清肝活血方及其拆方抗酒精性肝损伤大鼠肝细胞内质网应激性凋亡的作用及机制

Attenuation and Mechanism of Endoplasmic Reticulum Stress-mediated Hepatocyte Apoptosis in Rats with Alcohol-induced Liver Injury by Qinggan Huoxue Recipe and Its Disassembled Formulas

目的研究中药复方清肝活血方及其拆方抗酒精性肝损伤大鼠肝细胞内质网应激(endoplasmic reticulum stress,ERS)性凋亡的作用及机制。方法利用酒精-玉米油-吡唑复合试剂制备大鼠慢性酒精性肝损伤模型。将造模大鼠随机分为模型组、清肝活血方组、清肝方组及活血方组,另设CCl4对照组和正常对照组,每组10只。造模各组大鼠每天上午以酒精复合试剂灌胃,下午各中药组分别给予清肝活血方9.5g/(kg·d)、清肝方3.0g/(kg·d)、活血方6.5g/(kg·d)灌胃,模型组给予等体积生理盐水灌胃;CCl4组每周1次腹腔注射CCl40.3mL/kg,正常对照组给予生理盐水灌胃,连续治疗2周。组织病理学观察肝脏病理变化;ELISA法检测血清总同型半胱氨酸(total homocysteine,tHCY)水平;流式细胞术检测肝细胞凋亡率;RT-PCR和Westernblot法检测大鼠肝脏ERS凋亡相关因子真核生物翻译起始因子(eukaryotic translation initia-tion factor2alpha,eIF-2α)、磷酸化eIF-2α(p-eIF-2α)、葡萄糖调节蛋白78(glucose-regulated protein78,GPR78)、Caspase-3基因和蛋白表达。结果与正常对照组比较,模型大鼠出现了典型的慢性酒精性肝损伤病理改变如脂肪变性、炎症甚至纤维化;肝细胞凋亡明显增加,凋亡率达到了正常大鼠的5倍以上,且以早期凋亡为主;血清tHCY水平显著升高;p-eIF-2α、GRP78及Caspase-3蛋白表达明显增高(P<0.01);GRP78及Caspase-3mRNA表达量显著增高(P<0.01,P<0.05)。与模型组比较,清肝活血方及拆方组大鼠肝损伤及肝细胞凋亡程度明显减轻,血清tHCY水平显著降低,p-eIF-2α、GRP78及Caspase-3蛋白表达明显降低(P<0.01);清肝活血方组GRP78和Caspase-3mRNA表达明显降低(P<0.01,P<0.05);清肝方组仅GRP78mRNA表达明显降低(P<0.05)。结论清肝活血方及其拆方可能通过降低血清tHCY水平及肝脏ERS凋亡相关因子的表达抑制酒精性肝损伤动物肝细胞ERS性凋亡。

Objective To explore attenuation and mechanism of endoplasmic reticulum stress （ERS）- mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by Qinggan Huoxue Recipe （QGHXR） and its disassembled formulas （Qinggan Recipe and Huoxue Recipe respectively）. Methods A rat model of chronic alcoholic liver injury was successfully established using a compound reagent of alcohol, corn oil, and pyrazol. The modeled rats were randomly divided into the model group, the QGHXR group, the Qinggan Recipe （QGR） group, and the Huoxue Recipe group （HXR）. The CCI4 control group and the normal control group were also set up. There were ten rats in each group. All rats of modeled groups were gastrogavaged with alcohol compound re- agent every morning. Rats in the QGHXR group （ at the daily dose of 9.5 g/kg, QGR group （at the daily dose of 3. 0 g/kg）, and HXR group （at the daily dose of 6.5 g/kg） were administered with corresponding medicines by gastrogavage every afternoon. Equal volume of normal saline was given to rats of the model group by gastrogavage. CCl4 was intraperitoneally injected at the dose of 0.3 mL/kg to rats in the CCl4 control group, once per week. Normal saline was given to rats in the normal control group by gastrogavage. The treatment was lasted for two weeks. Pathological changes of the liver were observed by histopathology. Serum total homocysteine （tH- CY） level was detected by ELISA. The hepatocyte apoptosis rate was detected using flow cytometry. The gene and protein expressions of eukaryotic translation initiation factor 2 alpha （elF-2α）, phosphorylation elF-2α （p- elF-2α）, glucose-regulated protein 78 （GRP78）, and Caspase-3 in the liver were examined using Real-time PCR and Western blot respectively. Results Compared with the normal control group, typical pathological chan- ges of chronic alcoholic liver injury such as steatosis, inflammation, and even fibrosis occurred in model rats. The hepatocyte apoptosis obviously increased, with the apoptosis rate reaching the five-fold of that in normal rats. Be- sides, early apoptosis dominated. The serum tHCY level significantly increased. The expressions of p-elF-2α, GRP78, and Caspase-3 protein obviously increased （P〈0.01）. Expressions of GRP78 and Caspase-3 mRNA significantly increased （ P〈0.05, P〈0. 01 ）. Compared with the model group, the degrees of the liver injury and the hepatocyte apoptosis in the QGHXR group, the QGR group, and the HXR group were significantly alleviated. The serum tHCY level was significantly lowered. The protein expressions of p-elF-2α, GRP78, and Caspase-3 obviously decreased （P 〈 0.01 ）. mRNA expressions of GRP78 and Caspase-3 obviously decreased in the QGHXR group （P〈0.05, P 〈0.01 ）. Only GRP78 mRNA expression obviously decreased in the QGR group （P 〈0.05）. Cenclusien QGHXR and its disassembled formulas could attenuate ERS-mediated hepatocyte ap- optosis in alcohol-induced liver injury rats by lowering the serum tHCY level and expressions of ERS apoptosis correlated factors.