丹瓜方对ApoE~(-/-)糖尿病模型小鼠糖脂代谢及血管细胞黏附分子-1及mRNA表达水平的影响
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摘要
目的研究丹瓜方对载脂蛋白E基因敲除(ApoE~(-/-))小鼠复合糖尿病模型糖脂代谢指标、血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)及其mRNA表达水平的影响,探讨丹瓜方防治糖尿病合并血管病变的部分机制。方法 Apo E~(-/-)小鼠皮下注射链脲佐菌素(STZ)制备糖尿病模型。将32只成模鼠按照体重分层,再按血糖水平以随机数字表法分为模型组[无菌水(SW),15 m L/(kg·d)]、丹瓜方组[丹瓜方(DG),15 m L/(kg·d)]、联合用药组[吡格列酮(PG),4.3 mg/(kg·d)加DG1 5 mL/(kg·d)]、吡格列酮组[PG,4.3 mg/(kg·d)],每组8只。选血糖正常的8只同龄同品系C 57小鼠作为对照组。灌胃干预12周。监测各组小鼠空腹血糖(FBG)、体重、摄食量及饮水量;测量皮温及鼠尾长度,并通过肉眼观察肝脂肪变程度;测定各组小鼠血清HbA1 c、TC、LDL-C水平,采用放射免疫法测定ET-1,生化法—硝酸还原酶法测定NO,E LISA法测定VCAM-1,应用实时荧光定量PCR(qPCR)检测肾组织VCAM-1 mRNA表达。结果与对照组比较,模型组小鼠体重及摄食量减少,饮水量增加(P<0.05),且血糖值曲线全程高于对照组(P<0.01)。与模型组比较,丹瓜方组小鼠体重增量、HbA1 c、TC、LDL-C、ET-1、VCAM-1水平显著降低,皮温升高(P<0.05或P<0.01)。与吡格列酮组比较,丹瓜方组小鼠体重、体重增量、FBG、TC及LDL-C水平降低(P<0.05或P<0.01)、饮水量和摄食量增加,鼠尾较长(P<0.01);各组小鼠NO水平比较,差异无统计学意义。与对照组比较,模型组肝脂肪变程度较重(P<0.05);与模型组及吡格列酮组比较,丹瓜方组脂肪变程度明显减轻(P<0.05,P<0.01),吡格列酮组肝脂肪变程度重于模型组(P<0.01),而联合用药组肝脂肪变程度处于丹瓜方组和吡格列酮组之间。丹瓜方组VCAM-1 mRNA的表达显著低于模型组(P<0.01),且低于联合用药组和吡格列酮组(P<0.05)。结论丹瓜方对自发性动脉粥样硬化和高脂血症的ApoE~(-/-)转基因小鼠的复合糖尿病模型有降低血糖、血脂及抗脂肪肝作用,并可减轻吡格列酮致脂肪肝不良作用,降低ET-1、VCAM-1及其mRNA的表达,表现出综合调控糖脂代谢、改善血管功能的效果,有助于防治糖尿病血管病变。
Objective To study the effect of Dangua Recipe(DGR) on glycolipid metabolism,vascular cell adhesion molecule-1(VCAM-1) and its mRNA expression level of transgenic Apo E~(-/-)mouse with spontaneous atherosclerosis,thus revealing its partial mechanism for curing diabetes mellitus(DM) with angiopathy.Methods Diabetic model was prepared by peritoneally injecting streptozotocin(STZ) to Apo E~(-/-)mouse.Totally 32 modeled mice were stratified by body weight,and then divided into 4 groups referring to blood glucose levels from low to high by random digit table,i.e.,the model group(MOD,fed with sterile water,at the daily dose of 15 mL/kg),the DGR group(fed with DGR at the daily dose of 15 mL/kg),the combination group(COM,fed with DGR at the daily dose of 15 mL/kg and pioglitazone at the daily dose of 4.3 mg/kg),and the pioglitazone group(PIO,at the daily dose of 4.3 mg/kg),8in each group.Another 8 normal glucose C57 mouse of the same age and strain were recruited as the control group.All interventions lasted for 12 weeks by gastrogavage.The fasting blood glucose(FBG),body weight,food intake,water intake,skin temperature,the length of tail,and the degree of fatty liver were monitored.The hemoglobin A1c(HbA1c),total cholesterol(TC),and LDL-C were determined.Endothelin-1(ET-1) was determined by radioimmunoassay.Nitrogen monoxidum(NO) was determined by nitrate reductase.The kidney tissue VCAM-1 level was analyzed with ELISA.The expression of VCAM-1mRNA in the kidney tissue was detected with real time quantitative PCR.Results Compared with the control group,the body weight and food intake decreased,water intake increased in all the other model groups(P<0.05).Besides,the curve of blood glucose was higher in all the other model groups than in the control group(P <0.01).Compared with the model group,the body weight increased;levels of HbA1 c,TC,LDL-C,ET-1,and VCAM-1 were significantly lower;and skin temperature was higher in the DGR group(P <0.05,P <0.01).Compared with the PIO group,body weight,the increment of body weight,FBG,TC,and LDL-C were lower(P <0.05,P <0.01);food intake and water intake increased more and the tail length was longer in the DRG group(P <0.01).There was no statistical difference in the level of NO among groups.The degree of fatty liver in the model group was significantly severer than that in the control group(P<0.05).It was obviously alleviated in the DGR group(P <0.05) when compared with the model group and the PIO group(P <0.05,P <0.01).But it was severer in the PIO group than in the model group(P <0.01).The degree