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Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.
Subject(s)
Humans , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pyroptosis , Podocytes/metabolism , Hepatitis B virus/genetics , Caspase 1/metabolism , Cytokines/metabolism , Carrier Proteins/metabolism , MicroRNAs/genetics , Glomerulonephritis/metabolism , RNA, Small InterferingABSTRACT
OBJECTIVE@#To investigate the effect of Chang'an II Decoction ( II ))-containing serum on intestinal epithelial barrier dysfunction in rats.@*METHODS@#Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium. Caco-2 monolayers were treated with blank serum and Chang'an II Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang'an II Decoction intragastrically at doses of 0.49, 0.98, 1.96 g/(kg·d) for 1 week, respectively. After preparation of containing serum, cells were divided into the normal group, the model group, the Chang'an II-H, M, and L groups (treated with 30 ng/mL TNF-α and medium plus 10% high, middle-, and low-doses Chang'an II serum, respectively). Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-labeled dextran. Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs). Immunofluorescence of zonula occludens-1 (ZO-1), claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution. The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction. The expression levels of MLCK, myosin light chain (MLC) and p-MLC were determined by Western blot.@*RESULTS@#Chang'an II Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α. It alleviated TNF-α-induced morphological alterations in TJ proteins. The increases in MLCK mRNA and MLCK, MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang'an II-H group. Additionally, Chang'an II Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus.@*CONCLUSION@#High-dose Chang'an II-containing serum attenuates TNF-α-induced intestinal barrier dysfunction. The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.
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<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of Chinese medicine (CM) decoction Chang'an I Recipe ( I ) in the treatment of irritable bowel syndrome with diarrhea (IBS-D).</p><p><b>METHOD</b>A multicenter, randomized, double-blind, placebo-controlled clinical trial was designed. Based on the order of inclusion, the IBS-D patients were randomly assigned to the treatment group or the placebo control group, administrated with Chang'an I Recipe or placebo, 150 mL/bag, 3 times daily, for 8 weeks. The primary indices of efficacy included the effective rates of IBS symptom severity score (IBS-SSS) and the differences in adequate relief (AR) responder; the secondary indexes of efficacy included the changes in scores of the IBS Quality of Life (IBS-QOL) and Hospital Anxiety and Depression (HAD) scales. The safety indices included adverse events and related laboratory tests.</p><p><b>RESULTS</b>A total of 216 patients were included, with 109 in the treatment group and 107 in the control group, and finally 206 were included in the full analysis set (FAS), 191 were included in the per protocol set (PPS). In FAS, the total effective rate was 67.6% and 40.2% for the treatment and control groups, respectively, with 95% confidence interval (CI) for difference in the effective rates between the two groups of 14.4%-40.2%; while in PPS, the total effective rate was 71.3% and 41.2% for the treatment and control groups, respectively (95% CI 16.6%-43.4%). The consistent conclusions of FAS and PPS showed a better efficacy in the treatment group. Both FAS and PPS showed higher AR responder in the treatment group (FAS: 59.6% vs. 35.5%; PPS: 62.8% vs. 38.1%). As for IBS-QOL, the total score and scores in various dimensions of IBS-QOL were not significantly different between the two groups (P>0.05). Both anxiety and depression scales of HAD were not significantly different between the two groups (P>0.05). No adverse events or laboratory abnormalities were found to be obviously related to the tested drugs or clinically significant.</p><p><b>CONCLUSION</b>Chang'an I Recipe was more effective than placebo in the treatment of IBS-D, with no obvious adverse reactions. (No.ChiCTR-TRC-09000328).</p>
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Chinese medicine (CM) decoction placebo is one of the key factors restricting the placebo-controlled clinical trial of CM. In this article, based on the analysis of the diffificulties in preparing CM placebo, the production requirements for placebo were put forward. Moreover, in accordance with clinical practices, a production method and evaluation process of CM decoction placebo was proposed, to provide a reference for clinical research.
