Department of Veterinary Medicine, College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, PR China.
Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand.
Biomed Pharmacother. 2024 Nov;180:117417. doi: 10.1016/j.biopha.2024.117417. Epub 2024 Sep 18.
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.
目的 研究黄芩素甲酯（BME）对炎症反应中小鼠肠道屏障调节的影响。
方法 将 36 只 C57/BL 小鼠随机分为 6 组（每组 6 只）：对照组；LPS 组（仅在研究第 7 天腹腔内给予 LPS 3.5mg/kg），PBS 组和 3 个 BME 组（低：50mg/kg；中：100mg/kg；高：200mg/kg），BME 灌胃 7d，第 7 天腹腔内给予 LPS。所有小鼠于第 8 天处死，收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）蛋白表达和 Western Blot 检测肠紧密连接蛋白（ZO-1、occludin、claudin-1 和 claudin-4）。
结果 与对照组相比，LPS 显著增加了血清 DAO（p<0.01）和 DLA（p<0.01），上调了促炎因子、MLCK 蛋白（p<0.05）和 MLCK/ZO-1 比值（p<0.001）。LPS 还降低了空肠中 Claudin-4 的表达（p<0.01），并诱导了炎症反应破坏了空肠黏膜屏障。BME（100-200mg/kg）预处理显著降低了血清中的 DAO（p<0.05）和 DLA（p<0.01），下调了空肠中的促炎因子，显著下调了 MLCK 表达（p<0.05）和 MLCK/ZO-1 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，从而恢复了肠组织结构，提示 BME 减轻了 LPS 诱导的肠道炎症。在体外，将 MODE-K 细胞（源自小鼠肠上皮细胞）暴露于 0（对照-无 LPS）、10、20 和 40μM BME 24h，然后加入 50μg/mL LPS 2h。LPS 显著增加了促炎因子、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（10-40μM）显著降低了促炎因子、MLCK（p<0.05）和 MLCK/ZO-1 比值（p<0.01）的表达，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。在加入外源性 TNF-α和 LPS 后，MODE-K 细胞中的 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 BME 直接与 MODE-K 细胞中的 P65 蛋白结合。
结论 BME 通过 P65/TNF-α/MLCK/ZO-1 信号通路恢复肠屏障。
Can J Physiol Pharmacol. 2015-4
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