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目的:探讨疏肝健脾方药对非酒精性脂肪性肝炎(NASH)大鼠肝细胞NF-κB信号通路相关基因及蛋白表达的影响。方法:将SD雄性大鼠随机分为8组:正常对照组、模型组、疏肝高/低剂量组、健脾高/低剂量组、合方高/低剂量组,每组15只。采用高脂饲料喂养复制大鼠NASH模型,16后检测肝脂、血脂,油红O和HE染色观察肝组织病理学变化。同时每组取6只采用离体循环灌注Ⅳ型胶原酶法分离肝细胞,Real time Q-PCR法Western blot法分别检测各组大鼠肝细胞IKKβ、NF-κB mRNA和蛋白及磷酸化IKKβ蛋白表达。结果:与正常对照组比较,模型组大鼠肝细胞存在显著的脂质代谢紊乱;血清TC,肝脂及肝细胞IKKβ、NF-κB mRNA和蛋白表达及磷酸化IKKβ蛋白表达较正常对照组均显著升高(P<0.01);与模型组比较,健脾高剂量组、合方高剂量组大鼠肝细胞IKKβ、NF-κB mRNA表达水平显著降低(P<0.05或P<0.01),各药物干预组大鼠肝细胞IKKβ、NF-κB及磷酸化IKKβ蛋白表达均显著下调(P<0.05或P<0.01),其中以健脾高剂量组、合方高剂量组下调趋势最为显著。结论:抑制肝细胞IKKβ/NF-κB信号通路相关基因及蛋白表达可能是疏肝健脾方药防治NASH的药理作用机制之一;上述蛋白可能是其药物作用的有效靶点。疏肝健脾方药对NASH的干预可能存在一定的量效关系。
Objective: To investigate the effect of Shuganjianpi Recipe on the expression of NF-κB signaling pathway related genes and proteins in hepatocytes of nonalcoholic steatohepatitis (NASH) rats. Methods: SD male rats were randomly divided into 8 groups: normal control group, model group, Shugan high / low dose group, spleen high / low dose group, and combined high / low dose group, 15 rats in each group. The rats were fed with high-fat diet to replicate the rat model of NASH. After 16 hours, hepatic lipid, blood lipid, oil red O and HE staining were observed to observe the pathological changes of liver. At the same time, 6 rats in each group were subjected to ex vivo perfusion with type Ⅳ collagenase, and the expression of IKKβ, NF-κB mRNA and phosphorylated IKKβ protein were detected by Real time Q-PCR Western blotting . Results: Compared with the normal control group, there was a significant disturbance of lipid metabolism in rat hepatocytes in model group. The expressions of IKKβ, NF-κB mRNA and protein in serum TC, hepatic lipid and hepatocytes were higher than those in normal control group (P <0.01 or P <0.01). Compared with the model group, the expressions of IKKβ and NF-κB mRNA were significantly decreased in high-dose spleen group and high-dose Hefei group (P <0.05 or P <0.01) The expression of IKKβ, NF-κB and phosphorylated IKKβ protein were significantly down-regulated in the hepatocytes in the drug-treated group (P <0.05 or P <0.01). The highest down-regulation was observed in the high-dose Jianpi group and the high-dose Hefei formula group. CONCLUSION: Inhibition of IKKβ / NF-κB signaling pathway-related genes and protein expression in hepatocytes may be one of the pharmacological mechanisms of Shuganjianpi Decoction in preventing and treating NASH. The above proteins may be effective targets of its drug action. Shuganjianpi Fang intervention in NASH may have some dose-effect relationship.