{"title":"Baicalin methyl ester prevents the LPS – induced mice intestinal barrier damage in vivo and in vitro via P65/TNF-α/MLCK/ZO-1 signal pathway","authors":"","doi":"10.1016/j.biopha.2024.117417","DOIUrl":null,"url":null,"abstract":"<div><p>The effect of baicalin methyl ester (BME) on the regulation of mice intestinal barrier in the inflammatory response was studied in vivo and in vitro. Thirty six C57/BL mice were randomly divided into six groups (n = 6): control group; LPS group (LPS 3.5 mg/kg given intraperitoneal [ip] on day 7 of the study only), PBS group, and three BME groups (low: 50 mg/kg; medium: 100 mg/kg; high: 200 mg/kg) orally dosed with BME for 7d and LPS ip on day 7. All mice were sacrificed on day 8, and jejunum tissue collected for histopathology (H&E and PAS staining), protein expression of pro-inflammatory factors (TNF-α, IL-6, IL-8, IFN-γ) by ELISA, and intestinal tight junction proteins (ZO-1, occludin, claudin-1 and claudin-4) by Western Blot. Compared with the control group, LPS significantly increased the serum cytokines DAO (<em>p</em> < 0.01) and DLA (<em>p</em> < 0.01), u<em>p</em>regulated the expression of pro-inflammatory factors, MLCK proteins (<em>p</em> <0.05) and increased the MLCK/ZO-1ratio (<em>p</em> <0.001). LPS also decreased the expression of claudin-4 (<em>p</em> < 0.01) in the jejunum and induced an inflammatory response damaging the jejunal mucosal barrier. Pretreatment with BME (100–200 mg/kg) significantly decreased the cytokines DAO (<em>p</em> < 0.05) and DLA (<em>p</em> < 0.01) in the serum, pro-inflammatory factors in the jejunum, significantly down-regulated the expression of MLCK (<em>p</em> <0.05) and the ratio of MLCK/ZO-1(<em>p</em> <0.001) but upregulated the expressions of ZO-1(<em>p</em> < 0.01), occludin (<em>p</em> < 0.05), claudin-1(<em>p</em> < 0.05) and claudin-4 (<em>p</em> < 0.05), and thereby restored the intestinal tissue structure, suggestive of alleviation of LPS-induced intestinal inflammation by BME. In vitro, MODE-K cells (derived from mice intestinal epithelium) were exposed to BME at 0 (control group-No LPS), 10, 20 and 40 μM BME for 24 h prior to LPS addition at 50 μg/mL for 2 h. LPS significantly increased the expression of pro-inflammatory factors, MLCK (<em>p</em> < 0.01) and the ratio of MLCK/ZO-1(<em>p</em> <0.001), decreased the expressions of ZO-1 (<em>p</em> < 0.05), occludin (<em>p</em> < 0.01), claudin-1 (<em>p</em> < 0.01) and claudin-4 (<em>p</em> < 0.01) in MODE-K cells compared with the control group. Compared with the LPS group, BME (10 – 40 μM) significantly decreased the expression of pro-inflammatory factors, MLCK (<em>p</em> < 0.05) and the ratio of MLCK/ZO-1(<em>p</em> <0.01) but increased the expressions of ZO-1(<em>p</em> < 0.01), occludin (<em>p</em> < 0.05) and claudin-4(<em>p</em> < 0.01) indicating an up-regulation of the expression of tight junction proteins by BME. On addition of extrinsic TNF-α plus LPS, the TNF- α level increased (<em>p</em> < 0.001) in MODE-K cells and the protein expression of MLCK (<em>p</em> < 0.01) was markedly up-regulated. Molecular docking predicted BME interacted with P65 by forming hydrogen bonds. IP-WB further confirmed that BME was directly bound to P65 protein in MODE-K cells. In conclusion, BME was able to restore the intestinal barrier through the P65 / TNF-α / MLCK / ZO-1 signaling pathway.</p></div>","PeriodicalId":8966,"journal":{"name":"Biomedicine & Pharmacotherapy","volume":null,"pages":null},"PeriodicalIF":6.9000,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0753332224013027/pdfft?md5=b2452643e5a7a7b6390a9196678be563&pid=1-s2.0-S0753332224013027-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biomedicine & Pharmacotherapy","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0753332224013027","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
