扶正化瘀方影响转化生长因子β1/Smad信号通路的抗肝纤维化作用机制

Chinese herbal medicine Fuzheng Huayu recipe inhibits liver fibrosis by mediating the transforming growth factor-β1/Smads signaling pathway

目的:探讨扶正化瘀方影响转化生长因子β1(transforming growth factor-β1,TGF-β1)/Smad信号转导的抗肝纤维化作用机制。方法:本研究分体内实验和体外实验两部分。在体内实验中,37只雄性Wistar大鼠分为正常组(5只)、模型组(18只)和扶正化瘀方组(14只)。模型组和扶正化瘀组大鼠采用二甲基亚硝胺(dimethylnitrosamine,DMN)腹腔注射复制大鼠肝纤维化模型。灌胃治疗4周后,采用盐酸水解法检测肝组织中羟脯氨酸(hydroxyproline,Hyp)含量,蛋白质印迹法分析肝组织中TGF-β1、TGF-β1Ⅰ型受体(TGF-β1 type Ⅰ receptor,TβR-Ⅰ)、Smad2、Smad3及磷酸化Smad2/3蛋白的表达。体外实验采用培养4d的原代大鼠肝星状细胞(hepatic stellate cell,HSC),分为对照组、TGF-β1组、10%扶正化瘀方药物血清组及TβR-Ⅰ胞内激酶抑制剂(SB-431542)组。后3组用2.5ng/mLTGF-β1刺激24h后,对照组及TGF-β1组加10%正常大鼠血清,10%扶正化瘀方药物血清组加10%服用扶正化瘀方的大鼠血清,SB-431542组加10%正常大鼠血清及10μmol/LSB-431542。共孵育24h后,免疫荧光染色检测HSC中α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和Smad3蛋白的表达,蛋白质印迹法分析HSC中α-SMA蛋白的表达。结果:体内实验发现,扶正化瘀方可明显降低纤维化肝组织中异常升高的Hyp含量及TGF-β1、TβR-Ⅰ、磷酸化Smad2/3蛋白的表达。体外研究发现,正常HSC中α-SMA、TβR-Ⅰ表达较低,TGF-β1刺激可显著上调其表达;扶正化瘀方药物血清共孵育后,可明显抑制TGF-β1诱导的HSC中α-SMA的表达;正常HSCSmad3主要表达在细胞质中,细胞核中表达较低,TGF-β1可显著刺激Smad3向细胞核转移,扶正化瘀方药物血清可明显抑制TGF-β1诱导的Smad3核转位。结论:扶正化瘀方可下调纤维化大鼠肝脏及HSC中的TGF-β1/Smad病理信号转导通路,这可能是扶正化瘀方抗肝纤维化的部分作用机制。

OBJECTIVE： To investigate the mechanism of Fuzheng Huayu recipe （FZHY）, a compound traditional Chinese herbal medicine, against liver fibrosis related to transforming growth factor-β1 （TGF-β1）/Smads signaling transduction. METHODS： The research consisted of in vitro and in vivo experiments. In the in vivo experiment, 37 male Wistar rats were divided into 3 groups： 5 rats in normal group, 18 and 14 rats respectively in model and FZHY groups. Liver fibrosis was induced in rats of the model group and the FZHY group by intraperitoneal injection of dimethylnitrosamine with a dose of 10 μg/kg body weight for 4 weeks. Rats in the FZHY group were administered with FZHY for 4 weeks after liver fibrosis was induced. After the treatment of FZHY, hydroxyproline （Hyp） content in rat liver tissue was assayed by Jamall＇s method and protein expressions of TGF-β1, TGF-β1 receptor I （TμR-I）, Smad2, Smad3 and phosphorylated-Smad2/3 were analyzed by Western blotting. In the in vitro experiment, hepatic stellate cells （HSCs） were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. On the 4th day of cell culture, HSCs were stimulated by 2.5 ng/mL TGF-β1 for 24 h, then incubated with the medium containing 10% FZHY-medicated serum or 10 μmol/L SB-431542 （a potent and specific inhibitor of TGF-β1 receptor I kinase） for 24 h. And the HSCs without TGF-β1 stimulating were used as control group. Protein expressions and location of e-smooth muscle actin （a-SMA） and Smad3 in HSCs were assayed by immunofluorescent staining, and the image was analyzed by Image-Pro Plus 6.1 System. RESULTS： In the in vivo experiment, liver Hyp content in the FZHY group was reduced significantly compared with the model group. FZHY also down-regulated the protein expressions of TGF-β1, Trf- β1 and p-Smad2/3 in fibrotic liver tissue. In the in vitro experiment, FZHY-medicated serum incubated with TGF-β1- stimulated HSCs significantly down-regulated the protein expression of a-SMA. It also inhibited Smad3 nuclear translocation in TGF-β1-stimulated HSCs. CONCLUSION： The mechanism of FZHY against liver fibrosis is related to the regulation of TGF-β1signaling transduction pathway by inhibition of TGF-β1 and TβR-I expressions and Smads activation in fibrotic liver tissue and HSCs.