小檗碱对ApoE~(-/-)小鼠动脉粥样硬化的影响及机制研究
|
摘要
本文旨在高脂喂养的ApoE~(-/-)小鼠模型中研究小檗碱(berberine, BBR)抗动脉粥样硬化的作用及可能的作用机制,为BBR的临床研究打下基础。将7周龄的雄性ApoE~(-/-)小鼠,随机分为对照组(普通饲料)、模型组、BBR组(BBR-L:50 mg·kg~(-1)、BBR-H:150 mg·kg~(-1))、阿托伐他汀组(5 mg·kg~(-1))。高脂饲料喂养ApoE~(-/-)小鼠12周建立实验性高脂血症动物模型,同时给予不同剂量的BBR。12周后,分离主动脉进行油红染色,统计各组ApoE~(-/-)小鼠主动脉斑块情况;采用悬液芯片试剂盒检测血清中炎症因子水平;应用试剂盒测定肝脏总胆固醇、甘油三酯和游离脂肪酸的含量, Western blot方法检测肝脏中炎症信号通路相关蛋白以及胆固醇转运相关基因的表达情况。所有动物实验均按照医药生物技术研究所实验室动物管理条例进行。结果表明,与模型组相比, BBR可以显著降低主动脉全长中的斑块面积,抑制血清中促炎性因子IL-1β等的表达,减少肝脏中总胆固醇等的聚积;机制研究结果显示, BBR能显著抑制MAPKs和NF-κB信号通路的活化,显著上调胆固醇代谢调节蛋白LDLR、ABCA1等的表达。总之,BBR能显著抑制高脂饮食喂养的ApoE~(-/-)小鼠动脉粥样硬化斑块的形成,其作用机制与抑制炎症和降低胆固醇的聚积有关。
The purpose of this research is to study the anti-atherosclerotic effects and mechanisms of berberine(BBR) in high fat diet(HFD) fed ApoE~(-/-)mice, and then to lay a solid foundation of the clinical studies of BBR treatment. The hyperlipidemic ApoE~(-/-)mice model was established by feeding HFD for 12 weeks. Mice were randomly divided into control group(chow diet), model group, BBR group(BBR-L: 50 mg · kg~(-1), BBR-H: 150 mg · kg~(-1)) and atorvastatin(5 mg · kg~(-1)) group. Mice were intragastric administration with BBR in 0.5% sodium salt of caboxy methyl cellulose. After 12 weeks, enface aortas were stained with oil red O, and the lesions area wereanalyzed by Image J software. The inflammatory factor levels were detected by suspension microarray kits. Liver total cholesterol(TC), triglyceride(TG) and free fatty acids(FFA) were determined by commercial kits. Western blot was performed to examine the inflammatory pathway related and cholesterol and lipid transport related proteins' expression. All animal experiments were performed in accordance with the Regulation on the Administra-tion of Laboratory Animals of Institute of Medicinal Biotechnology. After 12 weeks treatment, compared with model group, BBR treatment significantly reduced the lesions area of en face aortas and obviously inhibited serum proinflammatory factors such as IL-1β and IL-6 compared with model group. In addition, BBR treatment obviously reduced liver TC, TG and FFA levels compared with model group. Furthermore, mechanic study showed that BBR significantly inhibited MAPKs and NF-κB pathways, and increased cholesterol and lipid regulated proteins expres-sion such as p-AMPK, LDLR, ABCA1 and SR-BI. In conclusion, BBR can obviously reduce enface aortas lesions in ApoE~(-/-)mice, which is related to inhibit inflammation and liver cholesterol and lipid accumulation.
