凉血化瘀方对肝细胞损伤中自噬与凋亡相关蛋白的影响

Discussion on the Effects of Liangxue Huayu Fang on Autophagy and Apoptosis of Protein Associated Hepatocyte Injury and Its Molecular Mechanism

目的研究凉血化瘀方对肿瘤坏死因子α(TNF-α)联合D-氨基半乳糖(D-GalN)所致肝细胞损伤中自噬和凋亡相关途径的影响,并探讨其分子调控机制。方法体外培养人正常肝LO2细胞,用TNF-α(100ng/mL)和D-GalN(44μg/mL)诱导肝细胞损伤模型,荧光显微镜观察细胞的形态学变化;MTT法检测细胞活力;Western blot检测凋亡和自噬标志蛋白caspase-3、Beclin1、LC3Ⅰ、LC3Ⅱ,凋亡和自噬调节蛋白Bax、Bcl-2和mTOR信号通路调控途径mTOR蛋白表达水平的变化。结果凉血化瘀方能改善肝细胞病理损伤,并表现有剂量依赖性;治疗组细胞凋亡和自噬标志蛋白caspase-3、Beclin1、LC3Ⅱ表达下调,凋亡和自噬调控蛋白中负相关的Bcl-2表达上调、正相关的Bax表达下调,并呈现一定的量效关系,同时mTOR蛋白的表达也体现了剂量相关的下调作用。结论凉血化瘀方对TNF-α+D-GalN所致肝细胞损伤的保护作用,与抑制细胞自噬和凋亡相关,其机制主要是参与自噬和凋亡相关途径中蛋白的表达调控。

OBJECTIVE To study the effects of Liangxue Huayu Fang on pathways related to autophagy and apoptosis of hepatocyte injury induced by tumor necrosis factor alpha （TNF-α） combined with D- galactosamine（D-GalN）, and to explore the molecular regulative mechanism. METHODS The normal human liver cells LO2 were cultured in vitro. The hepatocyte in- jury model was induced by TNF- α （100 ng/mL） and D-GaIN（44μg/mL）. Fluorescence microscope was used to observe the morphological changes of cells. The cell viability was detected by MTT; Western blot was adopted to detect the apoptosis and autophagy marker, such as proteins caspase-3, Beclinl, LC3 I , LC3 Ⅱ ; changes of mTOR protein expression level of apop- tosis and autophagy regulating protein Bax, Bcl-2 and mTOR signal pathway were also detected. RESULTS Liangxue Huayu Fang can improve pathological injury of hepatocyte with a dose-dependence; cell apoptosis was decreased and the expression of autophagy markers, such as protein caspase-3, Beclinl, LC3Ⅱ of the treatment group showed decreased and the expression of negative correlative Bcl-2 protein was up-regulated while the positive correlative Bax expression was down-regulated. All the data showed a dose - response relationship to some degree and the expression of roTOR protein showed down-regulated with dose - response. CONCLUSION The protective effects of Liangxue Huayu Fang on hepatocyte injury induced by TNF-α and D -GAIN may be related to inhibition of autophagy and apoptosis. Its main mechanism may relate to regulation of the expression of proteins participating in autophagy and apoptosis.