摘要
目的:探讨滋膵通脉饮对糖尿病心肌病大鼠心肌微血管损伤的影响及其作用机制。方法:将51只SD雄性大鼠随机分为空白对照组(9只)和造模组(42只)。造模组大鼠采用长期高糖高脂喂养联合腹腔注射链脲佐菌素(STZ,35 mg/kg)方法建立糖尿病心肌病大鼠模型,将成模大鼠随机分为模型组、滋膵通脉饮高剂量组、滋膵通脉饮低剂量组、特异性βⅡ-蛋白激酶C(PKC-βⅡ)抑制剂[Ruboxistaurin(LY333531)HCl,Rx抑制剂]组,每组9只。滋膵通脉饮高、低剂量组大鼠分别予相应剂量[60.84、15.21 g/(kg·d)]滋膵通脉饮灌胃,Rx抑制剂组大鼠予PKC-βⅡ抑制剂混悬液灌胃[1.00 mg/(kg·d)]。空白对照组和模型组大鼠均予蒸馏水灌胃[10 mL/(kg·d)]。2次/d,连续给药4周。给药结束后测量体质量、空腹血糖及糖化血清蛋白;采用蛋白质印迹(Western blotting)法检测心肌组织PKC-βⅡ、血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR2)及血管内皮细胞钙粘连蛋白(VE-cadherin)表达;定量逆转录PCR(RT-qPCR)检测心肌组织PKC-βⅡmRNA、VE-cadherin m RNA、VEGF mRNA、VEGFR2 mRNA表达;透射电镜观察心肌微血管的超微结构。结果:模型组大鼠空腹血糖、糖化血清蛋白、心肌组织PKC-βⅡ、VEGF蛋白相对表达量及心肌组织PKC-βⅡmRNA、VEGF mRNA相对表达量均高于空白对照组(P<0.01),体质量、心肌组织VE-cadherin、VEGFR2蛋白相对表达量及心肌组织VE-cadherin mRNA、VEGFR2mRNA相对表达量均低于空白对照组(P<0.01)。滋膵通脉饮高、低剂量组大鼠空腹血糖、糖化血清蛋白均低于模型组(P<0.05或P<0.01),体质量均高于模型组(P<0.01);Rx抑制剂组大鼠空腹血糖、糖化血清蛋白、体质量与模型组比较,差异无统计学意义(P>0.05);滋膵通脉饮低剂量组、Rx抑制剂组大鼠心肌组织PKC-βⅡ、VEGF蛋白相对表达量及心肌组织PKC-βⅡmRNA、VEGF mRNA相对表达量均低于模型组(P<0.01),VE-cadherin、VEGFR2蛋白相对表达量及VE-cadherin mRNA、VEGFR2 mRNA相对表达量均高于模型组(P<0.01);滋膵通脉饮高剂量组大鼠心肌组织VE-cadherin、VEGFR2蛋白相对表达量及VE-cadherin mRNA、VEGFR2 m RNA相对表达量高于模型组(P<0.05或P<0.01),心肌组织PKC-βⅡmRNA、VEGF mRNA相对表达量低于模型组(P<0.01)。透射电镜显示空白对照组大鼠微血管管腔圆滑规整,内皮细胞附着在基底膜内,基底膜均匀连续,胞间紧密连接完整;模型组大鼠心肌微血管损伤明显,基底膜及胞间连接断裂,细胞间隙增宽,管腔通透性增加;Rx抑制剂组、滋膵通脉饮高剂量组、滋膵通脉饮低剂量组大鼠微血管损伤均有不同程度改善,基底膜及胞间连接断裂改善或消失,细胞间隙轻微增宽或不明显。结论:滋膵通脉饮可抑制糖尿病心肌病大鼠PKC-βⅡ激活,影响VEGF的表达,从而调节心肌微血管细胞间正常连接,改善心肌微血管损伤。这可能是其治疗糖尿病心肌病的机制之一。
Objective:To investigate the possible mechanisms of Zicui Tongmai Yin on myocardial microvascular injury in rats with diabetic cardiomyopathy.Methods:Totally 51 male SD rats were randomly divided into blank control group(n=9)and modeling group(n=42).In the modeling group,rats were long-term fed with high glucose and high fat combined with intraperitoneal injection of streptozotocin(STZ,35 mg/kg)to establish diabetic cardiomyopathy rat model.The successfully modeled rats were randomly divided into model group,Zicui Tongmai Yin high-dose group,Zicui Tongmai low-dose group and Ruboxistaurin(Rx)inhibitor group,with 9 rats in each group.The Zicui Tongmai Yin high and low-dose groups were orally administered Zicui Tongmai Yin[60.84 and 15.21 g/(kg·d)]respectively.The Rx inhibitor group was orally administered PKC-βⅡinhibitor[1.00 mg/(kg·d)],and the blank control group and model group were orally administered distilled water[10 mL/(kg·d)]for 4 consecutive weeds of twice daily.After the end of administration,body weight,fasting blood glucose and glycosylated serum protein were measured.Western blotting was used to detect the protein expression of PKC-βⅡ,vascular endothelial growth factor(VEGF),vascular endothelial growth factor receptor 2(VEGFR2)and vascular endothelial-cadherin(VE-cadherin)in myocardial tissue.Quantitative reverse transcription PCR(RT-qPCR)was used to detect the expression of PKC-βⅡmRNA,VE-cadherin mRNA,VEGF mRNA and VEGFR2 mRNA in rat myocardium,and transmission electron microscopy was used to observe the ultrastructure of myocardial microvessels.Results:The model group showed higher fasting