The purpose of this research is to study the anti-atherosclerotic effects and mechanisms of berberine(BBR) in high fat diet(HFD) fed ApoE~(-/-)mice, and then to lay a solid foundation of the clinical studies of BBR treatment. The hyperlipidemic ApoE~(-/-)mice model was established by feeding HFD for 12 weeks. Mice were randomly divided into control group(chow diet), model group, BBR group(BBR-L: 50 mg · kg~(-1), BBR-H: 150 mg · kg~(-1)) and atorvastatin(5 mg · kg~(-1)) group. Mice were intragastric administration with BBR in 0.5% sodium salt of caboxy methyl cellulose. After 12 weeks, enface aortas were stained with oil red O, and the lesions area wereanalyzed by Image J software. The inflammatory factor levels were detected by suspension microarray kits. Liver total cholesterol(TC), triglyceride(TG) and free fatty acids(FFA) were determined by commercial kits. Western blot was performed to examine the inflammatory pathway related and cholesterol and lipid transport related proteins' expression. All animal experiments were performed in accordance with the Regulation on the Administra-tion of Laboratory Animals of Institute of Medicinal Biotechnology. After 12 weeks treatment, compared with model group, BBR treatment significantly reduced the lesions area of en face aortas and obviously inhibited serum proinflammatory factors such as IL-1β and IL-6 compared with model group. In addition, BBR treatment obviously reduced liver TC, TG and FFA levels compared with model group. Furthermore, mechanic study showed that BBR significantly inhibited MAPKs and NF-κB pathways, and increased cholesterol and lipid regulated proteins expres-sion such as p-AMPK, LDLR, ABCA1 and SR-BI. In conclusion, BBR can obviously reduce enface aortas lesions in ApoE~(-/-)mice, which is related to inhibit inflammation and liver cholesterol and lipid accumulation.
引文
[1]Frostegard J.Immunity,atherosclerosis and cardiovascular disease[J].BMC Med,2013,11:117.
[2]Weber C,Noels H.Atherosclerosis:current pathogenesis and therapeutic options[J].Nat Med,2011,17:1410-1422.
[3]Jia YJ,Li JJ.Anti-atherosclerosis effect of berberine and its mechanism[J].Chin J Arterioscler(中国动脉硬化杂志),2013,21:364-368.
[4]Jawien J.The role of an experimental model of atherosclerosis:ApoE-knockout mice in developing new drugs against atherogenesis[J].Curr Pharm Biotechnol,2012,13:2435-2439.
[5]Chang XX,Yan HM,Xu Q,et al.The effects of berberine on hyperhomocysteinemia and hyperlipidemia in rats fed with a long-term high-fat diet[J].Lipids Health Dis,2012,11:86.
[6]Kong W,Wei J,Abidi P,et al.Berberine is a novel cholesterollowering drug working through a unique mechanism distinct from statins[J].Nat Med,2004,10:1344-1351.
[7]Lee YS,Kim WS,Kim KH,et al.Berberine,a natural plant product,activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states[J].Diabetes,2006,55:2256-2264.
[8]Zhang M,Lv X,Li J,et al.Sodium caprate augments the hypoglycemic effect of berberine via AMPK in inhibiting hepatic gluconeogenesis[J].Mol Cell Endocrinol,2012,363:122-130.
[9]Wang Q,Zhang M,Liang B,et al.Activation of AMP-activated protein kinase is required for berberine-induced reduction of atherosclerosis in mice:the role of uncoupling protein 2[J].PLoS One,2011,6:e25436.
[10]Viola J,Soehnlein O.Atherosclerosis-a matter of unresolved inflammation[J].Semin Immunol,2015,27:184-193.
[11]Chen J,Cao J,Fang L,et al.Berberine derivatives reduce athero-sclerotic plaque size and vulnerability in ApoE-/-mice[J].JTransl Med,2014,12:326.
[12]Feng M,Zou Z,Zhou X,et al.Comparative effect of berberine and its derivative 8-cetylberberine on attenuating atherosclerosis in ApoE-/-mice[J].Int Immunopharmacol,2017,43:195-202.
[13]Zhu L,Zhang D,Zhu H,et al.Berberine treatment increases Akkermansia in the gut and improves high-fat diet-induced atherosclerosis in ApoE-/-mice[J].Atherosclerosis,2018,268:117-126.
[14]Li H,He C,Wang J,et al.Berberine activates peroxisome prolif-erator-activated receptor gamma to increase atherosclerotic plaque stability in ApoE-/-mice with hyperhomocysteinemia[J].J Diabetes Investig,2016,7:824-832.
