According with the Maker fringes method, oriented polymer films showing an order parameter of ∼0.23 can display outstanding and stable Second Harmonic Generation (SHG) effects under off-resonant conditions (SHG-532 nm). Also, the macroscopic non-linear optical (NLO)-coefficients were evaluated under the rod-like molecular approximation, obtaining: zzzχ(2) and zxxχ(2) in the order of 280 ± 10 and 100 ± 10 pm V−1, respectively. The mechanical and chemical properties, in addition to the large ONL-2 coefficients exhibited by this polymer, make it a promising organic material in the development of optoelectronic/photonic devices.Excellent quadratic non-linear optical (ONL-2) properties of the poly(2,5- Polydiacetylenes (PDAs) are a unique class of linear conjugated polymers derived from the topochemical polymerization of diacetylene (DA) monomers. One important aspect of their behavior is that they can aggregate into large-dimensional crystals. ,2,3. WegThese compounds are synthesized via a 1,4–addition reaction of monomer crystals of the form R–C≡C–C≡C–R, where the polymer backbone consists of alternating their alkene–alkyne structures. Owing to their intriguing stress-induced chromic transition (blue to red) and non-linear optical properties (NLO), PDAs have been extensively investigated as potential chemosensors and photonic materials. Recently, the embedment of PDA and its role in guest-host supramolecular chemistry has been used advantageously for the assembly of well-defined structures with unique optical, electronic, and mechanical properties . NLO in Considering quadratic NLO-applications such as SHG within the dipolar approximation, rod-like organic compounds display NLO-activity if they include conjugated molecules within their molecular architectures. Such conjugated moieties must contain substituents that bestow an important degree of push-pull effect unto them via electron donating (D) and electron accepting (A) functional groups. Besides this, in the case of polymers, these conjugated portions should be contained peripherally. This molecular design is commonly implemented for obtaining functionalized polymers suitable for second-order SHG/NLO-applications. Here, due to the push-pull effect induced upon photoexcitation , a modification in the charge distribution is forced; thus, this results in an intramolecular charge transfer and the subsequent NLO-response ,11. d33 SHG macroscopic coefficients. The main obstacle in this area has been the chromophore aggregation in the solid state (film samples), since the NLO chromophores are often highly polarized via the DA push-pull interactions. This effect causes the phenomenon that during the film formation, chromophores with large dipole moments tend to compactly pack, leading to the diminishment or cancellation of the dipoles and their assembling in centrosymmetric arrangements, which decreases their NLO properties. Besides, steric factors, polarization of the polymerizable groups by substituents and the ability of the substituents to participate in resonance stabilization, influence the reactivity. Additionally, a high glass transition temperature leads to a lower degree of conversion /[S-In/P-Out] signal ratio of ~3.3. This fact evidences the prominence of the mophores ,29,33, aFinally, in order to estimate the tensorial coefficients of the NLO-chromophore, the SHG-signal calibration was performed according to the relative Maker fringes method. Here, an α-quartz crystal was used as NLO-reference material. In this way, by modeling the film sample as a four-layer structure (Air-Film-Substrate-Air), and considering the optical absorbance negligible at both fundamental and SHG-frequencies, non-resonant experimental conditions are assumed c. Iω2(θ) as function of the incident angle iθ may obey the expression (1) [Thus, classical, theoretical models can be implemented assuming the Kleinman symmetry relations ,39,40. Hsion (1) ,37,38.α-quartz plate, the signal ratio was fixed for both the reference crystal and film samples. Thus, the evaluation of the tensorial NLO-coefficients was performed by fitting the respective theoretical expressions to the experimental data  = zzzχ(2) ≈ 280 ± 10 pm V−1 and 31χ(2) = zxxχ(2) ≈ 100 ± 10 pm V−1. The ijχ(2) values are relative high considering the film thickness, and larger than those reported for typical poly(acetylene), poly(diacetylene), poly(styrene), polybenzoates and poly(methylmethacrylate)-based polymers containing azo-compounds and tolan-units, as NLO-chromophores [10, containing polar diacetylene-units as pendant NLO-active chromophores, have been reported. Finally, as shown in the gT value; for instance, the materials herein described were able to exhibit SHG-conversion efficiencies of ~80% at temperatures close to 160 °C, demonstrating the rigidity, strength and thermal stability of the polymeric main chain.After simultaneous fitting, we obtained: mophores ,33,41,4210 and other polymers reported ClxTMEDA in polymer 10 is affecting the NLO values. Currently, we are doing experiments to know what is the role of copper in the polymer 10 in NLO values.Analyzing NLO-coefficients, the second order of polymer reported  are showsiveness . In the 1H NMR and 13C NMR-spectra were recorded on a Bruker Advance 400-MHz spectrometer using TMS as an internal standard. UV-Vis spectroscopy was performed in a Varian Cary 400 Cone UV-Vis spectrometer. The molecular weight of the obtained polymer was determined by gel permeation chromatography (GPC) relative to polymetylmetacrylate standards in DMF at 25 °C on a Waters 2695 ALLIANCE separation module. TGA-analysis was carried out with an SDT-Q600 apparatus . To determine the melting point of the processed materials, a Fisher-Johns apparatus was used. These latter measurements were performed with a heating rate of 25 °C min−1.All products were characterized by spectroscopic techniques. MS was performed implementing an Orbitrap Fusion TM Tribrid spectrometer and Jeol JMS-5 X 102 A instrument. IR-spectra were acquired in a FT-IR/ATR Nicolet 6700 spectrometer using a SeZn glass in contact with the sample surface. 6, which was purchased directly from Aldrich). In addition, we describe how we performed the deprotection of 4, which was carried out with a solution of AgNO3.The detailed description of the methods of the preparation of analog compounds has been previously described , and we 1), as white crystals. 1H NMR  δ (ppm): 1.13 , 1.77 , 3.39 , 3.41 , 3.75 , 6.51 , 7.43 . 13C NMR  δ (ppm): 11.9 (CH3), 45.8 (CH2-N), 52.6 (CH2), 60.3 (CH2-OH), 77.4 (OH), 114.9 (CH-Ar-N), 137.9 (CH-Ar-I), 148 (C-N). FT-IR, maxυ (cm−1): 3103, 1587, 804 , 3323, 1047 , 2968, 1587 (CH3), 2933, 1496 (CH2), 1356 (Ar-NO2), 503 (C-I). mp = 47–48 °C.The solid was purified by crystallization (hexane) to provide the desired product in 73% yield (from 2) 1H NMR  δ (ppm): 0.20 , 1.15 , 1.85 , 3.42 , 3.47 , 3.78 , 6.62 , 7.32 . 13C NMR  δ (ppm): 0.19 (Si-CH3), 12.1 (CH3), 45.7 (N-CH2), 52.5 (CH2), 60.4 (CH2-OH), 91.5 (C≡C-Si), 106.6 (Ar-C≡C), 110.2 (C-Ar), 119.9 (CH-Ar-N), 133.5 (CH-Ar-C≡C), 148.3 (C-N). FT-IR, υmax (cm−1): 3043, 1606, 818 , 3381, 1047 , 2962, 1516 (CH3), 2895, 1456 (CH2), 2148, 1398 (C≡C), 1249, 843 (C-Si), 1356 (C-NO2).The compound was purified by a wash of hot hexane to obtain a light yellow liquid with a yield: 90%  was dissolved in a homogeneous reaction mixture using dichloromethane (24 mL), methanol (14 mL) and water (4 mL) in the following relation 7:4:1, respectively. Thereafter, silver nitrate  was added to the mixture reaction. In this mixture, the deprotection reaction was achieved at 40 °C for 24 h. Finally, an aqueous saturated solution of ammonium chloride (NH4Cl) was added. A brown mixture was obtained and extracted with dichloromethane (150 mL × 6). The obtained organic phase was dried with MgSO4, concentrated and vacuum dried. The product was purified by crystallization (95:5 hexanes/CH2Cl2) to provide the desired product in 73% yield (from 4) as light yellow needle crystals. 