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
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摘要: 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（100 ng/mL）和D-GalN（44 μg/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。Abstract: 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 injury model was induced by TNF- α (100 ng/mL) and D-GalN(44 μg/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， Beclin1， LC3 Ⅰ， LC3 Ⅱ; changes of mTOR protein expression level of apoptosis 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， Beclin1， LC3 Ⅱof 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 mTOR protein showed down-regulated with dose - response.CONCLUSION The protective effects of Liangxue Huayu Fang on hepatocyte injury induced by TNF-α and D-GalN 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.
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Key words:
- Liangxue Huayu Fang /
- hepatocyte /
- apoptosis /
- autophagy
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