of fatty liver in the combination group ranged between that of the DGR group and the PIO group(P <0.05).The level of VCAM-1 mRNA expression was significantly lower in the DGR group than in the model group,the PIO group,and the combination group(P <0.05).Conclusions DGR had effect in lowering blood glucose and blood lipids,and fighting against fatty liver of transgenic Apo E~(-/-)mouse with spontaneous atherosclerosis.DGR played an effective role in preventing and treating DM with angiopathy by comprehensively regulating glycolipid metabolism and promoting the vascular function.
Objective To study the effect of Dangua Recipe(DGR) on glycolipid metabolism,vascular cell adhesion molecule-1(VCAM-1) and its mRNA expression level of transgenic Apo E~(-/-)mouse with spontaneous atherosclerosis,thus revealing its partial mechanism for curing diabetes mellitus(DM) with angiopathy.Methods Diabetic model was prepared by peritoneally injecting streptozotocin(STZ) to Apo E~(-/-)mouse.Totally 32 modeled mice were stratified by body weight,and then divided into 4 groups referring to blood glucose levels from low to high by random digit table,i.e.,the model group(MOD,fed with sterile water,at the daily dose of 15 mL/kg),the DGR group(fed with DGR at the daily dose of 15 mL/kg),the combination group(COM,fed with DGR at the daily dose of 15 mL/kg and pioglitazone at the daily dose of 4.3 mg/kg),and the pioglitazone group(PIO,at the daily dose of 4.3 mg/kg),8in each group.Another 8 normal glucose C57 mouse of the same age and strain were recruited as the control group.All interventions lasted for 12 weeks by gastrogavage.The fasting blood glucose(FBG),body weight,food intake,water intake,skin temperature,the length of tail,and the degree of fatty liver were monitored.The hemoglobin A1c(HbA1c),total cholesterol(TC),and LDL-C were determined.Endothelin-1(ET-1) was determined by radioimmunoassay.Nitrogen monoxidum(NO) was determined by nitrate reductase.The kidney tissue VCAM-1 level was analyzed with ELISA.The expression of VCAM-1mRNA in the kidney tissue was detected with real time quantitative PCR.Results Compared with the control group,the body weight and food intake decreased,water intake increased in all the other model groups(P<0.05).Besides,the curve of blood glucose was higher in all the other model groups than in the control group(P <0.01).Compared with the model group,the body weight increased;levels of HbA1 c,TC,LDL-C,ET-1,and VCAM-1 were significantly lower;and skin temperature was higher in the DGR group(P <0.05,P <0.01).Compared with the PIO group,body weight,the increment of body weight,FBG,TC,and LDL-C were lower(P <0.05,P <0.01);food intake and water intake increased more and the tail length was longer in the DRG group(P <0.01).There was no statistical difference in the level of NO among groups.The degree of fatty liver in the model group was significantly severer than that in the control group(P<0.05).It was obviously alleviated in the DGR group(P <0.05) when compared with the model group and the PIO group(P <0.05,P <0.01).But it was severer in the PIO group than in the model group(P <0.01).The degree of fatty liver in the combination group ranged between that of the DGR group and the PIO group(P <0.05).The level of VCAM-1 mRNA expression was significantly lower in the DGR group than in the model group,the PIO group,and the combination group(P <0.05).Conclusions DGR had effect in lowering blood glucose and blood lipids,and fighting against fatty liver of transgenic Apo E~(-/-)mouse with spontaneous atherosclerosis.DGR played an effective role in preventing and treating DM with angiopathy by comprehensively regulating glycolipid metabolism and promoting the vascular function.
