龙胆苦苷对非酒精性脂肪肝病大鼠AMPK信号通路的调节作用
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摘要
目的分析龙胆苦苷对高脂高糖饮食构建的非酒精性脂肪肝病(NAFLD)大鼠的干预作用及其机制。方法 60只SD大鼠按照随机数字法分为对照组、模型组、阳性药物多烯磷脂酰胆碱组及龙胆苦苷低、中、高剂量组,高脂高糖饮食12周建立NAFLD大鼠模型,龙胆苦苷低、中、高剂量组分别ig给予龙胆苦苷50、100、200 mg/kg,多烯磷脂酰胆碱组ig给予多烯磷脂酰胆碱23 mg/kg,对照组和模型组给予500μL/kg生理盐水,治疗8周后,收集各组大鼠血清,检测肝功能、血脂、血清氧化和抗氧化能力以及炎性因子相关指标,进行肝脏HE染色观察病理学改变,Western blotting和q RT-PCR检测肝脏组织中腺苷酸激活蛋白激酶α(AMPKα)和磷酸化的腺苷酸激活蛋白激酶α(p-AMPKα)的表达特点。结果 HE染色显示对照组大鼠肝脏细胞大小均一,细胞核呈均匀分布,模型组有明显脂肪空泡,且有一定的炎症反应浸润,与模型组相比,多烯磷脂酰胆碱和龙胆苦苷均有明显的改善作用,特别是龙胆苦苷中剂量和高剂量组已明显好转,但与对照组仍有一定差异;与对照组相比,模型组大鼠肝功能指标天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)、血脂高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)、氧化指标丙二醛(MDA)以及炎性因子白细胞介素-1(IL-1)和IL-6水平明显升高(P<0.05),抗氧化指标过氧化物歧化酶(SOD)活性明显下降(P<0.05),龙胆苦苷治疗后,AST、ALT、MDA、IL-1和IL-6明显降低(P<0.05),SOD明显升高(P<0.05),HDL-C和LDL-C也有一定的降低,但没有统计学意义,多烯磷脂酰胆碱则对上述指标均有明显的改善作用;与对照组相比,模型组p-AMPKα蛋白和AMPKm RNA明显降低(P<0.05);使用多烯磷脂酰胆碱和龙胆苦苷治疗后,均明显升高(P<0.05),且龙胆苦苷3个剂量组有明显的剂量依赖性,同时中剂量和高剂量组要好于多烯磷脂酰胆碱组(P<0.05),而AMPKα蛋白表达在整个治疗过程中没有明显差异。结论高脂高糖饮食12周构建的NAFLD大鼠有明显肝脏脂肪浸润,同时肝功能指标升高、血脂异常、炎性因子升高和抗氧化能力降低,龙胆苦苷则可以明显改善上述症状,这可能是龙胆苦苷增加大鼠肝脏组织中p-AMPKα蛋白表达实现的。
Objective To investigate the therapeutic effect of gentiopicroside has a on rats with non-alcoholic fatty liver disease(NAFLD) induced by high-fat and high-sucrose diet,and explore its mechanism.Methods After 10 d adaptive feeding,60 male SD rats were randomly divided into the normal group,model group,gentiopicroside low,medium,high dose treatment groups,and positive drug polyeno phosphoryl choline(PPC) intervention group.Except for the normal group,the rats in other groups were received a high fat and glucose diet for 12 weeks to establish NAFLD model;After model successfully established,gentiopicroside low,medium,and high dosetreatment groups were given 50,100,and 200 mg/(kg·d) gentiopicroside,PPC group was ig given 23 mg/(kg·d) PPC,and 500 μL/(kg·d) saline was given to the normal and model groups.After treated for eight weeks,the rats were sacrificed,and the serum was collected from rats to detect the liver function,blood lipid,serum oxidation,antioxidant capacity,and inflammatory factors.HE staining was used to observe pathological changes of liver.In addition,western blotting and qRT-PCR were used to detect the expression of AMPKα and p-AMPKα.Results HE staining showed that the size of liver cells in the normal group was uniform and the nuclei were evenly distributed,there were obvious vacuoles and a certain inflammatory reaction in the model group.Compared with the model group,gentiopicroside treatment group and PPC group(especially the gentiopicroside middle and high dose group) had a significant improvement,but there were still some differences compared with the normal group;Compared with the normal group,the AST,ALT,HDL-C,LDL-C,MDA,IL-1,and IL-6 in the model group were significantly increased(P<0.05),the levels of AST,ALT,MDA,IL-1,and IL-6 were significantly decreased(P<0.05),SOD was significantly increased(P<0.05),and HDL-C and LDL-C were decreased in the model group(P>0.05);The results of Western blotting and qRT-PCR showed that compared with the normal group,the expression of p-AMPKα protein and AMPKα m RNA in the model group was significantly decreased(P<0.05).After gentiopicroside and PPC administration,they were significantly increased(P<0.05),and gentiopicroside groups showed a significant dose-dependent manner,and the middle dose and high dose of gentiopicroside groups were better than the PPC group(P<0.05),while the expression of AMPKα protein has no significant difference in each group(P<0.05).Conclusion The NAFLD rats showed a obvious hepatic fat infiltration and dyslipidemia,the liver function index and inflammatory factors levels were elevated,and the anti-oxidant capacity was decreased.Gentiopicroside significantly improved above symptoms,which may be associated with the increased expression of p-AMPKα in liver tissue of NAFLD rats by gentiopicroside.
