二甲双胍对非酒精性脂肪肝小鼠的内质网应激相关凋亡基因的调控
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摘要
目的观察非酒精性脂肪性肝病(NAFLD)小鼠使用二甲双胍干预后CHOP、Caspase12通路相关基因的表达变化,探讨二甲双胍影响NAFLD的可能机制。方法将C57BL/6J雄性8周小鼠30只分为对照组、模型组和实验组,每组10只。对照组小鼠常规饲料喂养;模型组和实验组小鼠均给予高脂饮食喂养,喂养8周。脂肪肝模型建立成功后,实验组给予二甲双胍150mg·kg~(-1)·d~(-1),灌胃干预4周;对照组和模型组均予等体积生理盐水灌胃处理。末次用药后禁食12 h,麻醉状态下,称取小鼠体重、肝重、双侧附睾周围脂肪组织,并计算脂肪指数;心脏采血检测血清谷丙转氨酶(ALT)、三酰甘油(TG);肝组织分别用于组织病理切片、液氮速冻后转-80℃保存待测肝组织中GRP78、IRE-1α、CHOP、Caspase12、Bcl-2、Bim等基因与蛋白表达水平;以TUNEL法测肝细胞凋亡情况。结果与对照组相比,模型组小鼠体重及脂肪重量、脂肪指数均升高,差异均有统计学意义;与模型组相比,实验组小鼠体重、脂肪重量及脂肪指数明显著下降。与对照组相比,模型组小鼠AL、TG明显升高,差异有统计学意义;与模型组比较,实验组血清ALT、TG明显下降,差异有统计学意义。与对照组比较,模型组肝组织可见明显肝脂肪变性;与模型组相比,实验组小鼠肝脂肪变程度有减轻。与对照组相比,模型组小鼠肝组织中GRP78、IRE-1α、CHOP、Caspase12、Bim mRNA及蛋白表达增加,而Bcl-2基因表达下降,差异均有统计学意义;与模型组相比,实验组肝组织中GRP78、IRE-1α、CHOP、Caspase12、Bim mRNA及蛋白表达下降,而Bcl-2基因表达上升,差异均有统计学意义。对照组可见少量凋亡细胞,凋亡指数为30. 63±5. 25;模型组凋亡细胞明显增多,凋亡指数为58. 32±4. 16;实验组仅见少量凋亡细胞,凋亡指数为20. 26±5. 41。模型组凋亡指数与对照组和实验组比较,差异均有统计学意义(均P <0. 05)。结论二甲双胍可改善内质网应激,可能通过CHOP/Caspase12凋亡信号通路,上调Bcl-2、下调Bim表达,从而减轻肝细胞凋亡,达到治疗脂肪肝作用。
Objective To observe the expression changes of CHOP and Caspase12 pathway-related genes in mice with nonalcoholic fatty liver disease( NAFLD) treated with metformin,and to explore the possible mechanism of metformin affecting NAFLD. Methods Thirty C57 BL/6 J male 8-week mice were divided into control group,model group and experimental group,with 10 rats in each group. Control group was fed with regular diet; model group and experimental group were fed with high-fat diet for 8 weeks. After the fatty liver model was established successfully,experimental group began to give metformin 150 mg·kg~(-1)·d~(-1) for 4 weeks. Control group and model group were treated with equal volume of normal saline. After the last dose,the rats were fasted for 12 h. Under anesthesia,the body weight,liver weight and adipose tissue around the epididymis in each group were weighted and the fat index was calculated. The blood samples were taken for blood serum alanine aminotransferase( ALT) and triglyceride( TG). The liver tissues were used for histopathological section. After liquid freezing,the mRNA and protein expression levels of GRP78,IRE-1α,CHOP,Caspase12,Bcl-2 and Bim were detected at-80 ℃. The apoptosis of hepatocytes was detected by TUNEL method. Results Compared with control group,the body weight,fat weight and fat index in model group were increased,and the difference was statistically significant. Compared with model group,the body weight,fat weight and fat index of experimental group were significantly decreased. Compared with control group,the ALT and TG of model group were significantly increased,and the difference was statistically significant. Compared with model group,the serum ALT and TG of experimental group decreased significantly,and the difference was statistically significant. Compared with control group,hepatic steatosis was observed in the liver tissue of model group; compared with model group,the degree of hepatic steatosis in experimental group was alleviated. Compared with control group,the expression of GRP78,IRE-1α,CHOP,Caspase12 and Bim mRNA and protein in the liver tissue of model group increased,while the Bcl-2 gene expression decreased,and the difference was statistically significant. Compared with the model group,the mRNA and protein expressions of GRP78,IRE-1α,CHOP,Caspase12 and Bim in liver tissue of experimental group decreased,while the expression of Bcl-2 gene increased,and the difference was statistically significant. A small number of apoptotic cells were observed in control group,the apoptotic index was 30. 63 ± 5. 25. The apoptotic cells in model group increased significantly,and the apoptotic index was 58. 32 ± 4. 16. In experimental group,only a few apoptotic cells were observed,and the apoptotic index was 20. 26 ± 5. 41. Compared with control group and experimental group,the apoptotic index of model group were statistically significant( all P < 0. 05).Conclusion Metformin can improve endoplasmic reticulum stress,and may up-regulate Bcl-2 and down-regulate Bim expression through CHOP/Caspase12 apoptosis signaling pathway,thereby reducing hepatocyte apoptosis and achieving therapeutic effect on fatty liver.