引文
[1]中华医学会糖尿病学分会.中国2型糖尿病防治指南[M].北京:北京大学医学出版社,2011:9-11.
[2]Riddle MC,Ambrosius WT,Brillon DJ,et al.Epidemiologic relationships between A1C and allcause mortality during a median 3.4-year followup of glycemic treatment in the ACCORD trial[J].Diabetes Care,2010,33(5):983-990.
[3]ACCORD Study Group,Gerstein HC,Miller ME,et al.Long-term effects of intensive glucose lowering on cardiovascular outcomes[J].N Engl J Med,2011,364(9):818-828.
[4]ACCORD Study Group,Custhman WC,Evans GW,et al.Effects of intensive blood-pressure control in type 2 diabetes mellitus[J].N Engl J Med,2010,362(17):1575-1585.
[5]ACCORD Study Group,Ginsberg HN,Elam MB,et al.Effects of combination lipid therapy in type 2diabetes mellitus[J].N Engl J Med,2010,362(17):1563-1574.
[6]衡先培,黄国良,修玲玲主编.糖尿病大血管病变[M].北京:人民军医出版社,2011:序一,序二,前言,311-351.
[7]衡先培,黄苏萍,程心玲,等.丹栝方干预糖尿病动脉粥样硬化大鼠糖脂代谢及氧化应激研究[J].中国中西医结合杂志,2013,33(2):244-251.
[8]Xian-pei H,Ke-ji C,Zheng-feng H,et al.Glucose endothelial cytotoxicity and protection of Dan Gua-Fang,a Chinese herb prescription in HUVEC in hyperglycemia medium[J].J Diabetes Complications,2009,23(5):297-303.
[9]Heng XP,Chen KJ,Hong ZF,et al.Toxicity features of high glucose on endothelial cell cycle and protection by Dan Gua-Fang in ECV-304 in high glucose medium[J].Chin J Integr Med,2013,19(8):596-602.
[10]Heng XP,Chen KJ,Hong ZF,et al.Anticolchicine cytotoxicity enhanced by Dan Gua-Fang,a Chinese herb prescription in ECV304 in mediums[J].Chin J Integr Med,2011,17(2):126-133.
[11]黄苏萍,林如辉,陈文列,等.丹瓜方对糖尿病大鼠胸主动脉超微结构影响的初步观察[J].福建中医药大学学报,201 1,21(2):19-22.
[12]Tamarat R,Silvestre JS,Huijberts M,et al.Blockade of advanced glycation end-product formation restores ischemia-induced angiogenesis in diabetic mice[J].Proc Natl Acad Sci USA,2003,100(14):8555-8560.
[13]Buzello M,Temig J,Faulhaber J,et al.The apolipoprotein E knockout mouse:A model documenting accelerated atherogenesis in Uremia[J].Circulation,2003,14(2):311-316.
[14]章元沛主编.药理实验方法学[M].第2版.北京:人民卫生出版社,1996:238.