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<p><b>OBJECTIVE</b>To assess the effectiveness of Tongjiang Granule (TJG) in treating non-erosive reflux disease (NERD) of Gan-Wei incoordination syndrome (GWIS).</p><p><b>METHODS</b>Totally 128 NERD patients of GWIS were recruited from outpatients or inpatients at Department of Digestive Disease, Xiyuan Hospital, China Academy of Chinese Medical Sciences from February 2009 to November 2010. They were randomly assigned to two groups using the block group in the ratio of 1:1, 64 cases in each group. Patients in the experiment group were treated with TJG, 10 g each time, three times a day, while those in the control group were treated with Omeprazole Tablet, 20 mg each time, two times a day. The treatment course of both groups was 4 weeks. The symptoms questionnaires and SF-36 quality of life scale were observed.</p><p><b>RESULTS</b>Finally 114 patients completed the trial. There was statistical difference in epigastric upset, pharyngeal foreign body sensation, abdominal swelling or abdominal pain, and integral of excrement between the two groups before treatment (P < 0.05). However, there was no statistical difference in the rest indices between the two groups (P > 0.05). Compared with before treatment in the same group, the scores of each symptom or the total symptoms were somewhat improved in the two groups (P < 0.01, P < 0.05). There was statistical difference in the rest scores (P > 0.05) except the score of mental health in the experiment group (P < 0.01, P < 0.05). There was statistical difference in the rest scores (P > 0.05) except the score of physical function in the control group (P < 0.01, P < 0.05). There was statistical difference in post-treatment acid reflux, irritability, depression, body pain, roles of emotions (P < 0.01, P < 0.05). The total effective rate was higher in the experimental group, showing no statistical difference when compared with that of the control group (P > 0.05).</p><p><b>CONCLUSIONS</b>TJG had confirmative efficacy in treating NERD patients of GWIS. Meanwhile, it could improve their quality of life, with no obvious adverse reaction.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Drugs, Chinese Herbal , Therapeutic Uses , Gastroesophageal Reflux , Drug Therapy , Phytotherapy , Quality of Life , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>The pathogenesis of irritable bowel syndrome (IBS), a functional gastrointestinal disorder, is poorly understood. Treatment by Chinese medicine and pharmacy (CMP) is superior to treatment by Western medicine (WM). Therefore, we aimed to analyze Chinese medical experts' experiences in treating IBS by data mining.</p><p><b>METHODS</b>By retrieving related literatures from 1994 to 2012 from CNKI, we chose proved cases and screened effective medical records to establish a database using structural electronic medical case collecting system and data mining. The thinking ways of modern physicians in diagnosis and treatment of IBS were analyzed.</p><p><b>RESULTS</b>Totally 116 medical cases from 46 Chinese medical experts were recruited and analyzed. Results showed that Gan depression and Pi deficiency were most basic syndrome elements. In addition, cold dampness, damp heat, yang deficiency, and Shen deficiency were main elements for the development of diarrhea predominant IBS (IBS-D). Qi stagnation, yin deficiency, qi deficiency, and blood stasis were main elements for the development of constipation predominant IBS (IBS-C). Soothing Gan-qi and strengthening Pi was the most important treatment method. White peony root, prepared atractylodes, prepared licorice, tangerine peel, poria, bupleurum, Radix sileris, lanceolata, and Radix aucklandiae were the most common Chinese herbs used in treating IBS.</p><p><b>CONCLUSIONS</b>(1) Gan depression and Pi deficiency was the main pathogenesis for IBS. Diagnosis and treatment of IBS should be performed clinically according to its development. Tongxie Yao-fang should be used as the basic prescription with modification according to syndrome differentiation. (2) Data mining will have a wide application prospect in studying medical cases.</p>
Subject(s)
Humans , Data Mining , Irritable Bowel Syndrome , Diagnosis , Drug Therapy , Medicine, Chinese Traditional , Methods , Phytotherapy , MethodsABSTRACT
<p><b>OBJECTIVE</b>To assess the effectiveness of tongjiang granule (TJG) on the patients with nonerosive reflux disease (NERD) of Gan-Wei incoordination syndrome, its impact on their quality of life, and its safety.</p><p><b>METHOD</b>A randomized, controlled, double-blinded, and double-dummy method was adopted in the trial. There were 120 NERD patients enrolled in the study and randomly divided into the experiment and control groups, each with 60 patients; drugs were distributed according to the drug number by patients' inclusion sequences. In the experiment group, patients were given TJG 10 g and mosapride citrate dummy 5 mg three times a day, and in the control group, patients were given mosapride citrate 5 mg and TJG dummy 10 g three times a day. The treatment courses of both groups were 4 weeks.</p><p><b>RESULTS</b>Among 120 included patients, 112 were screened for full analysis set (FAS), and 105 were screened per-protocol set (PPS). The results were as follows: (1) the improvement of total scores of symptom in the experiment group (0-4 week) were 15.93±7.88 scores by FAS and 16.22 ±7.75 scores by PPS, and they were 10.43±10.16 scores and 10.79±10.27 scores in the control group, respectively. The 95% CI of net scores improvement between the two groups were 2.10-8.90 scores and 1.92-8.94 scores in FAS and PPS; it was significantly better in the experiment group than that in the control group (P<0.05). (2) The improvement of scores of major symptom in the experiment group (0-4 week) were 10.68±5.35 by FAS and 10.89±5.29 by PPS and 7.40±7.41 and 7.60±7.46 in the control group, respectively. The 95% CI of net scores improvement in the two groups were 0.85-5.71 and 0.71-5.69 in FAS and PPS separately, and the improvement in the experiment group was significantly better than that in the control group (P<0.05). (3) The total effective rates were 86.0% and 61.8% in the experiment and the control group separately, and the Ridit analysis results showed that it was better in the experiment group (P<0.05). (4) The improvement quality of life in the domain of physical functioning and general health in the experiment group was better than that in the control group (P<0.05). (5) One case of experiment group caught a cold and recovered in six days without drug suspension. No adverse event was found in the other cases. There was no meaningful safety examination indices change in pretreatment and posttreatment periods in both groups.</p><p><b>CONCLUSION</b>TJG showed a definite effect on the treatment of NERD with Gan-Wei incoordination syndrome, and it could improve the quality of life of NERD patient without obvious toxic and side effects.</p>