[15]Feng M,Kong SZ,Wang ZX,et al.The protective effect of coptisine on experimental atherosclerosis ApoE-/-mice is mediated by MAPK/NF-κB-dependent pathway[J].Biomed Pharmacother,2017,93:721-729.
[16]Ponziani FR,Pecere S,Gasbarrini A,et al.Physiology and patho-physiology of liver lipid metabolism[J].Expert Rev Gastroenterol Hepatol,2015,9:1055-1067.
[17]Song D,Fang G,Mao SZ,et al.Chronic intermittent hypoxia induces atherosclerosis by NF-κB-dependent mechanisms[J].Biochim Biophys Acta,2012,1822:1650-1659.
[18]Liu Y,Major AS,Zienkiewicz J,et al.Nuclear transport modula-tion reduces hypercholesterolemia,atherosclerosis,and fatty liver[J].J Am Heart Assoc,2013,2:e000093.
[19]Lee S,Lim HJ,Park HY,et al.Berberine inhibits rat vascular smooth muscle cell proliferation and migration in vitro and improves neointima formation after balloon injury in vivo.Berberine improves neointima formation in a rat model[J].Atherosclerosis,2006,186:29-37.
[20]Hu Y,Chen X,Duan H,et al.Chinese herbal medicinal ingredients inhibit secretion of IL-6,IL-8,E-selectin and TXB2 in LPS-induced rat intestinal microvascular endothelial cells[J].Immunopharmacol Immunotoxicol,2009,31:550-555.
[21]Wang QZ,Guo Y,Han LM,et al.The inhibitory effect of berberine on COX-2 by ERK and JNK signal transduction pathway[J].J Fourth Mil Med Univ(第四军医大学学报),2009,30:2935-2939.
[22]Xu WT,Huang JJ,Zhu LB,et al.Effect of berberine on vulnerable plaque and blood lipid in rabbit atherosclerosis[J].Suzhou Univ J Med Sci(苏州大学学报(医学版)),2010,2:280-283.
[23]Qiu HM,Yang FJ,Zhang Q,et al.Effect of berberine on lipid metabolism and atherosclerosis and its mechanism[J].J Chongqing Med Univ(重庆医科大学学报),2012,37:661-664.
[24]Li YH,Wang L,Hong B,et al.Synthesis of 13-hexyl berberine analogues and structure-activity relationship of CD36 antagonistic activity[J].Acta Pharm Sin(药学学报),2010,45:1128-1133.
[25]Li XM,Wang QZ,Shi J,et al.Berberine improves vascular inflammatory response and calcification in atherosclerotic mice[J].Basic Clin Med(基础医学与临床),2018,38:163-168.
[2]Weber C,Noels H.Atherosclerosis:current pathogenesis and therapeutic options[J].Nat Med,2011,17:1410-1422.
[3]Jia YJ,Li JJ.Anti-atherosclerosis effect of berberine and its mechanism[J].Chin J Arterioscler(中国动脉硬化杂志),2013,21:364-368.
[4]Jawien J.The role of an experimental model of atherosclerosis:ApoE-knockout mice in developing new drugs against atherogenesis[J].Curr Pharm Biotechnol,2012,13:2435-2439.
[5]Chang XX,Yan HM,Xu Q,et al.The effects of berberine on hyperhomocysteinemia and hyperlipidemia in rats fed with a long-term high-fat diet[J].Lipids Health Dis,2012,11:86.
[6]Kong W,Wei J,Abidi P,et al.Berberine is a novel cholesterollowering drug working through a unique mechanism distinct from statins[J].Nat Med,2004,10:1344-1351.
[7]Lee YS,Kim WS,Kim KH,et al.Berberine,a natural plant product,activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states[J].Diabetes,2006,55:2256-2264.
[8]Zhang M,Lv X,Li J,et al.Sodium caprate augments the hypoglycemic effect of berberine via AMPK in inhibiting hepatic gluconeogenesis[J].Mol Cell Endocrinol,2012,363:122-130.
