五灵胶囊调节TGF-β/Smad信号通路蛋白对胶原Ⅰ、Ⅲ型表达的影响

Effects of TGF-β/Smad signal pathway regulated by wu-ling capsules on the expression of collagen Ⅰ and collagen Ⅲ

目的探讨五灵胶囊(WL)调节肝纤维化大鼠肝组织及星状细胞(HSC)TGF-β/Smads信号转导蛋白对胶原Ⅰ和Ⅲ型(COL-Ⅰ、COL-Ⅲ)表达的影响。方法SD大鼠以高脂低蛋白饲养、饮用10%乙醇,皮下注射40%CCl4,隔4天注射1次,连续6周制备肝纤维化模型,灌胃给予WL3.86g/kg和秋水仙碱0.1mg/kg治疗1月;另体外分离HSC,以不同剂量的WL与HSC培养24h,最后3h加入TGF-β1 10ng/ml。实验结束后镜检肝细胞变性和肝组织纤维化程度,同时ELISA法测定血清COL-Ⅰ、COL-Ⅲ和培养液COL-Ⅰ含量,RT-PCR检测肝组织COL-Ⅰ、COL-Ⅲ、TGF-β1mRNA的表达。Western blot检测肝组织及HSC内COL-Ⅰ、ProCOL-Ⅰ、LN、α-SMA、ERK、p-ERK、TβRⅠ、TβRⅡ、Smad2/3、Smad7蛋白表达。结果WL组大鼠肝细胞变性和肝纤维化程度明显减轻,血清COL-Ⅰ、COL-Ⅲ含量增加,COL-Ⅰ、COL-ⅢmRNA表达降低。Western blot结果显示,WL能明显下调肝组织α-SMA、LN、p-ERK、TβRⅠ、COL-Ⅰ、ProCOL-Ⅰ蛋白表达,对ERK、TβRⅡ、Smad2/3、Smad7蛋白表达无影响;显著下调HSCα-SMA、TβRⅡ、p-ERK、Smad7表达,呈浓度依赖性抑制HSC分泌COL-Ⅰ。结论WL抑制肝纤维化大鼠肝组织COL-Ⅰ、COL-Ⅲ合成并增加COL-Ⅰ降解,其作用位点是下调肝组织和HSC TGF-β/Smad、Ras/ERK信号通路蛋白p-ERK、TβRⅡ表达。

Objective To investigate the effects of wu-ling capsules （WL） on the TGF-β/Smad signal pathway, which in turn regulated the expression level of COL I and COL-Ⅲ in the tissues and hepatic stellate cells （HSC） from the hepatic fi brotic rats. Methods SD rats were fed with 10% ethanol and food with high-fat and low-protein, and were injected subcutaneously with carbontetrachloride once every four days for 6 weeks to establish hepatic fibrotic rats models. Then, the rats were treated with WI. 3.86 g/kg and colchicine 0.1 mg/kg intragastrically. HSC were separated in vitro, incubated with WI. at different dosage for 24 hours, and TGF-β was added to the medium at the 21 hours. The degree of hepatocyte denaturalization and hepatic fibrosis was evaluated by microscope. The level of COL- I and COL- Ⅲ in serum and the level of COL- I in medium were assessed by EIASA. The mRNA expression level of COL- I , COL-Ⅲ , TGF-β1 in hepatic tissues was assessed by RT-PCR. The protein expression level of COL I, ProCOL-I , LN, α-SMA, ERK, p-ERK, TβR I, TβRII , Smad2/3 and Smad7 in hepatic tissues and HSC was assessed by Western blot. Results In the WE treated group, the degree of hepatocyte denaturalization and hepatic fibrosis decreased significantly, the content of COL- I and COL-Ⅲ in serum increased notably, and the mRNA expression level of COL- I and COL-Ⅲ in the hepatic tissues decreased. The results of Western-blot showed that WL could down-regulate the protein expression level of α-SMA, LN, p-ERK, TβR I , COL-I and ProCOL-I , while it had no effects on the protein expression level of ERK, TβRⅡ , Smad2/3 and Smad7. WL also could down-regulate the expression level of α-SMA, TβR Ⅲ, p-ERK and Smad7 in HSC, and inhibit the secretion of COL- I by HSC in a concentration-dependent manner. Conclusions WL could inhibit the synthesis of COL- I and COL-Ⅲ, and promote the degradation of COL-I through down-regulating the expression level of p ERK and TβRⅢ in hepatic tissues and HSC.