基于TLR4/NF-κB/MAPK信号通路探讨大黄灵仙方对胆管细胞炎性反应的影响效果

Analysis of Dahuang Lingxian Formula(大黄灵仙方)Regulating and Controlling TLR4/NF-κB/MAPK Signaling Pathway to Affect Inflammatory Response of Bile Duct Cells

目的通过观察大黄灵仙方调控TLR4/NF-κB/MAPK信号通路相关因子基因及蛋白的影响,探讨其对胆管细胞炎性反应的作用。方法采用大鼠胆总管注射脂多糖(lipopolysaccharide,LPS)构建原发性胆管结石胆管慢性炎症状态,取大鼠肝内胆管内皮细胞,通过qPCR技术同时结合使用信号通路阻断剂,观察胆管细胞炎性状态下TLR4/NF-κB/MAPK信号通路相关因子基因及蛋白影响变化及大黄灵仙方的干预作用。结果①Myd88、p-p38、TLR4、NF-κB mRNA变化情况:与空白组比较,L组、L+P组、L+S组、L+P+S组的各指标,均增加明显,剩余各组则降低明显,差异均具统计学意义(P<0.05)。与L组比较,L+P组、L+S组、L+P+S组的各指标有所降低,但差异无统计学意义(P>0.05),L+T组、L+P+T组、L+S+T组、L+P+S+T组则降低明显,差异均具统计学意义(P<0.05);与L+T组比较,L+P+T组、L+S+T组、L+P+S+T组的各指标有所降低,差异无统计学意义(P>0.05)。②Myd88、p-p38、TLR4、NF-κB蛋白变化情况:变化趋势与mRNA相同。结论大黄灵仙方可有效逆转胆管细胞炎性反应过程中存在的TLR4/NF-κB/MAPK信号通路相关因子及蛋白表达异常现象,其机制可能与NF-κB、MAPK信号阻断有关。

Objective To observe the effect of Dahuang Lingxian Formula(大黄灵仙方)regulating and controlling TLR4/NF-κB/MAPK signaling pathway related factor genes and discuss inflammatory response of bile duct cells.Methods We adopted inject LPS in rat choledochal duct to structure bile duct chronic inflammatory condition of primary bile duct stone,took rat intrahepatic bile duct endothelial cells and got through qPCR technic combined with signaling pathway blocker to observe the changes of bile duct cells'TLR4/NF-κB/MAPKsignaling pathway related factor protein and gene expression under inflammatory state and Dahuang Lingxian Formula's intervention effect.Result①Gene expressions of Myd88,p-p38,TLR4 and NF-κB:Compared with those of the blank group,those in L group,L+P group,L+S group and L+P+S group were all obviously increased.Those in L+T group,L+P+T group,L+S+T group and L+P+S+T group were obviously decreased.The difference all had statistical significance(P<0.05).Compared with those of the L group,protein and gene expressions of Myd88,p-p38,TLR4 and NF-κB in L+P group,L+S group and L+P+S group were all decreased,but had no obvious difference(P>0.05).Those in L+T group,L+P+T group,L+S+T group and L+P+S+T group were obviously decreased.The difference all had statistical significance(P<0.05).Compared with those in L+T group,protein and gene expressions of Myd88,p-p38,TLR4 and NF-κB in L+P+T group,L+S+T group and L+P+S+T group were all decreased,but had no obvious difference(P>0.05).②Protein expressions of Myd88,p-p38,TLR4 and NF-κB:The change trend was the same as mRNA.Conclusion Dahuang Lingxian Formula can effectively reverse TLR4/NF-κB/MAPK signaling pathway related factors and proteins in the process of cholangiocyte inflammatory response.The mechanism may be related to NF-κB and MAPK signal blocking.