栀子苷通过TGF-β1/Smad信号通路抑制肝纤维化和肝星状细胞活化

Geniposide inhibits hepatic fibrosis and hepatic stellate cell activation through blocking the TGF-β1/Smad signaling pathway

本研究旨在观察栀子苷对肝纤维化和肝星状细胞活化的影响,并探讨其可能机制。人肝星状细胞LX-2用5 ng/mL转化生长因子-β1 (transforming growth factor-β1, TGF-β1)处理,并用不同浓度(0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100μmol/L)的栀子苷联合培养,MTT法进行细胞活力测定,LX-2分别经5 ng/mL TGF-β1、TGF-β1+栀子苷(20μmol/L)处理后,用qPCR和Western blot分别检测I型胶原蛋白(collagen I)、纤连蛋白(fibronectin)、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、p-Smad2和p-Smad3基因和蛋白的表达。BALB/c小鼠用CCl_(4)(25%, 1 mL/kg)诱导肝纤维化,设立对照组、CCl_(4)组和CCl_(4)+栀子苷组(40 mg/kg),4周后检测肝功能及血清肝纤维化指标,用HE和Masson染色进行组织学观察,免疫组织化学分析组织α-SMA表达,Western blot检测α-SMA、TGF-β1、p-Smad2和p-Smad3蛋白的表达。结果显示,栀子苷显著抑制TGF-β1诱导的LX-2细胞增殖、collagen I、fibronectin和α-SMA的基因和蛋白表达及TGF-β1/Smad信号相关蛋白表达。组织学观察显示栀子苷显著抑制CCl_(4)诱导的小鼠肝纤维化,显著抑制肝星状细胞活化和TGF-β1/Smad信号相关蛋白表达。以上结果提示,栀子苷抑制肝纤维化和肝星状细胞活化,其机制可能是通过抑制TGF-β1/Smad信号传导。

The purpose of this study was to investigate the effect of Geniposide on hepatic fibrosis and activation of hepatic stellate cells(HSCs) and to explore possible underlying mechanism. Human HSCs(LX-2) were treated with 5 ng/mL transforming growth factor-β1(TGF-β1), followed by co-culture with Geniposide at various concentrations(0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 μmol/L).Cell viability was determined by MTT assay. Then, LX-2 cells were divided into control, TGF-β1(5 ng/mL) and TGF-β1 + Geniposide(20 μmol/L) groups, and the gene and protein expression of collagen I, fibronectin, α-smooth muscle actin(α-SMA), p-Smad2 and p-Smad3 was detected by qPCR and Western blot, respectively. BALB/c mice were treated with CCl_(4)(25%, 1 mL/kg) to generate a model of hepatic fibrosis(CCl_(4)group), and the control group and CCl_(4)+ Geniposide group were administered with olive oil and CCl_(4)+40 mg/kg Geniposide, respectively. After 4 weeks of treatment, the liver function and serum hepatic fibrosis indexes of mice were detected, histological observation was performed by HE and Masson staining, and α-SMA expression in the tissue was analyzed by immunohistochemistry. Western blot was utilized for the determination of the protein expression of α-SMA, TGF-β1, p-Smad2 and p-Smad3. The results showed that Geniposide inhibited LX-2 cell proliferation. In addition, Geniposide significantly downregulated the gene and protein expression of collagen I, fibronectin and α-SMA and the expression of TGF-β1/Smad signaling-related proteins induced by TGF-β1 in vitro. Histological observations showed that Geniposide significantly inhibited CCl_(4)-induced hepatic fibrosis,HSC activation and expression of TGF-β1/Smad signaling-related proteins in mice. In summary, Geniposide prevents the hepatic fibrosis and HSC activation possibly through the inhibition of the TGF-β1/Smad signaling pathway.