[15]中华医学会糖尿病学分会.中国2型糖尿病防治指南[M].北京:北京大学医学出版社,2011:15.
[16]高凌云,何作云,牟娇.罗格列酮对apoE基因敲除小鼠血清NOS/NO的影响[J].第三军医大学学报,2006,28(11):1204-1205.
[17]范建高,丁晓东,王国良,等.高脂饮食性非酒精性脂肪性肝病大鼠肝脏PPAR-γ表达增强[J].肝脏,2004,9(4):225-228.
[18]Yu S,Matsusue K,Kashireddy P,et al.Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gammal(PPARgammal)over-expression[J].J Biol Chem,2003,278(1):498-505.
[19]Li P,Shibata R,Unno K,et al.Evidence for the importance of adiponectin in the cardioprotective effects of pioglitazone[J].Hypertension,2010,55(1):69-75.
[20]Zhou M,Xu A,Lam KS,et al.Rosiglitazone promotes fatty acyl CoA accumulation and excessive glycogen storage in livers of mice without adiponectin[J].J Hepatol,2010,53(6):1108-1116.
[21]Da-Wa CR,Zhao F,Qi YF,et al.Role of adiponectin and its receptors in anti-atherosclerotic effects of pioglitazone on ApoE knocked out mice[J].J Peking Univ,2009,41(2):174-178.
[22]Alam S,Noor-E-Alam SM,Chowdhury ZR,et al.Nonalcoholic steatohepatitis in nonalcoholic fatty liver disease patients of Bangladesh[J].World J Hepatol,2013,5(5):281-287.
[23]Lazo M,Hernaez R,Eberhardt MS,et al.Prevalence of nonalcoholic fatty liver disease in the United States:The Third National Health and Nutrition Examination Survey,1988-1994[J].Am J Epidemiol,2013,178(1):38-45.
[24]李秀钧主编.代谢综合征胰岛素抵抗综合征[M].第2版.北京:人民卫生出版社,2007:201-202.
[25]Lan YL,Huang XP,Heng XP,et al.Dangua Fang improves glycolipid metabolic disorders through promoting hepatic AMPK expression in diabetic GK rats[J].Chin J Integr Med,2013,19(7):891-895.
[26]Park MH,Sovio U,Viner RM,et al.Overweight in childhood,adolescence and adulthood and cardiovascular risk in later life:pooled analysis of three British birth cohorts[J].PLoS One,2013,8(7):e70684.
[27]Wang H,Wang AX,Aylor K,et al.Nitric oxide directly promotes vascular endothelial insulin transport[J].Diabetes,2013,62(12):4030-4042.
[28]Dai Y,Mehta JL,Chen M.Glucagon-like peptide-1receptor agonist liraglutide inhibits endothelin-1 in endothelial cell by repressing nuclear factor-Kappa B activation[J].Cardiovasc Drugs Ther,2013,27(5):371-380.
[29]De Miguel C,Pollock JS.Does endoplasmic reticulum stress mediate endothelin-1-induced renal inflammation?[J].Am J Physiol Regul Integr Comp Physiol,2013,305(2):R107-R109.
[30]Jung C,Rafnsson A,Brismar K,et al.Endothelial progenitor cells in relation to endothelin-1 and endothelin receptor blockade:A randomized,controlled trial[J].Int J Cardiol,2013,168(2):1017-1022.
[31]Scott DW,Vallejo MO,Patel RP.Heterogenic endothelial responses to inflammation:role for differential N-glycosylation and vascular bed of origin[J].J Am Heart Assoc,2013,2(4):e000263.
[32]Lacal PM,Petrillo MG,Ruffini F,et al.GITRL(glucocorticoid-induced TNF receptor family-related ligand)triggering up-regulates VCAM-1 and ICAM-1 and promotes leukocyte adhesion[J].J Pharmacol Exp Ther,2013,347(1):164-172.
[33]衡先培,褚克丹,林青,等.丹瓜方对血糖控制不良2型糖尿病患者TNF-α的干预研究[J].福建中医学院学报,2009,19(2):9-12.