Objective To investigate the therapeutic effect of gentiopicroside has a on rats with non-alcoholic fatty liver disease(NAFLD) induced by high-fat and high-sucrose diet,and explore its mechanism.Methods After 10 d adaptive feeding,60 male SD rats were randomly divided into the normal group,model group,gentiopicroside low,medium,high dose treatment groups,and positive drug polyeno phosphoryl choline(PPC) intervention group.Except for the normal group,the rats in other groups were received a high fat and glucose diet for 12 weeks to establish NAFLD model;After model successfully established,gentiopicroside low,medium,and high dosetreatment groups were given 50,100,and 200 mg/(kg·d) gentiopicroside,PPC group was ig given 23 mg/(kg·d) PPC,and 500 μL/(kg·d) saline was given to the normal and model groups.After treated for eight weeks,the rats were sacrificed,and the serum was collected from rats to detect the liver function,blood lipid,serum oxidation,antioxidant capacity,and inflammatory factors.HE staining was used to observe pathological changes of liver.In addition,western blotting and qRT-PCR were used to detect the expression of AMPKα and p-AMPKα.Results HE staining showed that the size of liver cells in the normal group was uniform and the nuclei were evenly distributed,there were obvious vacuoles and a certain inflammatory reaction in the model group.Compared with the model group,gentiopicroside treatment group and PPC group(especially the gentiopicroside middle and high dose group) had a significant improvement,but there were still some differences compared with the normal group;Compared with the normal group,the AST,ALT,HDL-C,LDL-C,MDA,IL-1,and IL-6 in the model group were significantly increased(P<0.05),the levels of AST,ALT,MDA,IL-1,and IL-6 were significantly decreased(P<0.05),SOD was significantly increased(P<0.05),and HDL-C and LDL-C were decreased in the model group(P>0.05);The results of Western blotting and qRT-PCR showed that compared with the normal group,the expression of p-AMPKα protein and AMPKα m RNA in the model group was significantly decreased(P<0.05).After gentiopicroside and PPC administration,they were significantly increased(P<0.05),and gentiopicroside groups showed a significant dose-dependent manner,and the middle dose and high dose of gentiopicroside groups were better than the PPC group(P<0.05),while the expression of AMPKα protein has no significant difference in each group(P<0.05).Conclusion The NAFLD rats showed a obvious hepatic fat infiltration and dyslipidemia,the liver function index and inflammatory factors levels were elevated,and the anti-oxidant capacity was decreased.Gentiopicroside significantly improved above symptoms,which may be associated with the increased expression of p-AMPKα in liver tissue of NAFLD rats by gentiopicroside.
引文
[1]周雪,宋怡,邓波,等.我国非酒精性脂肪肝病流行现状及相关膳食营养因素探讨[A]//第十二届全国营养科学大会论文汇编[C].北京:中国营养学会,2015.
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[3]陈菲,艾国,盛柳青,等.九味肝泰胶囊对高脂饮食诱导大鼠非酒精性脂肪肝的治疗作用[J].中草药,2015,46(9):1338-1342.
