人参皂苷Rg1干预非酒精性脂肪肝模型大鼠肝细胞的凋亡

Ginsenoside Rg1 protects against hepatocyte apoptosis in a rat model of non-alcoholic fatty liver disease

背景:人参皂苷Rg1可以减轻氧化应激并且抑制细胞凋亡,是三七和人参共有的有效活性成分。目的:分析人参皂苷Rg1对非酒精性脂肪性肝病大鼠模型肝细胞凋亡的抑制作用及分子机制。方法:SD大鼠40只,适应性喂养2周后随机分成4组(n=10),分别为空白对照组,模型对照组,人参皂苷Rg1低剂量组,人参皂苷Rg1高剂量组。空白对照组正常饮食,模型对照组高脂高糖饮食22周,人参皂苷Rg1组高脂高糖饮食14周后加用Rg1灌胃治疗8周。用显色法检测血清中氧化应激指标,苏木精-伊红染色观察肝组织病理形态变化,油红染色观察肝组织的脂滴,TUNEL染色观察肝细胞的凋亡情况。通过Q-PCR从m RNA水平检测肝脏组织中Bax、Bcl-2、Caspase-3的表达情况。通过免疫组织化学、Western Blotting从蛋白质水平检测肝脏组织中的凋亡关键因子Bax、Bcl-2、Procaspase-3、Caspase-3 cleavage p17表达情况。结果与结论:①苏木精-伊红染色:与空白对照组比较,模型组肝脏细胞排列紊乱出现大量脂肪空泡并且大量炎性细胞浸润,与模型对照组相比,人参皂苷Rg1组肝细胞排列形态逐渐恢复正常脂肪空泡减少以及炎性细胞浸润减少;②油红染色:与模型对照组比较,人参皂苷Rg1组肝细胞脂滴明显减少,并且随着Rg1的浓度升高而降低;③TUNEL染色:与模型对照组比较,人参皂苷Rg1组的凋亡程度明显降低;④显色法检测:与模型对照组比较,人参皂苷Rg1组与模型组比较,谷胱甘肽、超氧化物歧化酶升高(P <0.05),丙二醛降低(P <0.05);⑤与模型对照组比较,人参皂苷Rg1组Bax、Caspase-3的m RNA表达降低(P <0.05),Bcl-2 mRNA表达升高(P <0.05);⑥与模型对照组比较,人参皂苷Rg1组的Bax,Caspase-3 cleavage p17的蛋白表达降低(P <0.05),Bcl-2、Procaspase-3的蛋白表达升高(P <0.05),存在一定的剂量依赖性; ⑦结果表明,人参皂苷Rg1可能通过减轻氧化应激,抑制非酒精性脂肪肝大鼠模型的肝细胞凋亡,从而缓解非酒精性脂肪肝的进展。

BACKGROUND:Ginsenoside Rg1,the functional and active ingredient of Panax notoginseng and Panax ginseng,can alleviate oxidative stress and inhibit cell apoptosis.OBJECTIVE:To investigate the inhibitory effect of ginsenoside Rg1 on hepatocyte apoptosis in non-alcoholic fatty liver disease and the underlying mechanism.METHODS:Forty Sprague-Dawley rats were randomly assigned into four groups(n=10)after 2-week adaptive feeding,including control group(normal diet),model(22-week high-fat and high-sugar diet),low-and high-dose ginsenoside Rg1 groups.The rats in the ginsenoside Rg1 groups were fed with high-fat and high-sugar diet for 8 weeks,and then treated with low-and high-dose ginsenoside Rg1 for 8 weeks via gavage,respectively.The levels of oxidative stress indexes were tested by reagent color-developing method.The pathological changes of liver tissues were observed by heamtoxylin-eosin staining.The lipid droplet in the liver tissue was observed by oil red O staining.The apoptosis of hepatocytes was detected by TUNEL staining.The mRNA expression levels of Bax,Bcl-2,and Caspase-3 in the liver tissue were detected by Q-PCR.The protein expression levels of Bax,Bcl-2,Procaspase-3,Caspase-3 cleavage p17 in the liver tissue were determined by immunohistochemistry and western blot assay.RESULTS AND CONCLUSION:Hematoxylin-eosin staining results showed that compared with the control group,the hepatocytes in the model group arranged in disorder with numerous large fat lipid droplets,and inflammatory cell infiltration.After treatment with ginsenoside Rg1,the hepatocytes returned to normal arrangement and lipid droplets and inflammatory cell infiltration reduced.Oil red O staining results found that compared with the model and control groups,the lipid droplet in the hepatocytes were obviously reduced after treatment of ginsenoside Rg1 in a dose-dependent manner.TUNEL staining revealed the number of apoptotic hepatocytes in the ginsenoside Rg1s was significantly smaller than that in the model and control groups.Compared with the control and model groups,the contents of glutathione and superoxide dismutase in the ginsenoside Rg1 group were significantly increased,and the content of malondialdehyde was significantly decreased(P<0.05).Compared with the control and model groups,there was a significant decrease in the mRNA expression levels of Bax and Caspase-3,and a significant increase in the mRNA expression of Bcl-2(P<0.05).Meanwhile,the protein expression levels of Bax and Caspase-3 cleavage p17 were significantly increased,and the protein expression levels of Bcl-2 and Procaspase-3 were significantly decreased(P<0.05),which presented a dose-dependent manner.To conclude,ginsenoside Rg1 can relieve oxidative stress and suppress hepatocyte apoptosis in the rat model of non-alcoholic fatty liver disease,and further delays the disease development.