[9]Wang Q,Zhang M,Liang B,et al.Activation of AMP-activated protein kinase is required for berberine-induced reduction of atherosclerosis in mice:the role of uncoupling protein 2[J].PLoS One,2011,6:e25436.
[10]Viola J,Soehnlein O.Atherosclerosis-a matter of unresolved inflammation[J].Semin Immunol,2015,27:184-193.
[11]Chen J,Cao J,Fang L,et al.Berberine derivatives reduce athero-sclerotic plaque size and vulnerability in ApoE-/-mice[J].JTransl Med,2014,12:326.
[12]Feng M,Zou Z,Zhou X,et al.Comparative effect of berberine and its derivative 8-cetylberberine on attenuating atherosclerosis in ApoE-/-mice[J].Int Immunopharmacol,2017,43:195-202.
[13]Zhu L,Zhang D,Zhu H,et al.Berberine treatment increases Akkermansia in the gut and improves high-fat diet-induced atherosclerosis in ApoE-/-mice[J].Atherosclerosis,2018,268:117-126.
[14]Li H,He C,Wang J,et al.Berberine activates peroxisome prolif-erator-activated receptor gamma to increase atherosclerotic plaque stability in ApoE-/-mice with hyperhomocysteinemia[J].J Diabetes Investig,2016,7:824-832.
[15]Feng M,Kong SZ,Wang ZX,et al.The protective effect of coptisine on experimental atherosclerosis ApoE-/-mice is mediated by MAPK/NF-κB-dependent pathway[J].Biomed Pharmacother,2017,93:721-729.
[16]Ponziani FR,Pecere S,Gasbarrini A,et al.Physiology and patho-physiology of liver lipid metabolism[J].Expert Rev Gastroenterol Hepatol,2015,9:1055-1067.
[17]Song D,Fang G,Mao SZ,et al.Chronic intermittent hypoxia induces atherosclerosis by NF-κB-dependent mechanisms[J].Biochim Biophys Acta,2012,1822:1650-1659.
[18]Liu Y,Major AS,Zienkiewicz J,et al.Nuclear transport modula-tion reduces hypercholesterolemia,atherosclerosis,and fatty liver[J].J Am Heart Assoc,2013,2:e000093.
[19]Lee S,Lim HJ,Park HY,et al.Berberine inhibits rat vascular smooth muscle cell proliferation and migration in vitro and improves neointima formation after balloon injury in vivo.Berberine improves neointima formation in a rat model[J].Atherosclerosis,2006,186:29-37.
[20]Hu Y,Chen X,Duan H,et al.Chinese herbal medicinal ingredients inhibit secretion of IL-6,IL-8,E-selectin and TXB2 in LPS-induced rat intestinal microvascular endothelial cells[J].Immunopharmacol Immunotoxicol,2009,31:550-555.
[21]Wang QZ,Guo Y,Han LM,et al.The inhibitory effect of berberine on COX-2 by ERK and JNK signal transduction pathway[J].J Fourth Mil Med Univ(第四军医大学学报),2009,30:2935-2939.
[22]Xu WT,Huang JJ,Zhu LB,et al.Effect of berberine on vulnerable plaque and blood lipid in rabbit atherosclerosis[J].Suzhou Univ J Med Sci(苏州大学学报(医学版)),2010,2:280-283.
[23]Qiu HM,Yang FJ,Zhang Q,et al.Effect of berberine on lipid metabolism and atherosclerosis and its mechanism[J].J Chongqing Med Univ(重庆医科大学学报),2012,37:661-664.
[24]Li YH,Wang L,Hong B,et al.Synthesis of 13-hexyl berberine analogues and structure-activity relationship of CD36 antagonistic activity[J].Acta Pharm Sin(药学学报),2010,45:1128-1133.
[25]Li XM,Wang QZ,Shi J,et al.Berberine improves vascular inflammatory response and calcification in atherosclerotic mice[J].Basic Clin Med(基础医学与临床),2018,38:163-168.