摘要
目的 研究葛根芩连汤(GQD)对脂多糖(LPS)诱导的人结直肠腺癌细胞(Caco-2)炎症模型紧密连接(TJ)蛋白的影响,探讨其治疗溃疡性结肠炎(UC)的相关机制.方法 SD雄性大鼠制备空白及含药血清,培养Caco-2细胞并将其分为Control、LPS、GQD-L、GQD-M、GQD-H五组.除Control组外,其余组以1 μg/ml LPS建立肠上皮细胞炎症模型.药物处理后,CCK-8法检测各组细胞的增殖情况;实时荧光定量聚合酶链反应(RT-qPCR)法测定各组闭锁蛋白(Occludin)、闭合蛋白-1(Claudin-1)、白细胞介素(IL)-1β、IL-6、IL-10、肿瘤坏死因子-α(TNF-α)、p38丝裂原活化蛋白激酶(p38MAPK)、肌球蛋白轻链激酶(MLCK)、肌球蛋白轻链2(MLC2)mRNA表达水平;蛋白质免疫印迹法(WB)法测定 Occludin、Claudin-1、IL-1β、IL-6、IL-10、TNF-α、p-p38MAPK、p38MAPK、MLCK、p-MLC 蛋白表达水平;免疫荧光染色法观察 Occludin、Claudin-1、p-p38MAPK、MLCK、p-MLC的阳性表达情况.结果 与Control组相比,LPS组细胞增殖被明显抑制,Occludin、Claudin-1表达减少,IL-1β、IL-6、TNF-α表达增多,而 IL-10 表达减少,p-p38MAPK、p38MAPK、p-p38MAPK/p38MAPK、MLCK、MLC2、p-MLC表达增多(P＜0.05);与LPS组相比,GQD各剂量组,尤其GQD-M与GQD-H组细胞增殖明显促进,Occludin、Claudin-1 表达增多,IL-1β、IL-6、TNF-α 表达减少,而 IL-10 表达增多,p-p38MAPK、p38MAPK、p-p38MAPK/p38MAPK、MLCK、MLC2、p-MLC表达减少(P＜0.05).结论 葛根芩连汤可能通过抑制p38MAPK/MLCK通路过度激活,减轻肠道炎症反应,逆转肠上皮TJ损伤,保护肠黏膜屏障而发挥对UC治疗作用.
Abstract
OBJECTIVE To investigate the effect of Gegen Qinlian Decoction(GQD)on Tight Junction(TJ)protein in Lipopolysaccharides(LPS)-induced inflammatory model constructed from human colon epithelial cancer cell line Caco-2,and to explore its mechanisms involved in the treatment of ulcerative colitis(UC).METHODS Blank and drug-containing sera were extracted from SD male rats.Caco-2 cells were cultured and categorized into five groups:Control,LPS,GQD-L,GQD-M and GQD-H;except for the control one,other groups were cultured by 1 μg/ml LPS to establish the inflammation model.The cellular proliferation rates were detected by CCK-8 assay,the mRNA and protein expressions of Occludin,Claudin-1,interleukin(IL)-1β,IL-6,IL-10,tumor necrosis fac-tor-α(TNF-α),p-p38 mitogen-activated protein kinase(p-p38MAPK)/p38MAPK,myosin light-chain kinase(MLCK)and p-myosin light chain(MLC)were detected by quantitative real time polymerase chain reaction(RT-qPCR)and Western blot(WB)respectively,the densities of Occludin,Claudin-1,p-p38MAPK,MLCK and MLC were detected by immunofluorescence staining.RESULTS Compared with the control group,cell prolifera-tion was significantly inhibited in the LPS group,with low levels of Occludin and Claudin-1,and high levels of IL-1β,IL-6,TNF-α,low level of IL-10,with high levels of p-p38MAPK,p38MAPK,p-p38MAPK/p38MAPK,MLCK,MLC2 and p-MLC(P＜0.05).Compared with the LPS group,all GQD dose groups,especially GQD-M and GQD-H,showed a significant promotion of cell proliferation,with high levels of Occludin and Claudin-1,and low levels of IL-1β,IL-6,TNF-α,high level of IL-10,with low levels of p-p38MAPK,p38MAPK,p-p38MAPK/p38MAPK,MLCK,MLC2 and p-MLC(P＜0.05).CONCLUSION Gegen Qinlian Decoction may exert therapeutic effects on UC by inhibiting the overactivation of the p38MAPK/MLCK pathway,attenuating the inflammatory re-sponse,reversing intestinal epithelial TJ injury and protecting the intestinal mucosal barrier.
维普
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