1H NMR  δ (ppm): 1.17 , 1.69 , 2.98 , 3.43 , 3.48 , 3.80 , 6.66 , 7.35 . 13C NMR  δ (ppm): 11.9 (CH3), 45.5 (N-CH2), 52.3 (CH2), 60.2 (CH2-OH), 74.8 (Ar-C≡C), 84.7 (C≡C-H), 108.9 (Ar-C), 111.8 (CH-Ar-N), 133.5 (CH-Ar-C≡H), 148.3 (C-N). FT-IR, maxυ (cm−1): 3049, 1595, 815 , 3378, 1046 , 2967, 1516 (CH3), 2927, 1399 (CH2), 3289, 2095 (C≡H), 1355 (Ar-NO2). mp = 45–47 °C.Compound 5) as a dark orange solid. 1H NMR  δ (ppm): 1.11 , 1.60 , 3.48 , 3.51 , 3.81 , 6.61 , 7.35 , 7.63 , 8.19 . 13C NMR  δ (ppm): 12.2 (CH3), 45.5 (N-CH2), 52.7 (CH2), 60.8 (CH2-OH), 72.1 (C≡C-C≡C), 79.2 (C≡C-C≡C), 111.4 (N-Ar-C-C≡C), 112.3 (CH-Ar-N), 124.1 (CH-Ar-NO2), 129.9 (NO2-Ar-C-C≡C), 133.3 (NO2-Ar-CH-C≡C), 134.7 (N-Ar-CH-C≡C), 147.8 (C-NO2), 149.9 (C-N). FT-IR, maxυ (cm−1): 3100, 1597, 848, 814 , 3412, 1071 , 2952, 1458 (CH3), 2922, 1400 (CH2), 2201, 2135 , 1510, 1349 (Ar-NO2). UV-Vis, maxλ = 424 nm. mp = 168–170 °C.The product was purified by crystallization (1:1 hexanes/AcOEt) to provide the desired product in 78% yield  as an orange powder. 1H NMR  δ (ppm): 1.27 , 2.50 , 2.53 , 3.60 , 3.82 , 4.52 , 4.63 , 4.73 , 6.83–6.87 , 7.08-7.15 , 7.39 , 7.89–7.94 , 8.30–8.34 . 13C NMR  δ (ppm): 13.2 (CH3), 42.8 (N-CH2), 45.6 (CH2), 57.4 (O-CH2), 59.1 (O-CH2), 66.6(CH2-COO), 73.9 (C≡C-C≡C), 76.5 (CH), 77.1 (C≡C-C≡C), 79.9 (C≡CH), 113.0 (N-Ar-C-C≡C), 115.6 (CH-Ar-N), 116.2 (C-Ar-COO), 116.9 (CH-Ar-ortho-COO), 121.1 (CH-Ar-meta-COO), 123.2 (CH-Ar-para-COO), 124.7 (CH-Ar-NO2), 131.2 (NO2-Ar-C-C≡C), 134.1 (N-Ar-CH-C≡C), 135.2 (NO2-Ar-CH-C≡C), 146.6 (C-NO2), 147.3 (C-N), 150.7 (C-O-ortho-COO), 152.0 (C-O-meta-COO), 163.4 (COO). FT-IR, maxυ (cm−1): 3100, 1591, 854, 813 , 2960, 1456 (CH3), 2926, 1493 (CH2), 3289, 2121(C≡H), 2199(C≡C), 1719, 1196 (Ar-COO), 1518, 1340 (Ar-NO2) 1282, 1074, 1032 (Ar-CO). UV-Vis maxλ = 423 nm. (FAB+) (m/z); 546.1785. mp = 175 °C.The product was purified by column chromatography on silica gel  to afford the desired product in a 91% yield  δ (ppm): 1.26 , 3.18–3.27 , 3.48–3.54 , 3.78–4.76 , 7.24–8.16 . 13C NMR  δ (ppm): 11.6, 38.1, 42.1, 42.4, 44.5, 47.8, 56.3, 57.5, 58.9, 62.3, 64.6, 66.4, 70.0, 71.6, 75.8, 79.4, 87.0, 104.9, 111.1, 111.5, 113.7, 114.3, 116.7, 116.8, 119.8, 120.0, 123.5, 123.8, 127.7, 130.1, 132.0, 133.1, 134.1, 147.0, 148.6, 150.7, 151.0, 151.1, 151.3, 164.3, 164.8, 166.2. FT-IR, maxυ (cm−1): 3100, 1599.73, 853.38, 813.36 , 2963.7, 1456.05 (CH3), 2930, 1491 (CH2), 2197 (C≡C), 1719, 1194 (Ar-COO), 1518, 1339 (Ar-NO2), 1280, 1075, 1024 (Ar-CO). UV-Vis maxλ = 430 nm. Mw = 12560; Mw/Mn = 1.548.The polymerization procedure was performed according to the oxidative coupling polymerization process (Hay Reaction) . The subgT-values (heating rate: 90 °C h−1). The poling process was maintained for 30 min (the sample temperature was fixed at gT + 10 °C). The poled films were then cooled to 60 °C (DC-field state: ON) and then to room conditions . Afterwards, the film samples were characterized by UV-Vis spectroscopic measurements in order to determine the corresponding order parameter.Organic thin films were deposited onto ITO-coated glass substrates via spin coating procedures, implementing the obtained polymeric product (using NMP-based solutions at 10 wt %). Film depositions were carried out at 120 °C, and the obtained films were vacuum dried at ~80 °C for ~1 h in order to remove residual NMP-solvent. The electrical molecular alignment was performed implementing an iron needle-point cathode placed at a needle-film gap distance of ~1.4 cm. The applied DC-voltage was in the range of 8–9 kV. Polymer film samples were mounted on a temperature-controlled heating plate to start the poling process from 60 °C to temperatures just above the TM tip with typical force constant of 0.05 N and resonant frequency of 22 KHz, which specify the mechanical characteristics of the cantilever .AFM-surface analyses of deposited films were performed on poled and unpoled film samples to determine their structural morphology using a Park-AutoProbe CP apparatus. The acquisition of images was carried out in non-contact mode with an applied interaction force between the sample and the AFM-tip of 1.5 nN. The AFM system was equipped with a SiN sharpened MIcroleverχ(2)-NLO/SHG effects. The experimental set-up comprises a commercial Q-switched Nd:YAG-Laser system  used to provide the fundamental excitation. The pulse power was set to ~80 J, and the laser beam was focused by means of an f = 5 mm focusing lens, so that peak irradiances on the order of ~8 MW/cm2 were achieved at the focal spot on the sample. This value was below the damage threshold supported by the samples. The polarization of the fundamental beam  was selected by means of an IR-coated Glan-Laser polarizer and a λ/2-retarder plate. A second polarizer used as an analyzer allowed the characterization of the SHG-signals. The SHG-waves (2ωλ = 532 nm) were detected using a photomultiplier tube (PMT) placed after convenient interferential optical filters (532 ± 5 nm). The SHG experimental set-up was calibrated by means of an α-quartz crystal wedged along the d11(2)-direction (d11(2) ≈ 0.64 pm V−1 = 0.5 χ11(2)), which is frequently used as the NLO-reference standard via the Maker fringes method [Electrically poled organic films were tested as active media for quadratic s method ,34,35. NParal, and gPerp are the parallel and perpendicular components of the g-tensors of one Cu(II) center, respectively. The term HF,Cu is the hyperfine contribution from the copper nucleus, I(Cu) = 3/2, and HF,N is the hyperfine contribution from the nitrogen nucleus, I(N) = 1.EPR spectra at room temperature (295 K) were measured under non-saturating conditions of microwave power on a Bruker Elexys E500 spectrometer, at approximately 9.86 GHz (X-band) and 100 KHz modulation. The EPR spectra were evaluated and simulated using the easyspin ver. 5.1, and g-values were calculated by measuring accurately the magnetic field and the microwave frequency. For the interpretation of the EPR spectra, we use the usual spin Hamiltonian for Cu(II), S = 1/2, specie. 10 was achieved alongside the determination of quadratic NLO/SHG-measurements. The thermal properties, amorphous nature and good solubility of this polymer allowed the fabrication of high quality spin-coated optical films with a stable mechanical structure. The samples analyzed by this methodology exhibited low absorption within the visible spectral range, and the corresponding poled samples displayed outstanding SHG-signals. In fact, an efficient and stable chromophore alignment, together with an inherent rigidity of the polymer main chain, can support the final molecular configuration, preserving the polar diacetylene chromophore orientation. In agreement to the Maker fringes technique, this polymeric material exhibits an intense and stable SHG-response in off-resonant experimental conditions, evidencing low dipolar aggregate interactions, which enhances the NLO/SHG-response. The obtained NLO-coefficients are larger than those reported for common polymers containing azo-compounds and tolan-units as NLO-chromophores. The synthesis and characterization of polymer This behavior makes our push-pull polymer a promising material for NLO photonic/opto-electronic applications. The polymer we present in our work exhibits attractive mechanical and opto-electronic properties, making it a potential candidate for technological devices . Finally