Objective To observe the expression changes of CHOP and Caspase12 pathway-related genes in mice with nonalcoholic fatty liver disease( NAFLD) treated with metformin,and to explore the possible mechanism of metformin affecting NAFLD. Methods Thirty C57 BL/6 J male 8-week mice were divided into control group,model group and experimental group,with 10 rats in each group. Control group was fed with regular diet; model group and experimental group were fed with high-fat diet for 8 weeks. After the fatty liver model was established successfully,experimental group began to give metformin 150 mg·kg~(-1)·d~(-1) for 4 weeks. Control group and model group were treated with equal volume of normal saline. After the last dose,the rats were fasted for 12 h. Under anesthesia,the body weight,liver weight and adipose tissue around the epididymis in each group were weighted and the fat index was calculated. The blood samples were taken for blood serum alanine aminotransferase( ALT) and triglyceride( TG). The liver tissues were used for histopathological section. After liquid freezing,the mRNA and protein expression levels of GRP78,IRE-1α,CHOP,Caspase12,Bcl-2 and Bim were detected at-80 ℃. The apoptosis of hepatocytes was detected by TUNEL method. Results Compared with control group,the body weight,fat weight and fat index in model group were increased,and the difference was statistically significant. Compared with model group,the body weight,fat weight and fat index of experimental group were significantly decreased. Compared with control group,the ALT and TG of model group were significantly increased,and the difference was statistically significant. Compared with model group,the serum ALT and TG of experimental group decreased significantly,and the difference was statistically significant. Compared with control group,hepatic steatosis was observed in the liver tissue of model group; compared with model group,the degree of hepatic steatosis in experimental group was alleviated. Compared with control group,the expression of GRP78,IRE-1α,CHOP,Caspase12 and Bim mRNA and protein in the liver tissue of model group increased,while the Bcl-2 gene expression decreased,and the difference was statistically significant. Compared with the model group,the mRNA and protein expressions of GRP78,IRE-1α,CHOP,Caspase12 and Bim in liver tissue of experimental group decreased,while the expression of Bcl-2 gene increased,and the difference was statistically significant. A small number of apoptotic cells were observed in control group,the apoptotic index was 30. 63 ± 5. 25. The apoptotic cells in model group increased significantly,and the apoptotic index was 58. 32 ± 4. 16. In experimental group,only a few apoptotic cells were observed,and the apoptotic index was 20. 26 ± 5. 41. Compared with control group and experimental group,the apoptotic index of model group were statistically significant( all P < 0. 05).Conclusion Metformin can improve endoplasmic reticulum stress,and may up-regulate Bcl-2 and down-regulate Bim expression through CHOP/Caspase12 apoptosis signaling pathway,thereby reducing hepatocyte apoptosis and achieving therapeutic effect on fatty liver.
引文
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[2]任路平,宋光耀等.二甲双胍对高脂喂养诱导小鼠脂肪肝和肝脏内质网应激的干预作用[J].中华消化杂志,2012,32(12):843-846.
[3]谢汝佳,韩冰,杨婷,杨勤.内质网应激凋亡通路中Caspase12激活介导的肝纤维化大鼠肝细胞凋亡[J].世界华人消化杂志,2016,24(16):2470-2477.
[4] JUNG U J,CHOI M S. Obesity and its metabolic complications:the role of adipokines and the relationship between obesity,inflam-mation,insulin resistance,dyslipidemia and nonalcoholic fatty liver disease[J]. Int J Mol Sci,2014,15(4):6184-6223.
[5] YANG N,SUN R B,CHEN X L,et al. In vitro assessment of the glucose-lowering effects of berberrubine-9-O-β-D-glucuronide,an active metabolite of berberrubine[J]. Acta Pharmacol Sin,2017,38(3):351-361.
[6]张健,俞敏.二甲双胍对糖尿病患者血脂的影响[J].深圳中西医结合杂志,2015,25(1):21-22.
[7] EVA S,SUSAN E L,ADRIENNE M,et al. Mediators of endoplasmic reticulum stress-induced apoptosis[J]. EMBO Reports,2006,7(9):880-885.
[2]任路平,宋光耀等.二甲双胍对高脂喂养诱导小鼠脂肪肝和肝脏内质网应激的干预作用[J].中华消化杂志,2012,32(12):843-846.
[3]谢汝佳,韩冰,杨婷,杨勤.内质网应激凋亡通路中Caspase12激活介导的肝纤维化大鼠肝细胞凋亡[J].世界华人消化杂志,2016,24(16):2470-2477.
[4] JUNG U J,CHOI M S. Obesity and its metabolic complications:the role of adipokines and the relationship between obesity,inflam-mation,insulin resistance,dyslipidemia and nonalcoholic fatty liver disease[J]. Int J Mol Sci,2014,15(4):6184-6223.
[5] YANG N,SUN R B,CHEN X L,et al. In vitro assessment of the glucose-lowering effects of berberrubine-9-O-β-D-glucuronide,an active metabolite of berberrubine[J]. Acta Pharmacol Sin,2017,38(3):351-361.
[6]张健,俞敏.二甲双胍对糖尿病患者血脂的影响[J].深圳中西医结合杂志,2015,25(1):21-22.
[7] EVA S,SUSAN E L,ADRIENNE M,et al. Mediators of endoplasmic reticulum stress-induced apoptosis[J]. EMBO Reports,2006,7(9):880-885.