糜酶对肝星状细胞转化生长因子β1/Smad3信号通路的影响

Effect of chymase on transforming growth factor β1/Smad3 signaling pathway in hepatic stellate cells in vitro

目的研究糜酶对肝星状细胞转化生长因子β1(TGF-β1)/Smad3信号通路的影响。方法对大鼠肝星状细胞株HSC-T6进行体外培养,设定2组,空白对照组(加入等量磷酸盐缓冲液)及糜酶处理组(加入终浓度为10mg/L的糜酶),作用24h后收集细胞培养上清液,提取全细胞蛋白,收集细胞爬片。TGF-β1在培养上清液中的表达采用免疫印迹法及ELISA法检测,Smad3、p-Smad2/3、纤维连接蛋白(Fn)的表达采用免疫印迹法检测。结果免疫印迹法及ELISA检侧结果均表明在糜酶处理组培养上清液中TGF-β1蛋白表达量均较空白对照组高[免疫印迹法:(0.33±0.05)vs(0.18±0.03),t=3.826,P<0.05;ELISA法:(178.05±34.63)ng/L vs(138.42±20.17)ng/L,t=3.401,P<0.05],差异均有统计学意义;而糜酶处理组与空白对照组在培养上清液中Smad3相对表达量比较差异无统计学意义[(0.65±0.06)vs(0.62±0.05),t=0.086,P=0.891>0.05];p-Smad2/3蛋白相对表达量、p-Smad2/3与Smad3蛋白相对表达量的比值则明显高于空白对照组,差异均有统计学意义[(0.72±0.04)vs(0.45±0.03),t=8.101,P=0.001;(1.11±0.13)vs(0.70±0.04),t=5.210,P=0.006<0.05];糜酶处理组Fn的表达量较空白对照组明显升高,差异有统计学意义[(0.56±0.04)vs(0.34±0.02),t=6.141,P<0.05]。结论对体外培养的HSC-T6,糜酶可促进其产生TGF-β1,并使其中的Smad3的磷酸化增加。在糜酶作用下HSC-T6的细胞外基质成分Fn分泌明显增加。

Objective To study the effect of chymase on transforming growth factorβ1（TGF-β1）/Smad3 signaling pathway in hepatic stellate cells. Methods Protein expression were measured in 2groups of HSC-T6 cell samples,and cells untreated or treated with chymase（10mg/L）.Expression of TGF-β1protein was detected by Western blot and ELISA,Smad3,p-Smad2/3and fibronectin（Fn）expression were measured by Western blot. Results The immunoblotting and ELISA analyses showed that the expression of TGF-β1in chymase-treated group was significantly higher than that in control group [（0.33±0.05）vs（0.18±0.03）,（178.05±34.63）ng/Lvs（138.42±20.17）ng/L],（t=3.826,P〈0.05;t=3.401,P〈0.05）,respectively].There was no significant difference in the expression of Smad3 between chymase and control groups[（0.65±0.06）vs（0.62±0.05）,t=0.086,P=0.891,P〉0.05].The expression of p-Smad2/3and the ratio of p-Smad2/3and Smad3 in chymase group were significantly higher than that in control group [（0.72±0.04）vs（0.45±0.03）,t=8.101,P=0.001;（1.11±0.13）vs（0.70±0.04）,t=5.210,P=0.006,P〈0.05）],respectively.The expression of Fn in chymase group was significantly higher than that in control group [（0.56±0.04）vs（0.34±0.02）,t=6.141,P〈0.05]. Conclusions Chymase may promote the production of TGF-β1in HSC-T6 in vitro,and increase the phosphorylation of Smad3.Extracellular matrix components protein Fn regulates chymase secretion significantly increased in hepatic stellate cells.Chymase may be involved in hepatic stellate cells through transforming growth factorβ1（TGF-β1）/Smad3 signaling pathway.