Transcatheter aortic valve implantation (TAVI) is a safe and effective treatment for inoperable, intermediate- or high-risk patients with severe symptomatic aortic stenosis and has been associated with excellent clinical outcomes. A clinically relevant remaining problem is aortic regurgitation (AR) post-TAVI, which is associated with increased mortality. Therefore, we conducted a prospective randomised trial to assess the safety and efficacy of a first-generation self-expandable valve  and a third-generation balloon-expandable valve  with respect to clinical outcomes and AR as determined quantitatively by magnetic resonance imaging (MRI).The ELECT study was an investigator-initiated, single-centre trial involving patients with severe symptomatic aortic stenosis and with a clinical indication for transfemoral TAVI. Fifty-six patients were randomly assigned to the BEV or SEV group.p = 0.01]. Secondary endpoints according to the criteria of the Second Valve Academic Research Consortium (VARC-2) showed BEV to have better early safety  at 30 days and a lower risk of stroke , major adverse cardiac and cerebrovascular events  or death  in the 1st year compared with SEV.AR determined quantitatively by MRI was lower in the BEV than in the SEV group [regurgitant fraction: 1.1% (0–8.0) vs 8.7% (3.0–14.8) for SEV; The use of the latest generation of BEV was associated with less AR as quantitatively assessed by MRI. Although the use of MRI to quantify AR is not feasible in daily clinical practice, it should be considered as a surrogate endpoint for clinical outcomes in comparative studies of valves for TAVI.ClinicalTrials.gov number NCT01982032. We report on a randomised trial comparing the use of the latest-generation balloon-expandable valve and a first-generation self-expandable valve in patients undergoing transcatheter aortic valve implantation.The severity of aortic regurgitation (AR) was quantitatively assessed by magnetic resonance imaging (MRI).The latest-generation balloon-expandable valve was associated with less AR compared with the first-generation self-expandable valve.Assessing AR quantitatively by MRI should be considered as a surrogate endpoint for clinical outcomes in comparative studies of valves for TAVI.Transcatheter aortic valve implantation (TAVI) is a safe and effective treatment for inoperable, intermediate- or high-risk patients with severe symptomatic aortic stenosis and has been associated with excellent clinical outcomes , 2. A reOver the past decade, several improvements to address AR have been introduced in different valves with different expansion systems. Two often-used systems are the self-expandable valve (SEV) by Medtronic  and the balloon-expandable valve (BEV) by Edwards . The latter has been specifically designed with the aim of reducing paravalvular AR by an additional outer skirt. Numerous studies have been published on the safety, efficacy and post-procedural AR of the SEV and the latest-generation BEV –13. To oTherefore, we conducted a prospective randomised trial to assess the safety and efficacy of the latest-generation BEV and first-generation SEV with respect to clinical outcomes, and AR as determined by MRI.The study design and patient selection of the Edwards Sapien 3 periprosthetic leakage evaluation versus Medtronic CoreValve in transfemoral aortic valve implantation (ELECT) trial have been described previously . BrieflyPrimary endpoint was severity of post-procedural AR, quantitatively assessed by MRI, performed 4–5 days after the index procedure. The severity of AR with respect to regurgitant fraction was graded according to previously published definitions: none/trace <8%, mild 9–20%, moderate 21–39% and severe >40% . As thesSecondary clinical endpoints at 30 days and 1 year have been described previously and were defined as per the Second Valve Academic Research Consortium (VARC-2) definitions , 17. Allt-test or Mann-Whitney U test, depending on data distribution. Categorical variables were presented as counts and percentages and compared using Fisher’s exact test. Time-to-event curves were constructed using Kaplan-Meier curves and compared with the log-rank test. The paired t-test, repeated-measures analysis of variance and Wilcoxon signed rank test were used to assess the change in effective regurgitant orifice area and mean aortic gradient over time between and within groups. The correlations between continuous and ordinal variables were assessed using Spearman’s correlation coefficient. Sample size calculation has been described previously , as well as MACCE at 30 days and length of hospital stay  vs 5 (19%); p = 0.02; Fig. The composite endpoint 30-day early safety according to the VARC‑2 criteria was significantly better for the BEV group [0 (0%) vs 8 (30%); At 1 year, clinical New York Heart Association (NYHA) symptoms had improved more in BEV patients compared with the SEV group Tab. . Aortic p = 0.09).No significant correlation between NYHA class improvement and AR was found , the lower crossing profile of the Sapien 3 system and a better distal flexion of the delivery catheter, resulting in fewer aortic arch injuries and life-threatening bleedings in patients receiving this latest-generation BEV. The overall low complication and mortality rates of the BEV are also reflected in the composite endpoints according to the VARC‑2 criteria, with a high device success rate, better early safety and a lower rate of MACCE. The observed difference in clinical outcomes between the SEV and BEV groups in our study might represent the difference in valve generation. Studies of the newer generations of the SEV have found lower complication rates, which are in line with the latest-generation BEV with a 1-year all-cause mortality rate and stroke rate around 8.5% and 3.5% respectively, 30.The results of the clinical outcomes in the present study should be interpreted in the context of its small sample size and the lack of power to detect differences in endpoints—due to low recruitment—between the valves, resulting in wider confidence intervals and possible underestimation of differences. Nevertheless, the reported differences that were statistically significant can be considered to be a real difference and not a coincidence, since the statistical type I error  is not related to the power of a test. However, as stated by Button et al. , it is pThe results of this study show a high-quality comparison between two valve types regarding AR severity as quantitatively assessed by MRI. Although the use of MRI for the quantification of AR is not feasible in daily clinical practice due to its high costs, low availability and certain contra-indications (e.g. in patients with claustrophobia or < 6 weeks after implantation of pacemaker leads), it is of additive value as regards improving the accuracy and reproducibility of AR as well as reducing variability with respect to AR severity. In addition, studies with quantitative AR assessment are needed in order to improve the accuracy of AR estimation and to better understand the relationship between AR severity and clinical outcomes as well as differences between next-generation valves. Therefore, measuring AR by MRI should be considered in future studies comparing valves. Finally, this small study could be a preamble to a new randomised comparison between current-generation TAVI valves, but probably requires a larger sample size because of smaller differences in technical and clinical outcomes.The use of latest-generation BEV was associated with less AR as quantitatively assessed by MRI. Although the use of MRI to quantify AR is not feasible in daily clinical practice, it should be considered as a surrogate endpoint for clinical outcomes in comparative studies of valves for TAVI.


