基于Notch信号通路研究银杏内酯B对非酒精性脂肪肝病的影响

Effect of Ginkgolide B on Non-Alcoholic Fatty Liver Disease based on Notch Signaling Pathway

目的:基于Notch信号通路探讨银杏内酯B对非酒精性脂肪肝病(NAFLD)的干预作用,为银杏内酯B治疗NAFLD提供参考。方法:72只SD大鼠按照随机数字表法分为正常组和实验组,分别为12、60只。正常组采用普通饲料喂养12周,实验组采用高脂高糖饲料(83.25%基础饲料、10%猪油、5%蔗糖、1.5%胆固醇和0.25%胆酸钠)喂养12周,构建NAFLD动物模型(造模成功标准为:肝脏HE染色有明显大量脂肪空泡)。造模成功后,大鼠按随机数字表法分为模型组,辛伐他汀组,银杏内酯B低、中、高剂量组,每组10只。正常组、模型组均给予1 mL/d生理盐水灌胃;辛伐他汀组按2 mg/(kg·d)剂量给予辛伐他汀灌胃;低、中、高剂量组分别按0.5、1.0、2.0 mg/(kg·d)剂量给予98%银杏内酯B灌胃。干预4周后,比较血浆中谷草转氨酶(AST)、谷丙转氨酶(ALT)、血脂低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)指标的差异;比较肝脏和血浆中转化生长因子-β1(TGF-β1)以及肿瘤坏死因子-α(TNF-α)含量的差异;采用HE染色方法观察肝脏组织病理学变化;分别采用实时荧光定量PCR法、Western blot法检测Notch-3、Hes-1蛋白及mRNA的表达情况。结果:(1)生化指标结果:与正常组比较,模型组AST、ALT、LDL-C、HDL-C、TGF-β1和TNF-α明显升高(P<0.05);与模型组比较,辛伐他汀组、低剂量组、中剂量组和高剂量组AST、ALT、LDLC、TGF-β1、TNF-α均明显降低(P<0.05),而HDL-C无明显差异(P>0.05);与辛伐他汀组比较,低、中、高剂量组AST、ALT、LDL-C、HDL-C均无明显差异(P>0.05),TGF-β1和TNF-α明显降低(P<0.05)。(2)组织病理学结果:正常组几乎没有脂肪空泡出现,而模型组则出现有大量脂肪空泡;与模型组比较,辛伐他汀组,高、中、低剂量组脂肪空泡数量明显降低(P<0.05)。(3)Notch-3、Hes-1蛋白及mRNA表达结果:与正常组比较,模型组Notch-3和Hes-1蛋白及mRNA表达明显升高(P<0.05);与模型组比较,辛伐他汀组,低、中、高剂量组Notch-3、Hes-1蛋白及mRNA表达明显降低(P<0.05);与辛伐他汀组比较,中、高剂量组Notch-3、Hes-1蛋白及mRNA表达明显降低(P<0.05)。结论:银杏内酯B可以改善NAFLD大鼠肝功能,降低血脂水平,减轻炎症反应,其作用机制与调节Notch信号通路,抑制肝脏组织中Notch-3、Hes-1蛋白及mRNA表达有关。

Objective:To explore the interventional effect of Ginkgolide B on non-alcoholic fatty liver disease(NAFLD)based on the Notch signal pathway,and provide basis for Ginkgolide B in treating NAFLD.Methods:A total of 72 SD rats were divided into the normal group and the experiment group according to the random number method,with 12 cases in the normal group and 60 cases in the experiment group.The normal group were fed with normal fodder for 12 weeks,and the experiment group were fed with high-fat and high-sugar fodder(83.25%basic feed,10%lard,5%sucrose,1.5%cholesterol and 0.25%sodium cholate)for 12 weeks to construct NAFLD animal model(The success criterion for modeling is that:there are a large number of fat vacuoles in the liver).After successful modeling,the experiment group were randomly divided into the model group,the simvastatin group,the ginkgolide B low,medium and high dose group,with 10 cases in each group.The normal group and the model group were given 1 mL/d normal saline by gavage;the simvastatin group was given 2 mg/(kg·d)simvastatin by gavage;the low,medium and high doses group were given 98%ginkgolide B by gavage at doses of 0.5,1.0,2.0 mg/(kg·d)respectively.After intervention for four weeks,some indexs such as aspartate aminotransferase(AST),alanine aminotransferase(ALT),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were compared;the differences of transforming growth factor-β1(TGF-β1)and tumor necrosis factor-α(TNF-α)in liver and plasma were compared;HE staining method was used to observe liver histopathological changes;Real-time fluorescent quantitative PCR method and Western blot method were used to detect the protein and mRNA expression of Notch-3,Hes-1.Results:①The results of biochemical indicators:compared with the normal group,AST,ALT,LDL-C,HDL-C,TGF-β1 and TNF-αin the model group increased significantly(P<0.05);compared with the model group,AST,ALT,LDL-C,TGF-β1,TNF-αof the simvastatin group,low-dose,middle-dose and high-dose group decreased significantly(P<0.05),while there was no significant difference in HDL-C(P>0.05);compared with the simvastatin group,there were no significant difference in AST,ALT,LDL-C,HDL-C of the low,medium and high dose group(P>0.05),while TGF-β1 and TNF-αof the low,medium and high dose group decreased significantly(P<0.05).②Histopathological results:there were almost no fat vacuoles in the normal group,while a large number of fat vacuoles appeared in the model group.Compared with the model group,the number of fat vacuoles in the simvastatin group,high,medium,and low dose group decreased significantly(P<0.05).③The protein and mRNA expression of Notch-3 and Hes-1:compared with the normal group,the protein and mRNA expression of Notch-3 and Hes-1 in the model group increased significantly(P<0.05);compared with the model group,the protein and mRNA expression of Notch-3,Hes-1 in the simvastatin group,low and medium high-dose group decreased significantly(P<0.05);compared with the simvastatin group,the protein and mRNA expression of Notch-3,Hes-1 of the medium and high-dose group decreased significantly(P<0.05).Conclusion:Ginkgolide B can improve the liver function,reduce blood lipids,and reduce inflammation of NAFLD rats.Its mechanism may be that Ginkgolide B could regulate the Notch signaling pathway by inhibiting Notch-3,Hes-1 protein and mRNA expression in liver tissue.