[34]黄雯晖,杨柳青,衡先培.痰瘀同治法治疗2型糖尿病痰瘀证患者血浆TNF-α、CRP的临床观察[J].中医临床研究,2011,3(9):1-3.
[35]陈玲,衡先培,蓝元隆,等.丹瓜方对2型糖尿病模型大鼠骨骼肌脂联素的影响[J].福建中医药大学学报,2013,23(1):10-12.
[36]翁苓,衡先培,郑海霞.芎蒌通脉方治疗2型糖尿病痰瘀证患者尿MA及血、尿β2-MG的临床观察[J].光明中医,2010,25(12):2257-2259.
[2]Riddle MC,Ambrosius WT,Brillon DJ,et al.Epidemiologic relationships between A1C and allcause mortality during a median 3.4-year followup of glycemic treatment in the ACCORD trial[J].Diabetes Care,2010,33(5):983-990.
[3]ACCORD Study Group,Gerstein HC,Miller ME,et al.Long-term effects of intensive glucose lowering on cardiovascular outcomes[J].N Engl J Med,2011,364(9):818-828.
[4]ACCORD Study Group,Custhman WC,Evans GW,et al.Effects of intensive blood-pressure control in type 2 diabetes mellitus[J].N Engl J Med,2010,362(17):1575-1585.
[5]ACCORD Study Group,Ginsberg HN,Elam MB,et al.Effects of combination lipid therapy in type 2diabetes mellitus[J].N Engl J Med,2010,362(17):1563-1574.
[6]衡先培,黄国良,修玲玲主编.糖尿病大血管病变[M].北京:人民军医出版社,2011:序一,序二,前言,311-351.
[7]衡先培,黄苏萍,程心玲,等.丹栝方干预糖尿病动脉粥样硬化大鼠糖脂代谢及氧化应激研究[J].中国中西医结合杂志,2013,33(2):244-251.
[8]Xian-pei H,Ke-ji C,Zheng-feng H,et al.Glucose endothelial cytotoxicity and protection of Dan Gua-Fang,a Chinese herb prescription in HUVEC in hyperglycemia medium[J].J Diabetes Complications,2009,23(5):297-303.
[9]Heng XP,Chen KJ,Hong ZF,et al.Toxicity features of high glucose on endothelial cell cycle and protection by Dan Gua-Fang in ECV-304 in high glucose medium[J].Chin J Integr Med,2013,19(8):596-602.
[10]Heng XP,Chen KJ,Hong ZF,et al.Anticolchicine cytotoxicity enhanced by Dan Gua-Fang,a Chinese herb prescription in ECV304 in mediums[J].Chin J Integr Med,2011,17(2):126-133.
[11]黄苏萍,林如辉,陈文列,等.丹瓜方对糖尿病大鼠胸主动脉超微结构影响的初步观察[J].福建中医药大学学报,201 1,21(2):19-22.
[12]Tamarat R,Silvestre JS,Huijberts M,et al.Blockade of advanced glycation end-product formation restores ischemia-induced angiogenesis in diabetic mice[J].Proc Natl Acad Sci USA,2003,100(14):8555-8560.
[13]Buzello M,Temig J,Faulhaber J,et al.The apolipoprotein E knockout mouse:A model documenting accelerated atherogenesis in Uremia[J].Circulation,2003,14(2):311-316.
[14]章元沛主编.药理实验方法学[M].第2版.北京:人民卫生出版社,1996:238.
[15]中华医学会糖尿病学分会.中国2型糖尿病防治指南[M].北京:北京大学医学出版社,2011:15.
[16]高凌云,何作云,牟娇.罗格列酮对apoE基因敲除小鼠血清NOS/NO的影响[J].第三军医大学学报,2006,28(11):1204-1205.
[17]范建高,丁晓东,王国良,等.高脂饮食性非酒精性脂肪性肝病大鼠肝脏PPAR-γ表达增强[J].肝脏,2004,9(4):225-228.
[18]Yu S,Matsusue K,Kashireddy P,et al.Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gammal(PPARgammal)over-expression[J].J Biol Chem,2003,278(1):498-505.