Allergic rhinitis (AR) is an inflammatory disease of nasal mucosa caused by IgE mediated inflammatory mediators and various immune active cells and cytokines after exposure of specific individuals to allergens. In recent years, its prevalence rate has increased gradually. Therefore, we must pay attention to carry out early intervention. However, there are still some side effects in the current drug therapy of AR, and the recurrence of AR cannot be well controlled. Some Chinese herbs have anti-allergic, anti-inflammatory and immunomodulatory effects, and have a better effect on the nasal symptoms of perennial and persistent rhinitis. The curative effect of allergic decoction on AR has been confirmed clinically. However, due to the lack of reliable evaluation means for its safety and effectiveness, it is necessary to carry out a systematic evaluation of allergic decoction in the treatment of AR, so as to lay a foundation for further research in the future.The following databases will be searched from their inception to August 2020: Electronic database includes PubMed, Embase, Cochrane Library, Web of Science, Nature, Science online, Chinese Biomedical Database WanFang, VIP medicine information, and China National Knowledge Infrastructure. Primary outcomes: nasal symptoms  and ocular symptoms . It can be measured by any appropriate scales or other forms of tools, such as the Total Nasal Symptom Score. Data will be extracted by 2 researchers independently, risk of bias of the meta-analysis will be evaluated based on the Cochrane Handbook for Systematic Reviews of Interventions. All data analysis will be conducted by data statistics software Review Manager V.5.3. and Stata V.12.0.The results of this study will systematically evaluate the efficacy and safety of Guominjian for patients with AR.Through the systematic review of this study, the evidence of the treatment of AR by Guominjian has been summarized so far, so as to provide guidance for further promoting the application of Guominjian in patients with AR.This study is a systematic review, the outcomes are based on the published evidence, so examination and agreement by the ethics committee are not required in this study. We intend to publish the study results in a journal or conference presentations.https://osf.io/24w8n).September 12, 2020.osf.io/24w8n.( At present, the treatment of AR mainly includes environmental control, drug therapy, immunotherapy and surgical treatment. For AR medications including antihistamines , glucocorticoid , oral drug leukotriene, decongestants , nasal spray resistance to choline medicine and mast cell stabilizer and so on, these drugs by acting on allergic some molecular targets of mucous membrane inflammation, blocking immune inflammatory disease caused by allergens, so it can effectively control the symptoms of AR, in the process of the clinical use of less side effects, high security. One or more may be used alone or in combination depending on the characteristics or clinical severity of the lesion. Immunotherapy, on the other hand, is a treatment method that can effectively relieve the nasal symptoms and improve the quality of life of AR patients by giving high-dose specific allergen continuous stimulation to the body and finally achieve tolerance to allergen stimulation. It can also prevent the formation of new allergens and reduce the risk of AR progressing to asthma. Some Chinese herbs have anti-allergic, anti-inflammatory and immunomodulatory effects and are safe and effective in improving the nasal symptoms of perennial and persistent rhinitis. Guominjian is a classic prescription for treating AR in the field of traditional Chinese medicine. At the same time, it can also be used for other allergic diseases. This prescription has a remarkable anti-wind table, the effect of dispel evils. Modern studies have shown that the prescription also antagonizes the itching caused by dde, the increased vascular permeability caused by histamine, lowers the IgE level in the blood, down-regulates the expression of PAR-2mRNA, stabilizes mast cell membranes, and inhibits mast cell degranulation. Although its effectiveness and safety have been proven in clinical practice, but lack of exact theoretical basis. Therefore, this study intends to evaluate the efficacy and safety of Guominjian in the treatment of AR through systematic evaluation and meta-analysis, and to explore its possible theoretical basis.Allergic Rhinitis (AR) is a common type of Rhinitis. Epidemiological studies show that in Asia, AR affects a large number of people, ranging from 27% in South Korea to 32% in the United Arab Emirates.22.1https://osf.io/24w8n). The protocol will follow the statement guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMAP), Changes will be reported in the full review as required.The protocol has been registered in OSF(Open Science Framwork) Preregistration. September 12, 2020.osf.io/24w8n. and ocular symptoms . It can be measured by any appropriate scales or other forms of tools, such as the Total Nasal Symptom Score.The secondary outcomes are quality of life , global non-nasal symptoms , use of conventional medication , laboratory indicators, and any expected or unexpected adverse events.33.1The following data bases will be searched to identify eligible studies: PubMed, Embase, Cochrane Library, Web of Science, Nature, Science on line, Chinese Biomedical Database WanFang, VIP medicine information, and China National Knowledge Infrastructure. The time range is: the starting time is determined according to the first literature available, and the deadline is August 2020.3.2In order to get more complete evidence, we will also retrieve other related documents by manually, such as medical textbooks, clinical laboratory manuals and so on. If it is necessary we will contact with trail author to obtain the latest clinical data. Moreover, studies associated with the review will be identified via evaluating related conference proceedings. The research flowchart is shown in Figure 3.3The following search terms will be used: randomized controlled trial/ randomized controlled trial; AR/Affection of AR; traditional chinese medicine/TCM; Guominjian/Guominjian therapy/Guominjian treatment. different retrieval strategies in Chinese and foreign databases will be used. Language restrictions are Chinese and English. There is no publication restriction. Here we take the search strategy in PubMed as an example and list in Table 44.1All articles in the search results were independently evaluated by 2 independent researchers  according to inclusion and exclusion criteria. Reviewers will then independently extract and collect the data included in the study using pre-designed data collection forms. Discrepancies will be discussedand resolved by consensus with a third author (SZ).4.2The following informations will be extracted from each study:(1)Normal test characteristics: title, author, year.(2)baseline data: sample size, age, gender, diagnosticcriteria, course of disease.(3)interventions: acupuncture therapy, control of intervention details, intervention. If the information is not enough, we will contact experts and authors in this field to get relevant information.4.3 The Cochrane System Evaluator's Manual give the evaluation criteria for authors to evaluated the randomized controlled trials quality. Assessing the risk of bias:The risk of bias will be assessed by 2 independent authors (YX and HL), together with completing the STRICTA checklist.(1)random sequence generation;(2)allocation concealment;(3)blinding of participants and personnel;(4)blinding of outcome assessment;(5)incomplete outcome data;(6)selective outcome reporting;(7)other bias.Any disagreement will be discussed or consulted with a third reviewer. Each them will be described from 3 levels: “high risk,” “low risk” and “unclear.”4.4The dichotomous outcomes will be expressed by the Odds ratio(ORs), while the continuous data will use the Standardized mean difference(SMD). All these outcomes report 95% confidence intervals.4.5We will take the method of contacting corresponding authors to obtain the missing data. If there is no response, we will analyze only the available data and describe the reason and impact of this exclusion in the paper.4.6The bias of publication will be explored through funnel plot analysis. If the funnel plot show asymmetry, it will be evaluated via the Egger and Beg tests, and P value <.05 means the publication bias is significant.4.7Q value statistic test, I2 statistic test, H statistic test). The I2 statistic test method shows us When I2 is 0, it means that studies are completely homogeneous, If I2>50%, it indicates there is heterogeneity in studies.There are 2 main methods for testing heterogeneity, namely graphical method  and statistical test . To test the heterogeneity of the research results, when the 4.9We will undertake subgroup analysis based on the different types of interventions and controls, characteristics of study or patient, and different outcome measurements.4.10Sensitivity analysis can not only assess the stability and reliability of the conclusions of the Meta analysis, but also assess whether the changes in the results are related to the impact of a single study. If the stability of the conclusion is poor, we can achieve When the heterogeneity test results are heterogeneous, we need to clarify the source of the heterogeneity by subgroup analysis. The effects of different types of therapy including design scheme, severity of illness, age, sex, andmild or severe AR were analyzed. We will also delete low-quality and/or medium-quality studies to check the robustness of theresults the purpose of increasing stability by changing the analysis model, inclusion and exclusion criteria, or excluding a certain type of literature.4.11We will publish the system review results in peer-reviewed journals, disseminated in meetings or in peer-reviewed publications. Aggregated published data will be used to exclud data of individuals, so there is no need for obtaining the ethical approval or patients’ informed consent.5 In addition, a number of studies have also shown that allergy decoction can significantly improve inflammatory factors in antagonists,–8 effectively improve patients’ allergic symptoms, and thus improve their quality of life.AR is a kind of caused by specific individual after exposure to an allergen mediated by IgE release of inflammatory mediators and the activity of a variety of immune cells and cytokines involved in the occurrence of inflammation in nasal mucosa disease, the main clinical symptoms include nasal itching, sneezing, flow stuff, nasal congestion, in addition to the nasal symptoms, some patients can merge bronchial asthma, resulting in asthma, cough, shortness of breath, chest tightness and other symptoms, lung clinically AR often interact with asthma and its influence, endangering human health. Some patients may have itchy eyes, tears, red eyes and burning eyes and other eye symptoms. There is a certain theoretical basis for the clinical efficacy of AR decoction. Study has shown that both low dose and high dose of allergic decoction can significantly alleviate the allergic reactions in model mice.In recent years, clinical experience has proved that Guominjian is an effective and safe way to treat AR. Relevant studies have proposed its possible mechanism of action, but the mechanism is still unclear, and most of the literature has not clearly pointed out the mechanism of Guominjian's treatment of AR. Therefore, it is very necessary to carry out standardized and unified experimental design in clinical studies to evaluate the efficacy, which can not only guide the application of Guominjian in AR, but also explore the mechanism of Guominjian's action in the treatment of AR.In summary, systematic review and meta-analysis are helpful to determine the application of Guominjian in the treatment of AR, and reduce the waste of medical resources, patients’ time and money. This study can not only provide a basis for the introduction of treatment guidelines for AR, but also promote the application of Guominjian treatment to benefit more patients.We are grateful to Dr Zhang for technical support and advice.Conceptualization: Yi-min Xiong; Haoran Li; Shu-nan Zhang.Data curation: Yi-min Xiong; Haoran Li.Formal analysis: Yi-min Xiong; Haoran Li.Methodology: Yi-min Xiong; Haoran Li; Shu-nan Zhang.Project administration: Shu-nan Zhang.Resources: Yi-min Xiong; Haoran Li.Software: Yi-min Xiong; Haoran Li.Supervision: Shu-nan Zhang.Writing – original draft: Yi-min Xiong.Writing – review & editing: Shu-nan Zhang.


