泽茵丸对非酒精性脂肪性肝病小鼠细胞凋亡的影响及其机制

Effect of Zeyin pill on cell apoptosis in mice with non-alcoholic fatty liver disease and its possible mechanism

目的观察泽茵丸对非酒精性脂肪性肝病(NAFLD)小鼠细胞凋亡的影响及其可能机制。方法36只小鼠经高脂饲料喂养12周建立NAFLD模型,随机分为NAFLD组、实验A组、实验B组,每组12只,另10只小鼠采用普通饲料喂养,纳入对照组。实验A、B组分别以生药量2.25、4.5 g/kg泽茵丸浓缩液灌胃,每天1次。对照组、NAFLD组给予等容量生理盐水灌胃。连续干预4周。比较四组血清空腹血糖(FBG)、胰岛素、二肽基肽酶4(DPP4)酶活性及口服葡萄糖耐量试验(OGTT)结果;比较四组肝指数及肝组织总胆固醇(TC)、甘油三酯(TG)含量;原位末端转移酶标记(Tunel)染色法检测四组肝组织细胞凋亡率;油红O染色观察四组肝组织脂质沉积;蛋白免疫印迹法检测肝组织DPP4、核转录因子κB(NF-κB)p65、p-NF-κB p65、半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)蛋白表达。结果NAFLD组FBG及OGTT后30、60、120 min血糖均高于对照组(均P<0.05);实验A、B组FBG及OGTT后30、60、120 min血糖均低于NAFLD组(均P<0.05);实验B组FBG及OGTT后30、60、120 min血糖均低于实验A组(均P<0.05)。NAFLD组胰岛素、DPP4酶活性,肝指数及肝组织TC、TG含量,肝细胞凋亡率均高于对照组(均P<0.05);实验A、B组胰岛素、DPP4酶活性,肝指数及肝组织TC、TG含量,肝细胞凋亡率均低于NAFLD组(均P<0.05);实验B组胰岛素、DPP4酶活性,肝指数及肝组织TC、TG含量,肝细胞凋亡率均低于实验A组(均P<0.05)。油红O染色结果发现,对照组无明显脂质沉积,NAFLD组表现为弥漫性分布的橘红色脂滴及大泡性脂肪变,实验A、B组肝组织橘红色脂滴少于NAFLD组,且实验B组少于实验A组。蛋白免疫印迹法结果发现,NAFLD组肝组织DPP4、Caspase-3蛋白表达量及p-NF-κB p65/NF-κB p65均高于对照组(均P<0.05);实验A、B组肝组织DPP4、Caspase-3蛋白表达量及p-NF-κB p65/NF-κB p65均低于NAFLD组(均P<0.05);实验B组肝组织DPP4、Caspase-3蛋白表达量及p-NF-κB p65/NF-κB p65均低于实验A组(均P<0.05)。结论泽茵丸可降低NAFLD小鼠血糖,减少肝细胞凋亡及肝组织脂质沉积,可能通过抑制DPP4/NF-κB信号通路发挥作用。

Objective To observe the effect of Zeyin pill on cell apoptosis in mice with non-alcoholic fatty liver disease(NAFLD)and its possible mechanism.Methods A total of 36 mice were fed with high-fat diet for 12 weeks to establish a NAFLD model,and were randomly divided into NAFLD group,experimental group A,and experimental group B,with 12 mice in each group.Another 10 mice were fed with regular feed and included in the control group.Experimental groups A and B were orally administered with concentrated Zeyin pill at doses of 2.25 and 4.5 g/kg,respectively,once a day.The control group and the NAFLD group were given equal volume saline by gavage,and were intervened continuously for 4 weeks.Serum fasting blood glucose(FBG),insulin,dipeptidyl peptidase-4(DPP4)enzyme activity,and oral glucose tolerance test(OGTT)results of four groups were compared;Four groups were compared in terms of liver index,total cholesterol(TC),and triglyceride(TG)content in liver tissue;The apoptosis rate of four groups of liver tissue cells was detected by TdT-mediated dUTP nick end labeling(Tunel)staining method;Oil red O staining was used to observe lipid deposition in four groups of liver tissues;Protein immunoblotting was used to detect the protein expression of DPP4,nuclear transcription factor kappa B(NF-κB)p65,p-NF-κB p65,and caspase-3 in liver tissue.Results The blood glucose levels in the NAFLD group were higher than those in the control group at 30,60,and 120 minutes after FBG and OGTT(all P<0.05);The blood glucose levels in groups A and B after FBG and OGTT were lower than those in the NAFLD group at 30,60,and 120 minutes(all P<0.05);The blood glucose levels in group B were lower than those in group A at 30,60,and 120 minutes after FBG and OGTT(all P<0.05).The insulin and DPP4 enzyme activities,liver index,liver tissue TC and TG content,and liver cell apoptosis rate in the NAFLD group were all higher than those in the control group(all P<0.05);The insulin and DPP4 enzyme activities,liver index,liver tissue TC and TG content,and liver cell apoptosis rate in groups A and B were all lower than those in the NAFLD group(all P<0.05);The insulin and DPP4 enzyme activities,liver index,liver tissue TC and TG content,and liver cell apoptosis rate in experimental group B were all lower than those in experimental group A(all P<0.05).The Oil Red O staining results showed that there was no significant lipid deposition in the control group,while the NAFLD group exhibited diffuse distribution of orange red lipid droplets and large vesicular steatosis.The number of orange red lipid droplets in the liver tissue of experimental groups A and B was less than that of the NAFLD group,and the number of orange red lipid droplets in experimental group B was less than that of experimental group A.The results of protein immunoblotting showed that the expression levels of DPP4,Caspase-3 proteins and p-NF-κB p65/NF-κB p65 in liver tissue of NAFLD group were higher than those of the control group(all P<0.05);The expression levels of DPP4 and Caspase-3 proteins,as well as p-NF-κB p65/NF-κB p65,in the liver tissues of groups A and B were lower than those in the NAFLD group(all P<0.05);The expression levels of DPP4 and Caspase-3 proteins,as well as p-NF-κB p65/NF-κB p65,in the liver tissue of experimental group B were lower than those in experimental group A(all P<0.05).Conclusions Zeyin Pill can lower blood glucose levels,reduce liver cell apoptosis and lipid deposition in NAFLD mice,possibly by inhibiting the DPP4/NF-κB signaling pathway.