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

IF 6.9 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL Biomedicine & Pharmacotherapy Pub Date : 2024-09-18 DOI:10.1016/j.biopha.2024.117417
{"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&amp;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> &lt; 0.01) and DLA (<em>p</em> &lt; 0.01), u<em>p</em>regulated the expression of pro-inflammatory factors, MLCK proteins (<em>p</em> &lt;0.05) and increased the MLCK/ZO-1ratio (<em>p</em> &lt;0.001). LPS also decreased the expression of claudin-4 (<em>p</em> &lt; 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> &lt; 0.05) and DLA (<em>p</em> &lt; 0.01) in the serum, pro-inflammatory factors in the jejunum, significantly down-regulated the expression of MLCK (<em>p</em> &lt;0.05) and the ratio of MLCK/ZO-1(<em>p</em> &lt;0.001) but upregulated the expressions of ZO-1(<em>p</em> &lt; 0.01), occludin (<em>p</em> &lt; 0.05), claudin-1(<em>p</em> &lt; 0.05) and claudin-4 (<em>p</em> &lt; 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> &lt; 0.01) and the ratio of MLCK/ZO-1(<em>p</em> &lt;0.001), decreased the expressions of ZO-1 (<em>p</em> &lt; 0.05), occludin (<em>p</em> &lt; 0.01), claudin-1 (<em>p</em> &lt; 0.01) and claudin-4 (<em>p</em> &lt; 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> &lt; 0.05) and the ratio of MLCK/ZO-1(<em>p</em> &lt;0.01) but increased the expressions of ZO-1(<em>p</em> &lt; 0.01), occludin (<em>p</em> &lt; 0.05) and claudin-4(<em>p</em> &lt; 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> &lt; 0.001) in MODE-K cells and the protein expression of MLCK (<em>p</em> &lt; 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.

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
黄芩苷甲酯通过 P65/TNF-α/MLCK/ZO-1 信号通路预防体内和体外 LPS 诱导的小鼠肠屏障损伤
研究了黄芩苷甲酯(BME)在体内和体外对炎症反应中小鼠肠屏障调节作用的影响。将36只C57/BL小鼠随机分为6组(n = 6):对照组、LPS组(LPS 3.5 mg/kg,仅在研究的第7天腹腔注射[ip])、PBS组和3个BME组(低:50 mg/kg;中:100 mg/kg;高:200 mg/kg)。所有小鼠均于第8天处死,采集空肠组织进行组织病理学(H&E和PAS染色)、ELISA检测促炎因子(TNF-α、IL-6、IL-8、IFN-γ)的蛋白表达、Western Blot检测肠道紧密连接蛋白(ZO-1、occludin、claudin-1和claudin-4)。与对照组相比,LPS明显增加了血清细胞因子DAO(p <0.01)和DLA(p <0.01),上调了促炎因子、MLCK蛋白的表达(p <0.05),并增加了MLCK/ZO-1比率(p <0.001)。LPS 还降低了空肠中 claudin-4 蛋白的表达(p <0.01),并诱发了破坏空肠粘膜屏障的炎症反应。用 BME(100-200 mg/kg)预处理可明显降低血清中的细胞因子 DAO(p < 0.05)和 DLA(p < 0.01),它们是空肠中的促炎因子,可明显下调 MLCK 的表达(p < 0.05)和 MLCK/ZO-1 的比值(p < 0.001),但上调了ZO-1(p <0.01)、occludin(p <0.05)、claudin-1(p <0.05)和claudin-4(p <0.05)的表达,从而恢复了肠道组织结构。在体外,将 MODE-K 细胞(来源于小鼠肠上皮细胞)暴露于 0(对照组-无 LPS)、10、20 和 40 μM BME 中 24 小时,然后加入 50 μg/mL LPS 2 小时。与对照组相比,LPS明显增加了MODE-K细胞中促炎因子MLCK(p <0.01)和MLCK/ZO-1比值(p <0.001)的表达,降低了ZO-1(p <0.05)、occludin(p <0.01)、claudin-1(p <0.01)和claudin-4(p <0.01)的表达。与LPS组相比,BME(10 - 40 μM)明显降低了促炎因子MLCK(p <0.05)和MLCK/ZO-1比值(p <0.01)的表达,但增加了ZO-1(p <0.01)、occludin(p <0.05)和claudin-4(p <0.01)的表达,表明BME上调了紧密连接蛋白的表达。加入外源性 TNF-α 和 LPS 后,MODE-K 细胞中 TNF-α 水平升高(p < 0.001),MLCK 蛋白表达明显上调(p < 0.01)。分子对接预测 BME 通过形成氢键与 P65 相互作用。IP-WB进一步证实了BME在MODE-K细胞中直接与P65蛋白结合。总之,BME能够通过P65 / TNF-α / MLCK / ZO-1信号通路恢复肠道屏障。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
CiteScore
11.90
自引率
2.70%
发文量
1621
审稿时长
48 days
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.
期刊最新文献
Molecular mechanism through which Tripterygium hypoglaucum (Lévl.) Hutch alleviates psoriasis ZenoSWATH DIA proteomics and clustering analysis of the effect of cysteamine at the cellular level in cystinotic fibroblasts Protective mechanism of Prim-O-glucosylcimifugin in the treatment of osteoarthritis: Based on lncRNA XIST regulation of Nav1.7 Mitochondria in skeletal system-related diseases Accelerated remyelination and immune modulation by the EBI2 agonist 7α,25-dihydroxycholesterol analogue in the cuprizone model
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1