To the Editor,The COVID-19 pandemic has generated enhanced focus on the safety of healthcare providers and efforts to mitigate the risks of viral transmission.1The King LT(S)-D laryngeal tube , abbreviated hereafter as the King LT, is a new-generation extraglottic device  used as In 2016, Dodd and colleagues introduced a novel, nonsurgical approach to facilitate definitive airway management in ED patients with a King LT in place.We believe that the King LT exchange method described by Dodd and colleagues represents a safe and simple approach that can be readily performed by clinicians who are skilled at video laryngoscopy. Further, we believe that this method, used in combination with administration of a high-dose, paralytic medication to mitigate spontaneous patient respiration and cough during the procedure, represents the safest method for both patients and care providers to exchange a King LT for a cuffed tracheal tube in a patient with known or suspected COVID-19. Lastly, the airway equipment required is readily available to most emergency providers, critical care providers, and anesthesiologists. We describe this procedure, with updates accounting for risks inherent to the COVID-19 pandemic, in


The development of targeted therapies increases treatment options for metastatic castration resistant prostate cancer (mCRPC) patients. There is a need for strong predictive and prognostic signatures to guide physicians in treating mCRPC patients. In this review we unravel the possible actionability in the AR pathway, PI3K AKT signaling, and DNA repair pathways. Additionally, we make recommendations on biomarker trial design, and the clinical use of this new type of data. Prostate cancer (PCa) is not a uniform disease as its outcome is highly associated with the corresponding clinical risk category. In general, PCa is strongly driven by androgen receptor (AR) regulated transcription . HoweverMetastatic castration resistant prostate cancer (mCRPC) results from many genomic and transcriptomic changes. The heterogeneous appearance of these changes underly the multifocal nature of PCa. It is thought that AR insensitive clones will survive the therapeutic selection pressure by AR targeting treatments. As mCRPC patients have a bad prognosis, these patients need to be rapidly treated with drugs likely to be effective. Identifying patients that would benefit from specific treatments will need to be based on specific markers, which could be detected as genomic and/or transcriptomic signatures.In this review we will look at these genomic and transcriptomic alterations, with particular attention on those with prognostic and predictive potential. CRPC is diagnosed in PCa patients with castrate serum testosterone < 50 ng/dL and biochemical or radiological progression  Figure . mCRPC iBesides the standard of care, novel targeted therapies are entering clinic with PARP (poly-ADP ribose polymerase)-inhibitors being beneficial in PCa escaping from ARSI treatment on the condition that homologous recombination repair (HRR) alterations are present . Other pIt has been shown that the overall survival gain of consecutive treatments is not equal to the survival gain of each drug separately . CombinaMost recent guidelines advise to base the choice of first-line treatment on different patient characteristics including performance status, symptoms, comorbidities, location and extent of disease, patient preference, and previous treatments . The optIn mCRPC, genetic sequencing on different time point can guide the management throughout the course of the disease ,8. AlthoTCGA, a molecular classification initiative, aimed to classify different tumors, including prostate cancer, by their genomic alterations. In mCRPC, large sequencing initiatives identified the genomic alterations and confirmed possible actionable targets to be present in most patients ,11,12. AStudies confirmed a possible benefit of using genomic information to help treatment allocation ,13,14. HThe AR signaling pathway is essential for prostate development and throughout different stages of PCa . As ADT The AR dependent resistance mechanisms cover AR overexpression, AR gene amplifications, mutations, splice variants, and alterations of coregulators, with the majority being amplifications. Amplifications result in an increased sensitivity to androgens due to an overexpression of AR . In anotAR mutations are most often found in the Ligand Binding Domain (LBD) . These mAlthough still strongly debated, expression of splice variants, e.g., AR-V7, Arv567es, may cause the PCa cells to become insensitive to ADT or ARSI . AR-V7 hDifferent coregulators have been described to be involved in the AR axis, including: NCOA1, 2, 3, p300, transcription factor FOXA1, negative regulator NCOR1/2, and AR induced negative regulator ZBTB16 ,29. PrecSPOP is described as tumor suppressor gene, degrading oncogenic substrates like NCOA3/SRC3 (co-activator of AR), ERG, and DEK ,32. BesiETS) gene fusions are the most common genetic alteration in cancer and it is found in approximately 50% of localized PCa as well as in mCRPC [TMPRSS2 gene to the protein coding part of members of the oncogenic ETS gene family . This renders the ETS oncogene family AR dependent and highly expressed. TMPRSS2-ERG fusions are more resistant to treatment with taxane-chemotherapeutics and, as they are AR regulated, these patients are more susceptible to ADT and ARSI [Erythroblast Transformation Specific  upregulation as escape mechanism after ARSI treatment. As GR and AR have very similar mechanisms of action, the GR can take over part of the AR pathway . MoreoveGR antagonists were found to be beneficial as adjuvant treatment to ARSI in preclinical models, as the expression of GR and AR seemed to be inversely correlated . AndrogeHSD3B1 gene also affect the expression levels. This results in higher concentrations of androstenedione, and therefore DHT [Castration and abiraterone both target the AR axis by depleting its ligands, but this inhibition is overcome by the activation of the tumoral steroid synthesis. Cancer cells of local and metastatic disease can synthesize DHT from adrenal precursors, resulting in a release of the inhibited AR . For exafore DHT ,53. Precfore DHT . FurtherIn conclusion, as PCa is an androgen driven tumor, different escape mechanisms alter the AR pathway. Future efforts should not only be directed to the characterization of AR alterations and common other AR escape mechanisms, but also focus on targeting the steroid metabolism and the GR pathway.PTEN is a major regulator of the cell cycle and tumor suppressor gene, its loss is associated with poor clinical outcome and progression to mCRPC [PTEN loss is associated with ETS rearrangements (see above), enforcing their mutagenic capacities [As to mCRPC ,57,58,59to mCRPC ,36,60. Ipacities ,62.PTEN deleted models showed lower AR activity after activation of the PI 3K pathway. As AR and PI 3K compensate for each other’s inhibition, a dual inhibition of both pathways, consisting of an AKT inhibitor and an ARSI, seems promising [PTEN loss [The exact mechanisms explaining how PTEN leads to castration resistance are still debated. The inhibition of the PI 3K pathway , via AKT inhibition by PTEN, is considered an important contributor . As AKT romising . A phaseTEN loss .Of all germline variants found in metastatic cancers, 75% were related to defects in DNA repair confirming the importance of aberrant DNA repair in carcinogenesis . AlthougMutations in HRR render patients sensitive to PARP inhibitors (PARPi), as PARP is involved in the repair of single strand DNA breaks. If unrepaired, these are transformed to double strand DNA breaks during replication. PARPi are therefore called synthetically lethal because these are only effective when repair mechanisms fail ,68. ThisMLH1, MLH3, MSH2, MSH3, MSH6, PMS1, and PMS2. Tumors with MMR mutations typically accumulate large numbers of mutations and microsatellite instability. Such tumors can be relatively easily identified with immunohistochemistry (see below) and have been shown to respond well to both ADT and ARSI treatment and possibly immune checkpoint inhibitors [Mismatch repair (MMR) genes will correct nucleotide allocation mistakes by an excision and reallocation of the base by verifying the genetic code on the adjacent strand. Different genes have been described to be involved in MMR, including: hibitors ,70.CDK12 defines a distinct subtype of PCa and is present in 5% of mCRPC [ERG, SPOP, TP53, and PTEN [CDK12 loss show aberrant DNA replication during S-phase and have a high immune infiltration [CDK12 loss to form an aggressive PCa subtype with poor responses to hormonal and taxane treatment as well as PARPi. Surprisingly, good response of this subtype was seen after treatment with immune checkpoint inhibitors, possibly linked to their higher mutation burden [Cyclin dependent kinase 12 (CDK12) is a member of the kinases, involved in the regulation of the cell cycle. Biallelic loss of of mCRPC . When prand PTEN . Tumors ltration . A retron burden . FurtherIn this review we focused on adenocarcinoma of the prostate. Although this is not the only histopathologic form of mCRPC, it covers more than 95% of all mCRPC cases . PreclinIn mCRPC patients it is unclear what percentage of tumors develop into a neuroendocrine state, and what percentage result from a neuroendocrine population being positively selected as a result of the treatments. The latter has enriched losses of p53 and Rb1 and should be treated early on ,77.An overview of the discussed pathways from this section is represented in A good biomarker has reproducible predictive or prognostic characteristics in a well-defined disease state, and provides information that can impact the treatment allocation and the further care. As a biomarker should be reproducible in a certain disease state, different patients, and trial settings, a reliable predictive risk profile will most likely consist of different biomarkers. Here, we focus on the possible contribution of genomic biomarkers in future PCa care.Although there are many reports suggesting new biomarkers for different specific PCa settings, limited clinical improvements have been made ,79. One PTEN loss, SPOP, and ATM mutant) and can therefore reflect corresponding underlying biomarkers [PTEN and ERG) [Histopathology is an elegant way to detect protein-biomarkers. Firstly, although hematoxylin-eosin staining suffices to find malignant cells, some markers (like AR) can help to reduce doubt . Second,omarkers . Lastly,and ERG) .Most candidate biomarkers result from small, hypothesis generating, studies. Subsequent biomarker trials should therefore be run to validate them for specific indications. Besides treatment allocation, signatures could identify reasons for treatment intensification, drug selection, and duration of therapy. In further disease stages the role in defining the treatment-sequence becomes equally important . Good exMultiple attempts have already been done to categorize PCa based on its molecular characteristics, with the TCGA being the most promising ,79,87. HBesides genomic alterations, which were the topic of this review, there are multiple clinical biomarkers being studied. The overarching aim is that some of these factors will contribute to the identification of specific patient profiles that will allow clinicians to propose treatments likely to be effective .Additionally, by increasing knowledge on cancer biology throughout the following years, novel treatment options that are not included in this review will be suggested and investigated. These can be targeted towards mCRPC or act as an adjuvant treatment to block certain escape mechanisms.To increase PCa survival, multidisciplinary oncological PCa boards should be able to use all available diagnostic data for their discussions and treatment decisions . These dDefining better subclassifications of mCRPC populations that respond well to given treatments is therefore a high priority in mCRPC care. Predictive molecular biomarkers for the treatment of mCRPC are intensively investigated, but until now, only limited clinical implementation has been realized.


