cells , this nu07 cells , 35 and ls [iso1 , 37, areiso1Au/TNF, as suggested by the increased doxorubicin uptake, 2 h after treatment, and by the reduction of tumor perfusion at later time points.In previous studies, we have shown that low doses of NGR-TNF, a peptide-TNF fusion protein that targets the tumor vasculature, are sufficient to cause the disassembly VE-cadherin dependent-adherence junctions and gap formation in the tumor endothelium, thereby increasing vascular permeability . This coOther investigators have shown previously that 27-nm nanogold functionalized with TNF  can suppress tumor perfusion and increase the antitumor effects of chemotherapy in murine models of mammary carcinoma and in genetically engineered mice with pancreatic ductal adenocarcinoma, when administered at doses equivalent to 3–5 µg of bioactive TNF , 43. NotIso1Au/TNF and iso1Au/TNF + IL12 showed similar anti-tumor effects in the murine fibrosarcoma model, when used alone or in combination with chemotherapy, suggesting that the addition of IL12 does not increase the anti-tumor effects of iso1Au/TNF in this model. The rationale for combining TNF and IL12 in a single nanodrug (iso1Au/TNF + IL12) relies on the notion that IL12 can induce the release of IFNγ from lymphoid cells, such as NK cells and T cells, a cytokine crucial for the activity of low-dose targeted TNF [iso1Au/IL12 can significantly increase tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils [iso1Au/IL12 can also induce the release of sTNF-R2 in the tumor microenvironment, an inhibitor of TNF [iso1Au/TNF + IL12.eted TNF . Furthertrophils . Howeverr of TNF . We speciso1Au/TNF did not impair its synergism with chemotherapy in the WEHI fibrosarcoma model and considering our previous studies showing that iso1Au/IL12 and TNF can individually support the therapeutic efficacy of adoptive T-cell therapy in other tumor models [iso1Au/TNF + IL12 might exert synergistic effects with other chemo-immunotherapeutic combinations in different tumors, which is an approach that merits future investigation.In any case, given that the addition of IL12 to r models , 44, theisoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multicytokine delivery to cells of the tumor vasculature and that extremely low doses of isoDGR-coated nanoparticles functionalized with TNF or with TNF + IL12 can induce synergistic effects with doxorubicin. Furthermore, the present study establishes the conceptual feasibility of multifunctional cytokine nanoformulations using iso1-coated gold nanoparticles, a technology potentially applicable to other cytokines.We have shown that Additional file 1: Scheme S1. Schematic representation of the protocol used for preparation of iso1Au/TNF (A) iso1Au/IL12 (B) and iso1Au/TNF + IL12 (C). Figure S1. UV–Vis absorption spectra of Au/IL12, Au/TNF and Au/TNF + IL12. Figure S2. Characterization of iso1Au/TNF, iso1Au/IL12 and iso1Au/TNF + IL12 by transmission electron microscopy (TEM).