补虚化瘀方及其拆方对脂多糖诱导的人肝内胆管上皮细胞TLR4/MyD88/NF-κB信号通路的影响  

Effects of Buxu Huayu Recipe(补虚化瘀方)and Its Decomposed Prescriptions on TLR4/MyD88/NF-κB Signal Pathway of Human Intrahepatic Bile Duct Epithelial Cells Induced by Lipopolysaccharide

作　　者：景如斌 袁文洁 张玮[1] JING Rubin;YUAN Wenjie;ZHANG Wei(Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032

出　　处：《辽宁中医杂志》2024年第7期197-201,I0004,共6页Liaoning Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(81673926);上海中医药领军人才建设项目(lh01.22.007);上海市卫生和计划生育委员会面上项目(ZYKC201840234);上海市浦东新区名中医工作室建设项目(PDZYXK-3-2014020)。

摘　　要：目的 观察补虚化瘀方及其拆方对脂多糖(LPS)诱导的人肝内胆管上皮细胞(HIBEC)细胞外基质及TLR4/MyD88/NF-κB信号通路的影响,探讨其改善原发性胆汁性胆管炎(PBC)炎症病变的相关机制。方法 将48只SD大鼠随机分为空白组、补虚组、化瘀组、补虚化瘀方组,每组12只。补虚组、化瘀组、补虚化瘀方组分别灌以相应中药水煎液,空白组灌以等量的生理盐水,每日1次,连续灌胃2周,灌胃结束后取血制备含药血清;将不同处理因素的HIBEC分为正常组、模型组(5μg/mL脂多糖)、补虚组、化瘀组、补虚化瘀方组;CCK8法筛选最佳干预浓度,检测各组细胞活力;免疫荧光检测各组NF-κB激活-核转运状态;Western blot检测各组TLR4、MyD88、NF-κB p65、P-NF-κB p65目的蛋白的表达;酶联免疫吸附测定法(ELISA)测定各组细胞培养上清液中IL-6、IL-8、MCP-1、CXCL10的含量。结果 (1) CCK8结果显示,15%含药血清为最佳干预浓度;与正常组及模型组相比,化瘀组细胞活力增加(P<0.05),补虚化瘀方组细胞活力增加明显(P<0.01);(2)免疫荧光检测显示,与正常组比较,模型组NF-κB亚基从细胞浆大量转运到细胞核内,光密度明显增强(P<0.01);与模型组、补虚组、化瘀组相比,补虚化瘀方含药血清组转运到细胞核内的NF-κB亚基明显减少,光密度减弱(P<0.01);(3) Western blot结果显示,与正常组比较,模型组TLR4、MyD88、NF-κB p65、P-NF-κB p65蛋白表达增加(P<0.01);与模型组比较,补虚组NF-κB p65、P-NF-κB p65蛋白表达减少(P<0.01),化瘀组P-NF-κB p65蛋白表达减少(P<0.01),补虚化瘀方组TLR4、MyD88、NF-κB p65、P-NF-κB p65蛋白表达明显减少(P<0.01);(4) ELISA检测显示,与正常组比较,模型组IL-6、IL-8、MCP-1、CXCL10分泌增多(P<0.01);与模型组比较,补虚组、化瘀组IL-6、MCP-1、CXCL10分泌减少(P<0.01),补虚化瘀方组IL-6、IL-8、MCP-1、CXCL10分泌明显减少(P<0.05,P<0.01)。结论 相对于模型组、�Objective To observe the effects of Buxu Huayu Recipe(补虚化瘀方)and its disassembled prescriptions on the extracellular matrix and Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB)signal pathway of human intrahepatic bile duct epithelial cells(HIBEC)induced by lipopolysaccharide(LPS)and explore the relevant mechanisms of its improvement on inflammatory lesions of primary biliary cholangitis(PBC).Method Forty-eight SD rats were randomly divided into blank serum group,tonifying deficiency group,resolving blood stasis group and Buxu Huayu Recipe group,with 12 rats in each group.The corresponding decoction of traditional Chinese medicine was infused into the tonifying deficiency group,the resolving blood stasis group and the Buxu Huayu Recipe group respectively,and the blank serum group was infused with the same amount of normal saline once a day for two consecutive weeks.After the end of the gastric perfusion,the blood was taken otu to prepare the serum containing drugs.HIBEC with different treatment factors were divided into normal group,model group(5μg/mL lipopolysaccharide),tonifying deficiency group,resolving blood stasis group and Buxu Huayu Recipe group.CCK8 method was used to screen the best intervention concentration and detect the cell viability of each group.NF-κB activation nuclear transport status was detected by immunofluorescence.Western blot was used to detect the expressions of TLR4,MyD88,NF-κB p65 and P-NF-κB p65 in each group.The contents of interleukin-6(IL-6),interleukin-8(IL-8),monocyte chemokine-1(MCP-1)and CXCL10 in the supernatant of cell culture in each group were determined by enzyme-linked immunosorbent assay(ELISA).Result①CCK8 results showed that 15%medicated serum was the best intervention concentration.Compared with that of the normal group and the model group,the cell vitality of the resolving blood stasis group increased(P<0.05),and the cell vitality of the Buxu Huayu Recipe group increased significantly(P<

关 键 词：补虚化瘀方 脂多糖 TLR4/MyD88/NF-κB信号通路 HIBEC 细胞因子 

分 类 号：R285[医药卫生—中药学]