Background. Chronic kidney disease (CKD) is a global health burden with high mortality and morbidity. Clinical efficacy has been demonstrated for Shen Shuai II Recipe (SSR), an approved and widely used Chinese herbal medicine for over 20 years in China, to attenuate CKD progression. In this study, we explored the underlying molecular mechanisms of SSR benefits and studied its effects on apoptosis, a critical process in CKD development and progression. CKD was induced in rats with 5/6 renal ablation and infarction (A/I). Eight weeks after SSR treatment, we mainly assessed the severity of renal injury and fibrosis, the translocation of apoptotic factors in the mitochondrial apoptosis pathway, the degree of mitochondrial dysfunction, and the nuclear and mitochondrial translocation of p53. Furthermore, we detected the interaction of p53 with antiapoptotic Bcl-xL and Bcl-2 proteins. Our results showed that SSR significantly attenuated renal injury and fibrosis and inhibited the mitochondrial accumulation of proapoptotic proteins Bax and Puma and release of cytochrome c from mitochondria to the cytosol in a rat CKD model. In addition, SSR also improved the mitochondrial function and inhibited the nuclear and mitochondrial translocation of p53. In addition, SSR suppressed the p53 transactivation and the interaction of p53 with Bcl-xL and Bcl-2. These results suggested that SSR could block apoptosis in CKD by inhibiting p53 transcriptional-dependent and transcriptional-independent proapoptotic function and the mitochondrial pathway of apoptosis. Chronic kidney disease (CKD) has considerably increased worldwide and remains a major global health problem . DiffereApoptosis is a cell self-destruction process involved in a variety of biological events . AbnormaIt has been demonstrated that Chinese herbal medicine may be suitable for the treatment of CKD for its efficacy and safety of long-term treatment. Indeed, Shen Shuai II Recipe (SSR), a classic formula for the treatment of CKD in the clinic, has been widely used for more than 20 years and is an effective treatment option for CKD. Our previous studies have demonstrated that SSR attenuated renal fibrosis, increased renal blood flow, and improved oxygen consumption after renal injury in CKD induced by 5/6 renal ablation/infarction (A/I) in rats –9. In thg. The supernatant was merged and concentrated to prepare suspension containing 6 g/mL of the original drug by means of ethanol. The gavage dose  in this study was determined on the basis of the previous studies [Eight-week-old male Sprague-Dawley (SD) rats, weighing approximately 190 g-210 g, were purchased from SLAC Laboratory Animal Co., Ltd. . Rats were maintained at 23 ± 3°C and relative humidity of 55 ± 15% with a 12 h light/12 h dark cycle and had free access to food and water. SSR consists of Salvia miltiorrhiza Bge (15 g), Codonopsis pilosula (15 g), Angelica sinensis (15 g), Rheum palmatum (15 g), Folium perillae (15 g), Epimedium (15 g), peach kernel (15 g), Ligusticum chuanxiong (15 g), and Coptis chinensis (6 g). The raw herbs for preparation of SSR were obtained from Shanghai Kangqiao Chinese Medicine Tablet Co., Ltd.  and identified by Dr. Guanglin Xu from Pharmacy Department, Shuguang Hospital Affiliated to Shanghai University of TCM. The preparation of SSR was performed as previously described , 8. Brie studies , 8. Losan = 10), and 5/6 (A/I)+Losartan group (5/6 (A/I)+LOR, treated with 6 mL/kg of Losartan daily by gavage, n = 10). This study also included a group of 10 sham-operated rats. Eight weeks after treatment, rats were euthanized with sodium pentobarbital . Kidney tissues were collected for histology and molecular analysis. The animal procedures were approved by the Animal Experiment Ethics Committee of Shanghai University of Traditional Chinese Medicine in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals.A rat CKD model with 5/6 (A/I) was created as previously described –10. FourμL buffer B and then centrifuged at 8000 g for 5 min. The supernatant was further centrifuged at 16,000 g for 30 min to collect the pellet as a mitochondrial fraction.Using a tissue mitochondria isolation kit , the cytosolic and mitochondrial fractions were extracted at 4°C with a centrifugal column filter and by a multiple centrifugation method. About 30 mg of tissue was placed in a filter cartridge. The tissue was ground with a plastic rod for one minute by pushing the tissue against the surface of the filter repeatedly. The filter cartridge was centrifuged at 16,000 g for 30 seconds. The pellet was resuspended by vortexing briefly. The homogenates were first centrifuged at 700 g for one minute to remove cell debris and nuclei and then centrifuged at 16,000 g for 10 min to collect the supernatant as a cytosolic fraction. The pellet was resuspended in 200 μL cytoplasmic protein extraction reagent and then centrifuged at 16,000 g for 5 min to collect the supernatant as a cytosolic fraction. The pellet was resuspended in 50 μL nuclear protein extraction reagent and then centrifuged at 16,000 g for 10 min to collect the supernatant as a nuclear fraction.Cytosolic and nuclear fractions were extracted at 4°C by the multiple centrifugation method, using a nuclear and cytoplasmic protein extraction kit . About 50 mg of tissue was cut into pieces and homogenized gently in the cytoplasmic protein extraction reagent with a glass tissue grinder. The homogenates were centrifuged at 1500 g for 5 min to collect the supernatant as a cytosolic fraction. The pellet was resuspended in 200 μg protein was incubated with 50 μL reaction buffer and 5 μL caspase 3 substrate at 37°C for 4 h. The caspase 3 activity was measured at absorbance of 405 nm.The caspase 3 activity was detected with a caspase 3 activity assay kit . About 50 mg of tissue was ground in lysis buffer with a glass homogenizer and then centrifuged at 12,000 g for 15 min. The supernatant protein was collected and quantified by the Bradford method. The supernatant containing 200 μg of total protein was incubated overnight at 4°C with anti-P53  followed by precipitation with 70 μL of protein A/G-Plus-Agarose  for 4 h at 4°C. Nonspecific IgG (Proteintech) was used as the control. The precipitated complexes were washed in immunoprecipitation buffer and then resuspended in 30 μL of 2× loading buffer and boiled for 5 min.Immunoprecipitation analysis was performed as previously described . Brieflyμm sections were used for hematoxylin and eosin (HE) staining according to the standard protocol. Histopathological changes were scored as follows: 0, no damage; 1, <25%; 2, 25-50%; 3, 50-75%; and 4, >75%. IHC staining was performed as previously described [α-SMA . 4 random fields per section were selected for the analysis of semiquantification by Image-Pro Plus 6.0 software.After embedding kidney tissues into paraffin, the 3 escribed . The priThe Hoechst 33342 staining  was used to identify the morphology of apoptotic nuclei. The sections were deparaffinized and then exposed to the Hoechst 33342 solution for 5 min at 37°C in the dark. The positive nuclei were observed with a fluorescence microscope  at 200x magnification.An immunoblotting assay was performed as previously described , 8. The −ΔΔCT method. β-Actin was used as an internal control. The PCR reaction conditions were as follows: 95°C for 5 min, followed by 40 cycles for 10 s at 95°C and 30 s at 60°C. The primers used were as follows: Puma, forward ACTGCCAGCCTTGCTTGTC and reverse AGTCCTTCAGCCCTCCCTTC; Bax, forward GGCGATGAACTGGACAAC and reverse CCGAAGTAGGAAAGGAGG; Noxa, forward GTTACCGCCTGAATTCGCAG and reverse AGTTATGTCCGGTGCACTCC; COX I, forward CACATGAGCAAAAGCCCACT and reverse ACGGCCGTAAGTGAGATGAA; and β-actin, forward GAGAGGGAAATCGTGCGT and reverse GGAGGAAGAGGATGCGG.Total RNA was extracted from kidney tissues using the TRIzol reagent  and reverse transcribed to cDNA using a TaqMan RNA Reverse Transcription Kit  according to the manufacturer's instructions. qPCR was performed on an ABI StepOnePlus Real-Time PCR System  with SYBR Green Master Mix  according to the instructions provided with the kit. Relative expression was determined by the 2P < 0.05 was considered statistically significant.All data were presented as mean ± SEM and analyzed by one-way analysis of variance