blood glucose,glycosylated serum protein,protein expression of PKC-βⅡand VEGF,and expression of PKC-βⅡmRNA and VEGF mRNA than blank control group(P<0.01),while lower body weight,protein expression of VE-cadherin and VEGFR2,and expression of VE-cadherin mRNA and VEGFR2 mRNA than blank control group(P<0.01).The Zicui Tongmai Yin high and low-dose groups showed lower fasting blood glucose and glycosylated serum protein than model group(P<0.05 or P<0.01),while higher body weight both than model group(P<0.01).There was no statistically significant difference in fasting blood glucose,glycosylated serum protein and body weight between Rx inhibitor group and model group(P>0.05).The Zicui Tongmai Yin low-dose group and Rx inhibitor group showed lower protein expressions of PKC-βⅡand VEGF and expressions of PKC-βⅡmRNA and VEGF mRNA than model group(P<0.01),while higher protein expressions of VE-cadherin and VEGFR2 and expressions of VE-cadherin mRNA and VEGFR2 mRNA than model group(P<0.01).The Zicui Tongmai Yin high-dose group showed higher protein expressions of VE-cadherin and VEGFR2 and expressions of VE-cadherin mRNA and VEGFR2 mRNA than model group(P<0.05 or P<0.01),while lower expressions of PKC-βⅡmRNA and VEGF mRNA than model group(P<0.01).The transmission electron microscopy showed that the microvascular lumen was smooth and regular in blank control group,with the endothelial cells attach within the basement membrane.The basement membrane was uniform and continuous,and the intercellular tight junction was intact in blank control group.The myocardial microvascular damage was obvious,and the basement membrane and intercellular junction were broken in model group.The intercellular space was widened,and the luminal permeability was increased in model group.The microvascular injury was improved to varying degrees in the Rx inhibitor group and Zicui Tongmai Yin high and low-dose groups,and the basement membrane and intercellular junction rupture were improved or disappeared.The intercellular space was slightly widened or not obvious in the Rx inhibitor group and Zicui Tongmai Yin high and low-dose groups.Conclusion:Zicui Tongmai Yin can inhibit the activation of PKC-βⅡ,and affect the expression of VEGF,to regulate the normal connection between myocardial microvascular cells,and improve myocardial microvascular injury,which may be one of the mechanisms for the treatment of diabetic cardiomyopathy.
作者
李汶珊
黄昌锐
易晓利
卜献春
袁春云
吴刚强
刘佳琴
谭军
LI Wenshan;HUANG Changrui;YI Xiaoli;BU Xianchun;YUAN Chunyun;WU Gangqiang;LIU Jiaqin;TAN Jun(The Fifth People's Hospital of Longgang District,Shenzhen Guangdong 518111,China;Changsha Health Vocational College,Changsha Hunan 410100,China;Hunan Provincial Hospital of Integrated Traditional Chinese and Western Medicine/The Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha Hunan 410006,China)
出处
《中医药导报》
2024年第10期20-26,31,共8页
Guiding Journal of Traditional Chinese Medicine and Pharmacy
基金
湖南省自然科学基金青年基金项目(2022JJ40204)
湖南省教育厅科研项目(20C1414)
湖南省中医药管理局青年基金项目(2021183)
湖南中医药大学中西结合一流学科开放基金项目(2019ZXYJH11)。