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[8]余红军,张福生.龙胆泻肝汤联合水飞蓟宾胶囊治疗非酒精性脂肪肝疗效观察[J].新中医,2017(2):45-47.
[9]刘占文,陈长勋,金若敏,等.龙胆苦苷的保肝作用研究[J].中草药,2002,33(1):47-50.
[10]王露.龙胆苦苷对人肝细胞色素CYP450酶的影响[D].杨凌:西北大学,2011.
[11]李夏.龙胆苦苷基于AMPK-PPARα改善酒精性脂肪肝的干预机制[D].延吉:延边大学,2016.
[12]封欣婵.龙胆苦苷对肝细胞表面膜转运蛋白MRP3、MRP4的表达影响[D].重庆:第三军医大学,2014.
[13]郭清峰,王玮,韩文清.龙胆苦苷的药理作用进展[J].广东化工,2014,41(17):121.
[14]苏琳,刘玉兰.高糖饮食及高脂饮食建立非酒精性脂肪肝大鼠模型的比较[J].实验动物科学,2009,26(3):14-17.
[15]王振,杨涛,李世朋,等.脂肪肝动物模型研究进展[J].实用器官移植电子杂志,2016,4(2):105-108.
[16]Pi?a-Zentella R M,Rosado J L,Gallegos-Corona M A,et al.Lycopene improves diet-mediated recuperation in rat model of nonalcoholic fatty liver disease[J].J Med Food,2016,19(6):607.
[17]刘月环,吴旧生,应华忠,等.长爪沙鼠NAFLD模型的建立及其遗传学的研究[J].中国比较医学杂志,2017,27(5):9-11.
[18]喻琴,王丽.非酒精性脂肪性肝病动物模型研究进展[J].泸州医学院学报,2016,39(3):300-302.
[19]陈丽.亚麻油对高脂饲料喂养apoE基因敲除小鼠非酒精性脂肪肝的保护作用及其机制研究[D].武汉:华中科技大学,2015.
[20]毛志敏,宋海燕,杨丽丽,等.当药和水飞蓟提取混合物对毒性物质诱导的脂肪肝大鼠肝损伤的防治作用[J].J Integr Med,2012,10(2):193-199.
[21]Xin C,Liu J,Zhang J,et al.Irisin improves fatty acid oxidation and glucose utilization in type 2 diabetes by regulating the AMPK signaling pathway[J].Int J Obes,2016,40(3):443-451.
[22]Yu J W,Deng Y P,Xue H,et al.Metformin improves the angiogenic functions of endothelial progenitor cells via activating AMPK/eNOS pathway in diabetic mice[J].Cardiovasc Diabetol,2016,15(1):1-10.
[23]He T,Zhao J.Resveratrol inhibits renal interstitial fibrosis in diabetic nephropathy by regulating AMPK/NOX4/ROS signaling[J].Hong Kong J Nephrol,2015,17(2):S10-S10.
[24]Qiang X,Xu L,Zhang M,et al.Demethyleneberberine attenuates non-alcoholic fatty liver disease with activation of AMPK and inhibition of oxidative stress[J].Biochem Biophys Res Commun,2016,472(4):603-609.
[25]崔小萌,刘欣,史海涛,等.甘草酸二铵脂质配位体对非酒精性脂肪性肝病大鼠肝组织AMPK信号通路的影响[J].实用肝脏病杂志,2017,20(1):55-59.
[26]姚笑睿,夏凡,唐外姣,等.护肝清脂片对非酒精性脂肪肝大鼠肝脏中AMPK通路激活及NF-κB-p65蛋白的影响[J].南方医科大学学报,2017,37(1):56-62.
[2]武俊紫.艾塞那肽对非酒精性脂肪肝病的治疗作用研究[D].昆明:昆明理工大学,2014.
[3]陈菲,艾国,盛柳青,等.九味肝泰胶囊对高脂饮食诱导大鼠非酒精性脂肪肝的治疗作用[J].中草药,2015,46(9):1338-1342.
[4]武俊紫,牛世伟,贾亚敏,等.艾塞那肽通过调控PPARα及ACOX1改善大鼠非酒精性脂肪肝病症状[J].基础医学与临床,2014,34(4):464-469.
