大鼠肝纤维化与microRNA-181a调控肝星状细胞自噬的关系

Relationship between rat hepatic fibrosis and regulation of hepatic stel⁃late cell autophagy by microRNA-181a

目的:观察四氯化碳(CCl4)诱导肝纤维化(HF)大鼠肝脏结构的改变、肝组织中转化生长因子β1(TGF-β1)、微小RNA-181a(microRNA-181a)、自噬标志性蛋白LC3-Ⅱ/-Ⅰ和beclin-1水平及胶原沉积的变化,以及microRNA-181a对TGF-β1诱导大鼠肝星状细胞(HSCs)自噬的作用,探讨microRNA-181a调控HSCs活化及HF的可能机制。方法:(1)40只健康雄性Wistar大鼠随机分为空白对照(control)组和CCl4诱导HF模型2周(W2)、4周(W4)、6周(W6)及8周(W8)组,每组8只。control组给予皮下注射橄榄油3 mL/kg;W2、W4、W6及W8组给予皮下注射40%CCl4(4∶6混合橄榄油)3 mL/kg,每周2次,分别连续作用2、4、6及8周;Masson染色评估大鼠HF改变;ELISA法检测大鼠血清及肝组织中TGF-β1的水平;RT-qPCR检测肝组织中microRNA-181a的表达;Western blot检测肝组织中LC3-Ⅱ/-Ⅰ、beclin-1、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col Ⅰ)和Ⅲ型胶原(ColⅢ)的蛋白水平;(2)microRNA-181a inhibitor转染大鼠HSC-T6细胞,或采用自噬抑制剂3-甲基腺嘌呤(3-MA)预处理细胞后,以TGF-β1诱导细胞自噬,RT-qPCR和Western blot分别检测HSC-T6细胞中microRNA-181a的表达及LC3-Ⅱ/-Ⅰ、beclin-1、α-SMA、Col Ⅰ和ColⅢ的蛋白水平。结果:(1)大鼠肝组织中TGF-β1和micro RNA-181a表达、自噬标志性蛋白LC3-Ⅱ/-Ⅰ比值和beclin-1水平均随HF程度加重而呈上升趋势,microRNA-181a表达水平与细胞自噬呈正相关(P<0.01);(2)体外TGF-β1诱导HSC-T6细胞自噬及活化时伴有microRNA-181a表达显著上调;转染microRNA-181a inhibitor后HSC-T6细胞中microRNA-181a表达显著下降,细胞自噬及活化受到显著抑制(P<0.01),这一结果与3-MA作用相似。结论:CCl4呈时间依赖性地促进大鼠HF,上调肝组织microRNA-181a表达及自噬水平;降低HSC-T6细胞microRNA-181a表达可抑制TGF-β1诱导的细胞自噬;大鼠HF与microRNA-181a调控HSCs自噬有关。

AIM:To observe the changes of liver structure,the levels of transforming growth factor-β1(TGF-β1),microRNA-181 a,LC3-Ⅱ/-Ⅰ,beclin-1 and collagen deposition in hepatic fibrosis(HF)rats induced by carbon tetrachloride(CCl4),and the effect of microRNA-181 a on autophagy of rat hepatic stellate cells(HSCs)induced by TGF-β1,and to explore the possible mechanism of microRNA-181 a in regulating HSC activation and HF.METHODS:Wistar rats(n=40)were randomly divided into 5 groups(with 8 in each):control group(subcutaneous injection of olive oil,3 mL/kg,twice a week),and CCl4-induced HF groups of 2,4,6 and 8 weeks(subcutaneous injection of 40% CCl4,3 mL/kg,twice a week for 2,4,6 and 8 weeks,respectively).Masson staining was used to evaluate the changes of HF in rats.The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA.The level of microRNA-181 a in rat liver tissues was detected by RT-qPCR.The protein levels of LC3-Ⅱ/-Ⅰ,beclin-1,α-smooth muscle actin(α-SMA),collagen type I(Col I)and collagen type Ⅲ(Col Ⅲ)in rat liver tissues were measured by Western blot.HSC-T6 cells were transfected with microRNA-181 a inhibitor,or pretreated with the autophagy inhibitor 3-methyladenine(3-MA),before treatment with TGF-β1 to stimulate autophagy.The expression of microRNA-181 a,LC3-Ⅱ/-Ⅰ,beclin-1,α-SMA,Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot.RESULTS:The levels of TGF-β1,microRNA-181 a,LC3-Ⅱ/-Ⅰ ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF,and microRNA-181 a expression showed a positive correlation with autophagy-associated proteins(P<0.01).MicroRNA-181 a level was significantly increased,which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181 a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181 a inhibitor,along with suppression of autophagy and cell activation(P<0.01),which were similar to the effects of 3-MA treatment.CONCLUSION:CCl4 promotes rat HF,the microRNA-181 a expression of liver tissue,and autophagy in a timedependent manner.Reducing the expression of microRNA-181 a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1.The regulation of HSC autophagy by microRNA-181 a may be involved in rat HF.