引用次数: 0
Abstract
The effect of baicalin methyl ester (BME) on the regulation of mice intestinal barrier in the inflammatory response was studied in vivo and in vitro. Thirty six C57/BL mice were randomly divided into six groups (n = 6): control group; LPS group (LPS 3.5 mg/kg given intraperitoneal [ip] on day 7 of the study only), PBS group, and three BME groups (low: 50 mg/kg; medium: 100 mg/kg; high: 200 mg/kg) orally dosed with BME for 7d and LPS ip on day 7. All mice were sacrificed on day 8, and jejunum tissue collected for histopathology (H&E and PAS staining), protein expression of pro-inflammatory factors (TNF-α, IL-6, IL-8, IFN-γ) by ELISA, and intestinal tight junction proteins (ZO-1, occludin, claudin-1 and claudin-4) by Western Blot. Compared with the control group, LPS significantly increased the serum cytokines DAO (p < 0.01) and DLA (p < 0.01), upregulated the expression of pro-inflammatory factors, MLCK proteins (p <0.05) and increased the MLCK/ZO-1ratio (p <0.001). LPS also decreased the expression of claudin-4 (p < 0.01) in the jejunum and induced an inflammatory response damaging the jejunal mucosal barrier. Pretreatment with BME (100–200 mg/kg) significantly decreased the cytokines DAO (p < 0.05) and DLA (p < 0.01) in the serum, pro-inflammatory factors in the jejunum, significantly down-regulated the expression of MLCK (p <0.05) and the ratio of MLCK/ZO-1(p <0.001) but upregulated the expressions of ZO-1(p < 0.01), occludin (p < 0.05), claudin-1(p < 0.05) and claudin-4 (p < 0.05), and thereby restored the intestinal tissue structure, suggestive of alleviation of LPS-induced intestinal inflammation by BME. In vitro, MODE-K cells (derived from mice intestinal epithelium) were exposed to BME at 0 (control group-No LPS), 10, 20 and 40 μM BME for 24 h prior to LPS addition at 50 μg/mL for 2 h. LPS significantly increased the expression of pro-inflammatory factors, MLCK (p < 0.01) and the ratio of MLCK/ZO-1(p <0.001), decreased the expressions of ZO-1 (p < 0.05), occludin (p < 0.01), claudin-1 (p < 0.01) and claudin-4 (p < 0.01) in MODE-K cells compared with the control group. Compared with the LPS group, BME (10 – 40 μM) significantly decreased the expression of pro-inflammatory factors, MLCK (p < 0.05) and the ratio of MLCK/ZO-1(p <0.01) but increased the expressions of ZO-1(p < 0.01), occludin (p < 0.05) and claudin-4(p < 0.01) indicating an up-regulation of the expression of tight junction proteins by BME. On addition of extrinsic TNF-α plus LPS, the TNF- α level increased (p < 0.001) in MODE-K cells and the protein expression of MLCK (p < 0.01) was markedly up-regulated. Molecular docking predicted BME interacted with P65 by forming hydrogen bonds. IP-WB further confirmed that BME was directly bound to P65 protein in MODE-K cells. In conclusion, BME was able to restore the intestinal barrier through the P65 / TNF-α / MLCK / ZO-1 signaling pathway.
期刊介绍:
Biomedicine & Pharmacotherapy stands as a multidisciplinary journal, presenting a spectrum of original research reports, reviews, and communications in the realms of clinical and basic medicine, as well as pharmacology. The journal spans various fields, including Cancer, Nutriceutics, Neurodegenerative, Cardiac, and Infectious Diseases.