[5]Dahlhoff C,Worsch S,Sailer M,et al.Methyl-donor supplementation in obese mice prevents the progression of NAFLD,activates AMPK and decreases acyl-carnitine levels[J].Mol Metabol,2014,3(5):565-580.
[6]张林,梁茂新.龙胆草潜在功用的发掘与利用[J].世界科学技术-中医药现代化,2015,17(3):675-678.
[7]张立红.龙胆泻肝软胶囊治疗脂肪肝65例观察[J].实用中医药杂志,2010,26(3):180-181.
[8]余红军,张福生.龙胆泻肝汤联合水飞蓟宾胶囊治疗非酒精性脂肪肝疗效观察[J].新中医,2017(2):45-47.
[9]刘占文,陈长勋,金若敏,等.龙胆苦苷的保肝作用研究[J].中草药,2002,33(1):47-50.
[10]王露.龙胆苦苷对人肝细胞色素CYP450酶的影响[D].杨凌:西北大学,2011.
[11]李夏.龙胆苦苷基于AMPK-PPARα改善酒精性脂肪肝的干预机制[D].延吉:延边大学,2016.
[12]封欣婵.龙胆苦苷对肝细胞表面膜转运蛋白MRP3、MRP4的表达影响[D].重庆:第三军医大学,2014.
[13]郭清峰,王玮,韩文清.龙胆苦苷的药理作用进展[J].广东化工,2014,41(17):121.
[14]苏琳,刘玉兰.高糖饮食及高脂饮食建立非酒精性脂肪肝大鼠模型的比较[J].实验动物科学,2009,26(3):14-17.
[15]王振,杨涛,李世朋,等.脂肪肝动物模型研究进展[J].实用器官移植电子杂志,2016,4(2):105-108.
[16]Pi?a-Zentella R M,Rosado J L,Gallegos-Corona M A,et al.Lycopene improves diet-mediated recuperation in rat model of nonalcoholic fatty liver disease[J].J Med Food,2016,19(6):607.
[17]刘月环,吴旧生,应华忠,等.长爪沙鼠NAFLD模型的建立及其遗传学的研究[J].中国比较医学杂志,2017,27(5):9-11.
[18]喻琴,王丽.非酒精性脂肪性肝病动物模型研究进展[J].泸州医学院学报,2016,39(3):300-302.
[19]陈丽.亚麻油对高脂饲料喂养apoE基因敲除小鼠非酒精性脂肪肝的保护作用及其机制研究[D].武汉:华中科技大学,2015.
[20]毛志敏,宋海燕,杨丽丽,等.当药和水飞蓟提取混合物对毒性物质诱导的脂肪肝大鼠肝损伤的防治作用[J].J Integr Med,2012,10(2):193-199.
[21]Xin C,Liu J,Zhang J,et al.Irisin improves fatty acid oxidation and glucose utilization in type 2 diabetes by regulating the AMPK signaling pathway[J].Int J Obes,2016,40(3):443-451.
[22]Yu J W,Deng Y P,Xue H,et al.Metformin improves the angiogenic functions of endothelial progenitor cells via activating AMPK/eNOS pathway in diabetic mice[J].Cardiovasc Diabetol,2016,15(1):1-10.
[23]He T,Zhao J.Resveratrol inhibits renal interstitial fibrosis in diabetic nephropathy by regulating AMPK/NOX4/ROS signaling[J].Hong Kong J Nephrol,2015,17(2):S10-S10.
[24]Qiang X,Xu L,Zhang M,et al.Demethyleneberberine attenuates non-alcoholic fatty liver disease with activation of AMPK and inhibition of oxidative stress[J].Biochem Biophys Res Commun,2016,472(4):603-609.
[25]崔小萌,刘欣,史海涛,等.甘草酸二铵脂质配位体对非酒精性脂肪性肝病大鼠肝组织AMPK信号通路的影响[J].实用肝脏病杂志,2017,20(1):55-59.
[26]姚笑睿,夏凡,唐外姣,等.护肝清脂片对非酒精性脂肪肝大鼠肝脏中AMPK通路激活及NF-κB-p65蛋白的影响[J].南方医科大学学报,2017,37(1):56-62.