with LSD-t multiple comparison, using SPSS software . α-smooth muscle actin (α-SMA) protein, the marker of fibroblast activation. As shown in Figures α-SMA protein and SSR showed significant reduction in interstitial fibrosis when compared with the model group, which verified the renoprotective effects of SSR in kidney injury.Our previous studies have demonstrated that SSR markedly improved renal function and downregulated the expression of extracellular matrix (ECM) proteins . In thisAs a key executor of apoptosis, caspase 3 is implicated in the proteolysis of many critical proteins . In thisThe mitochondrial pathway of apoptosis is mainly triggered by Bax accumulation in the mitochondria and subsequent release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space . We enriβ-actin to estimate mitochondrial function in the remnant kidneys. Compared with the sham group, the SDHB and ATPB, the subunits of complex II and complex V [Furthermore, mitochondrial dysfunction is a well-known pathogenic factor in ischemic renal injury and the destruction of mitochondrial integrity is more sensitive to the insertion of Bax , 16. Theomplex V , and mitSince p53 is the most critical molecule upstream of Puma and Bax and that the nuclear translocation of p53 represents its activation , we extrIn addition to its transcriptional-dependent proapoptotic effect, p53 could also translocate to mitochondria and interact with Bcl-xL and Bcl-2 to inhibit their antiapoptotic effects , 19–21. In this study, we demonstrated that SSR blocked apoptosis induced by 5/6 (A/I) injury by inhibiting the accumulation of Bax and Puma in the mitochondria. As a result, release of cytochrome c into the cytosol and caspase 3 activity were decreased. In addition, SSR inhibited the nuclear translocation and transcriptional activity of p53 in 5/6 (A/I)-injured rats, which was shown by the decreased expression of Puma, Bax, and Noxa mRNA. Importantly, coimmunoprecipitation/immunoblotting analysis demonstrated that SSR not only significantly reduced the translocation of P53 to mitochondria but also inhibited the direct interaction of P53 with antiapoptotic Bcl-2 and Bcl-xL proteins.α release. Rheum and its extracts have nephroprotective effects in the CKD model induced by nephrotoxicity and adenine [Previous studies demonstrated that in different disease models, inhibition of apoptosis showed significant renal protective effects. Du et al.  reported adenine , 28. The adenine . Upon st adenine , 31. Our adenine . In this adenine . During  adenine . TherefoPrevious studies have reported that persistent hypoxia could be the main trigger of p53 activation in the process of renal injury and p53-mediated induction of Puma and Bax was responsible for apoptosis in ischemic kidneys –37. HoweIn conclusion, SSR could attenuate apoptosis in the rat CKD model by the mitochondrial pathway of apoptosis. Furthermore, activation of p53-induced target genes and transcriptional-independent apoptotic activity of p53 could amplify the intrinsic pathway of apoptosis, which was inhibited by SSR treatment for 8 weeks. However, the relationship between p53 transcriptional-dependent and transcriptional-independent proapoptotic function and the regulatory effects of SSR on them need to be further clarified in the future study.


Oxidative stress is a key player of the inflammatory cascade responsible for the initiation of ulcerative colitis (UC). Although the short chain quinone idebenone is considered a potent antioxidant and a mitochondrial electron donor, emerging evidence suggests that idebenone also displays anti-inflammatory activity. This study evaluated the impact of idebenone in the widely used dextran sodium sulphate (DSS)-induced mouse model of acute colitis. Acute colitis was induced in C57BL/6J mice via continuous exposure to 2.5% DSS over 7 days. Idebenone was co-administered orally at a dose of 200 mg/kg body weight. Idebenone significantly prevented body weight loss and improved the disease activity index (DAI), colon length, and histopathological score. Consistent with its reported antioxidant function, idebenone significantly reduced the colonic levels of malondialdehyde (MDA) and nitric oxide (NO), and increased the expression of the redox factor NAD(P)H (nicotinamide adenine dinucleotide phosphate) dehydrogenase quinone-1 (NQO-1) in DSS-exposed mice. Immunohistochemistry revealed a significantly increased expression of tight junction proteins, which protect and maintain paracellular intestinal permeability. In support of an anti-inflammatory activity, idebenone significantly attenuated the elevated levels of pro-inflammatory cytokines in colon tissue. These results suggest that idebenone could represent a promising therapeutic strategy to interfere with disease pathology in UC by simultaneously inducing antioxidative and anti-inflammatory pathways. Inflammatory bowel disease (IBD) is characterised as a chronic, relapsing, multifactorial disorder of the gastrointestinal tract with an unknown aetiology. It is primarily divided into two major sub-forms: ulcerative colitis (UC) and Crohn’s disease (CD) . IBD oftThe DSS mouse model is one of the well-established indispensable tools for studying intestinal inflammation that recapitulates the morphological and clinical symptoms of human IBD, such as weight loss, diarrhoea, rectal bleeding, shortening of the colon, depletion of goblet cells and ulcerations ,9. DSS iThe imbalance between the levels of radicals and antioxidative enzymes contributes to oxidative stress and initiates the peroxidation of fatty acids and lipoproteins in cell membranes. The end product of lipid peroxidation  is highly toxic for cells and tissues . In respThe short-chain quinone idebenone is a potent antioxidant and mitochondrial electron donor . IdebenoWe therefore hypothesised that idebenone might be a promising drug to alleviate DSS-induced inflammation due to the combination of anti-inflammatory and antioxidant activities. The current study demonstrated that idebenone successfully ameliorated intestinal inflammation and maintained tissue redox homeostasis, which was associated with an upregulation of the antioxidant enzyme NQO-1.p < 0.0001) increased in DSS-treated mice from day 2 onwards (p < 0.05) until the end of the observation period on day 8 (p < 0.0001) compared to DSS-treated animals.Administration of 2.5% DSS for 7 days A replica onwards C. Idebenp < 0.0001) shortened the colon length (5.25 cm ± 0.17), in contrast with HC (7.09 cm ± 0.08). Consistent with the reduction in disease severity, idebenone treatment significantly (p < 0.0001) normalised the colon length compared to DSS-treated animals (6.46 cm ± 0.14) (DSS-induced inflammation significantly ( ± 0.14) C,D. ThisThe histopathology of the proximal (PC) and distal colon (DC) was assessed using haematoxylin and eosin (H&E) staining of tissue sections A. Colon Tight junction (TJ) proteins are essential for the maintenance of the intestinal epithelial barrier that limits the entry of harmful molecules into the lamina propria. Therefore, the expression of TJ proteins occludin and ZO-1 was assessed using immunohistochemistry. In HC, occludin and ZO-1 were expressed homogenously and were mainly detected around the surface membrane and around the crypts A of the p < 0.05) increased mucus levels compared to the reduced mucus levels in DSS-treated animals  in the DC  to levels comparable to healthy animals (HC) (91.40%) (p < 0.01) increased by DSS-treatment, was significantly supressed by idebenone in the DC , a lipid peroxidation by-product, is one of the main indicators of oxidative damage to lipid membranes and proteins. Thus, the levels of MDA in colon tissue of the test animals were determined A. DSS-trn the DC A. We fur(91.40%) B. Nitric, −47.49 C.p < 0.05 vs. DSS)  B.DSS-induced inflammation is associated with the release of an array of pro-inflammatory cytokines. To investigate the anti-inflammatory effects of idebenone, the colon tissue levels of pro-inflammatory cytokines and chemokines were quantified in the test animals. In the distal colon (DC), idebenone consistently downregulated the levels of IL-1α, IL-6, TNF-α, GM-CSF (granulocyte macrophage colony stimulating factor), G-CSF (granulocyte colony stimulating