中文摘要：

目的研究凉血化瘀方对肿瘤坏死因子α（TNF—α）联合I）-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L02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；Westernblot检测凋亡和自噬标志蛋白caspase-3、Beclinl、LC3I、LC3Ⅱ，凋亡和自噬调节蛋白Bax、Bel-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、Beelinl、LC3lI表达下调，凋亡和自噬调控蛋白中负相关的Bcl一2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。

英文摘要：

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.