[19]Li P,Shibata R,Unno K,et al.Evidence for the importance of adiponectin in the cardioprotective effects of pioglitazone[J].Hypertension,2010,55(1):69-75.
[20]Zhou M,Xu A,Lam KS,et al.Rosiglitazone promotes fatty acyl CoA accumulation and excessive glycogen storage in livers of mice without adiponectin[J].J Hepatol,2010,53(6):1108-1116.
[21]Da-Wa CR,Zhao F,Qi YF,et al.Role of adiponectin and its receptors in anti-atherosclerotic effects of pioglitazone on ApoE knocked out mice[J].J Peking Univ,2009,41(2):174-178.
[22]Alam S,Noor-E-Alam SM,Chowdhury ZR,et al.Nonalcoholic steatohepatitis in nonalcoholic fatty liver disease patients of Bangladesh[J].World J Hepatol,2013,5(5):281-287.
[23]Lazo M,Hernaez R,Eberhardt MS,et al.Prevalence of nonalcoholic fatty liver disease in the United States:The Third National Health and Nutrition Examination Survey,1988-1994[J].Am J Epidemiol,2013,178(1):38-45.
[24]李秀钧主编.代谢综合征胰岛素抵抗综合征[M].第2版.北京:人民卫生出版社,2007:201-202.
[25]Lan YL,Huang XP,Heng XP,et al.Dangua Fang improves glycolipid metabolic disorders through promoting hepatic AMPK expression in diabetic GK rats[J].Chin J Integr Med,2013,19(7):891-895.
[26]Park MH,Sovio U,Viner RM,et al.Overweight in childhood,adolescence and adulthood and cardiovascular risk in later life:pooled analysis of three British birth cohorts[J].PLoS One,2013,8(7):e70684.
[27]Wang H,Wang AX,Aylor K,et al.Nitric oxide directly promotes vascular endothelial insulin transport[J].Diabetes,2013,62(12):4030-4042.
[28]Dai Y,Mehta JL,Chen M.Glucagon-like peptide-1receptor agonist liraglutide inhibits endothelin-1 in endothelial cell by repressing nuclear factor-Kappa B activation[J].Cardiovasc Drugs Ther,2013,27(5):371-380.
[29]De Miguel C,Pollock JS.Does endoplasmic reticulum stress mediate endothelin-1-induced renal inflammation?[J].Am J Physiol Regul Integr Comp Physiol,2013,305(2):R107-R109.
[30]Jung C,Rafnsson A,Brismar K,et al.Endothelial progenitor cells in relation to endothelin-1 and endothelin receptor blockade:A randomized,controlled trial[J].Int J Cardiol,2013,168(2):1017-1022.
[31]Scott DW,Vallejo MO,Patel RP.Heterogenic endothelial responses to inflammation:role for differential N-glycosylation and vascular bed of origin[J].J Am Heart Assoc,2013,2(4):e000263.
[32]Lacal PM,Petrillo MG,Ruffini F,et al.GITRL(glucocorticoid-induced TNF receptor family-related ligand)triggering up-regulates VCAM-1 and ICAM-1 and promotes leukocyte adhesion[J].J Pharmacol Exp Ther,2013,347(1):164-172.
[33]衡先培,褚克丹,林青,等.丹瓜方对血糖控制不良2型糖尿病患者TNF-α的干预研究[J].福建中医学院学报,2009,19(2):9-12.
[34]黄雯晖,杨柳青,衡先培.痰瘀同治法治疗2型糖尿病痰瘀证患者血浆TNF-α、CRP的临床观察[J].中医临床研究,2011,3(9):1-3.
[35]陈玲,衡先培,蓝元隆,等.丹瓜方对2型糖尿病模型大鼠骨骼肌脂联素的影响[J].福建中医药大学学报,2013,23(1):10-12.
[36]翁苓,衡先培,郑海霞.芎蒌通脉方治疗2型糖尿病痰瘀证患者尿MA及血、尿β2-MG的临床观察[J].光明中医,2010,25(12):2257-2259.