基于PERK/eIF2α/ATF4/CHOP信号通路探讨银杏内酯B治疗代谢性脂肪性肝病的作用

Exploration of the Effect of Ginkgolide B for Metabolic Associated Fatty Liver Disease Based on PERK/eIF2α/ATF4/CHOP Signaling Pathway

目的:通过动物实验验证银杏内酯B(GB)治疗代谢性脂肪性肝病(MAFLD)的作用是否与调控PERK/eIF2α/ATF4/CHOP信号通路,抑制内质网应激,减少肝细胞凋亡及炎症反应相关。方法:72只雄性SD大鼠随机分为正常组,模型组,辛伐他汀组,GB低、中、高剂量(GB-L、GB-M、GB-H)组(n=12),高脂高糖饲料喂养12周构建MAFLD模型(正常组除外)。治疗8周后,收集血清和肝组织。生化试剂盒检测血脂和肝脂[总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)]及肝功能[天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)];酶联免疫吸附法(ELISA)法检测血清和肝脏中的炎性因子[白细胞介素-6(IL-6)、白细胞介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)]水平;HE染色、油红O染色观察肝组织病理学;Western blot法检测肝脏组织中GRP78、PERK、p-eIF2α、ATF4、CHOP、Bcl-2和Bax的蛋白表达。结果:肝脏组织病理学显示,与正常组相比,模型组大鼠肝脏脂肪变性显著,与模型组相比,各治疗组脂肪变性情况有明显改善;生化指标和蛋白表达显示,与正常组相比,模型组大鼠肝脏及血清TC、TG、LDL-C、IL-1、IL-6、TNF-α,血清AST、ALT及肝脏GRP78、PERK、p-eIF2α、ATF4、CHOP、Bax水平显著升高(P<0.05),血清、肝脏HDL-C及肝脏Bcl-2水平显著降低(P<0.05);与模型组相比,各治疗组大鼠血清和肝脏TC、TG、LDL-C、IL-1、IL-6、TNF-α,血清ALT、AST及肝脏GRP78、PERK、p-eIF2α、ATF4、CHOP、Bax水平显著降低(P<0.05),肝脏Bcl-2水平显著升高(P<0.05),与模型组相比,GB-H组大鼠血清和肝脏HDL-C水平明显升高(P<0.05),GB-L组和GB-M组HDL-C水平呈上调趋势,但这2组相较于模型组,差异无统计学意义(P>0.05)。结论:GB对MAFLD具有治疗作用,其作用机制可能与调节PERK/eIF2α/ATF4/CHOP信号通路抑制内质网应激,缓解肝细胞凋亡及炎症反应有关。

Objective:The purpose of this study is to verify whether the mechanism of ginkgolide B(GB)in the treatment of metabolic associated fatty liver disease(MAFLD)is related to the regulation of the PERK/eIF2α/ATF4/CHOP signaling pathway to inhibit endoplasmic reticulum stress and alleviate hepatocyte apoptosis and inflammation through animal experiments.Methods:A total of 72 male SD rats were randomly divided into normal group,model group,Simvastatin group and GB low-,middle-and highdose(GB-L,GB-M and GB-H)groups(n=12).Except for the normal group,the other groups were fed a high-fat high-sugar diet to establish animal models for 12 weeks.After 8 weeks,serum and liver tissue were collected.Biochemical kit was used to detect blood lipids,liver lipids,[total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)]and liver function[aspartate aminotransferase(AST),alanine aminotransferase(ALT)].Detect serum and liver inflammatory factors[interleukin-1(IL-1),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)]levels by enzyme-linked immunosorbent assay(ELISA).Observation of liver histopathology by HE and Oil red O staining.Detect protein expressions of GRP78,PERK,p-eIF2α,ATF4,CHOP,Bcl-2 and Bax in liver tissue by Western blot.Results:Liver histopathology showed that hepatic steatosis in the model group were significantly higher than those in the normal group,and the condition were especially improved in all treatment groups.Biochemical indexes and protein expression showed that compared with the normal group,the levels of TC,TG,LDL-C,IL-1,IL-6 and TNF-αin liver and serum,AST and ALT in serum and GRP78,PERK,p-eIF2α,ATF4,CHOP and Bax in liver in model group were remarkably increased(P<0.05),the levels of HDL-C in liver and serum and liver Bcl-2 were significantly decreased(P<0.05).Compared with the model group,the levels of TC,TG,LDL-C,IL-1,IL-6 and TNF-αin serum and liver,ALT and AST in serum and GRP78,PERK,p-eIF2α,ATF4,CHOP and Bax in the liver in each treatment group were significantly decreased(P<0.05),while the level of Bcl-2 in liver was significantly increased(P<0.05).Compared with the model group,HDL-C levels in serum and liver of GB-H group were significantly increased(P<0.05).HDL-C levels in GB-L and GB-M groups showed an up-regulated trend,but there was no statistical difference between the two groups and model group(P>0.05).Conclusions:GB has a therapeutic effect on MAFLD.Its mechanism may be related to the regulation of the PERK/eIF2α/ATF4/CHOP signaling pathway to inhibit endoplasmic reticulum stress and alleviate hepatocyte apoptosis and inflammation.