factor), IL-17, IL-10 and IL-3 A. In addFor the first time, the current study revealed a protective role for idebenone in a well-described and frequently used pre-clinical murine model of acute colitis. Our results demonstrate the substantial therapeutic potential of idebenone in reducing the severity of DSS-induced disease by simultaneously exerting both antioxidant and anti-inflammatory activities, while concomitantly regulating the immune responses and barrier function.The current study used idebenone at a dose of 200 mg/kg that previously ameliorated oxidative stress, pro-inflammatory cytokines and DNA damage in a titanium-dioxide-induced hepatotoxicity model , and alsIt is well established that reactive oxygen species and reactive nitrogen species (RNS) at low concentrations and over short time intervals are essential signalling molecules involved in a multitude of physiological processes . HoweverHowever, it must be noted that idebenone has also been associated with a pro-oxidative activity in some reports , which aThe accumulation of ROS and RNS also disrupt the intestinal barrier integrity by redistributing TJ protein complexes involving occludin and ZO-1, which leads to a vicious cycle of intestinal inflammation ,68,69,70Gut inflammation is characterised by the overproduction of pro-inflammatory cytokines and chemokines, along with altered barrier integrity and an oxidative response ,16,69,75It is well known that the anti-inflammatory cytokine IL-10 is elevated in colitis patients ,84,85,86Our results also align with clinical studies that reported up-regulated chemokines, such as MIP-1α, MIP-1β, eotaxin and RANTES, in the colonic biopsies of UC patients ,87,88,89Apart from DC, idebenone administration also showed significant reductions of IL-6, TNF-α, GM-CSF, MIP-1α, eotaxin and IL-17 in the PC. Our results also indicated that the inflammation level varied between PC and DC. This has been substantiated by previous studies ,72,83, wVery few randomised controlled clinical trials have reported therapeutic effects of antioxidants in UC patients. Trials that tested compounds, such as curcumin and polyunsaturated fatty acids (PUFAs), were associated with methodical limitations, including short duration, small subject size or the absence of a healthy control group ,92. OtheAccumulation of oxidative stress not only damages the colon, but also stimulates the release of pro-inflammatory cytokines and increases intestinal permeability. It must be noted that at present, it is still unclear how the different idebenone-dependent events that protect against colitis are initiated and regulated, and whether there is a functional or temporal hierarchy between the different activities described in the current study. To our knowledge, the present study is the first to explore the antioxidative and anti-inflammatory properties of idebenone in attenuating the pathology of acute colitis. Idebenone provides effective cytoprotection against oxidative damage, likely by a SOD/NQO-1-dependent mechanism, maintaining intestinal barrier function and simultaneously preventing the up-regulation of pro-inflammatory cytokines in a mouse model of DSS-induced acute colitis. Therefore, our study strongly suggests that idebenone could be developed as a promising therapeutic alternative to treat the acute phases of UC.ad libitum. Animals were housed at a controlled temperature with 12-h day/night light cycle. Individual body weights were assessed daily over an acclimatisation period of 7 days before being included into any experiments. All procedures were approved by the Animal Ethics Committee of University of Tasmania .All female C57BL/6J mice were purchased from the University of Tasmania animal breeding facility. Mice were caged individually with access to standard chow and autoclaved drinking water n = 10/group): healthy controls without DSS and drug (HC), DSS treatment (DSS) and DSS + idebenone treatment (DSS + I). All animals were acclimatised for 1 week before the start of the experiment. Colitis was induced by administering 2.5% DSS   in autoclaved drinking water to all groups except HC, continuously over 7 days. Idebenone was prepared as wet food mash by mixing it with 0.5% of carboxymethylcellulose (CMC), 4% sucrose and autoclaved powdered chow pellets. Idebenone was administered orally at a dose of 200 mg/kg of body weight. HC were supplied with normal autoclaved water and standard autoclaved chow pellets in the form of food mash. DSS-treated and DSS + idebenone groups were treated with a drug vehicle and idebenone (200 mg/kg of body weight), respectively, for the treatment period of 7 days. Food mash was aliquoted as 2.5 g per dish and was stored at −20 °C until use.Female C57BL/6J mice aged 6–7 weeks with an average weight around 18 g were randomly divided into three groups (The disease activity index (DAI) was calculated as the sum of the individual scores for bloody stool, stool consistency and body weight loss, as previously described ,103. AllAn HRP/DAB detection IHC kit from Abcam  was used to perform immunohistochemical analysis, as previously described . BrieflyBriefly, distal colon tissue sections were homogenised and lysed in a RIPA buffer containing protease inhibitor cocktails . Protein quantification was done using a DC protein Assay Kit from Biorad. The samples were suspended in loading dye and boiled at 95 °C for 5 min. A total of 20 μg of protein was separated on 4–15% of SDS-PAGE gel (Mini-PROTEAN TGX Precast Gels (50 µL), Biorad, New South Wales, Australia) at 100 V for 60 min, then electro-transferred to a PVDF membrane at 250 A for 60 min. The membranes were blocked with 5% non-fat milk for 1 h at room temperature (RT) and then incubated with primary antibodies against NQO-1 (1:1000)  and β-actin (1:8000)  overnight at 4 °C. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody  for 1 h at RT. The bands were visualised with a chemiluminescence reagent  and imaged using a Fujifilm Luminescent Image Analyzer  .Alcian blue staining kit , Abcam, Australia) was utilised to visualise the sulphated and acidic mucopolysaccharides (MUC2). Staining was performed as described previously . Brieflyg for 10 min. The resulting supernatants were supplemented with thiobarbituric acid (TBA) and were boiled at 95 °C in a water bath for 60 min. The MDA-TBA adduct was formed, which was quantified colorimetrically at 532 nm. The amount of MDA in the samples was detected by plotting against an MDA standard (provided in the kit) calibration curve. The values were expressed as nmol/mg protein.The levels of MDA as a marker of lipid peroxidation was determined by using a commercially available lipid peroxidation colorimetric/fluorometric assay kit , as mentioned previously . Brieflyg for 5 min, supernatants  were assayed by adding WST-1 (water soluble tetrazolium-1) solution according to the manufacturer’s instructions. The data represents the percentage inhibition of superoxide production by SOD and was termed the SOD activity. The more SOD in the samples, more its inhibitory activity.Total superoxide dismutase (SOD) activity was measured using a commercially available superoxide dismutase activity assay kit , as described previously . BrieflyN-1-napthylethylenediamine dihydrochloride (NED) under acidic conditions. The sample absorbance was plotted against a nitrite standard reference curve at 550 nm. The values were expressed as a concentration in μM/gram of tissue. For NO generation, a Griess reagent kit  was used to measure nitrite, a stable by-product of NO, as described previously . Colon tProximal and distal colon samples were excised from the test animals, washed with PBS and cultured in 12–well plates containing 500 µL/well of RPMI1640 , supplemented with 10% Fetal Bovine Serum  and 1% antibiotics solution , as mentioned previously . After ip < 0.05 was considered significant.GraphPad Prism software version 6.0  was used to perform statistical analysis. Results were expressed as a mean ± SEM from at least 3 to 10 animals per group. Statistical differences between the groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc. Two-way ANOVA followed by Tukey’s post-hoc test was used to analyse DAI and body weight changes during the experimental period. A statistical difference of