动脉粥样硬化家兔蛋白质硝基化修饰及补肾抗衰片干预研究

Effect of Bushen Kangshuai Tablet on Protein Nitrative Modification in Atherosclerotic Rabbits

目的:研究补肾抗衰片对家兔动脉粥样硬化(AS)病变后血管组织蛋白质硝基化修饰的影响。方法:56只日本大耳白兔随机分为正常组(NOR)、模型组(CHOL)、补肾抗衰片组(BSKS,1 g·kg-1·d-1)、辛伐他汀组(SIMVA,5 mg·kg-1·d-1);各组分别于给药前、给药后8,12,16周采血,最后1次采血后处死动物,无菌条件下取主动脉,检测血清环氧合酶-2(COX-2)活性以及一氧化氮(NO)、3-硝基酪氨酸(3-NT)水平,检测主动脉诱导型一氧化氮合酶(iNOS)mRNA、p38 MAPK mRNA的表达,p38MAPK阳性面积以及eNOS蛋白水平。结果:与正常组比较,动脉粥样硬化造模的不同时期都可检测到iNOS表达和蛋白质酪氨酸的硝基化,模型组内膜明显增厚、纤维帽较薄,斑块内有大量脂质沉积,血清3-NT水平明显升高,补肾抗衰片干预后,血清3-NT水平下降,NO水平明显升高,而COX-2活性没有明显变化;免疫组化染色发现,主动脉p38 MAPK mRNA,iNOS mRNA基因在正常组仅有少量表达,AS病变组p38 MAPK mRNA,iNOS mRNA表达增加;与模型组比较,补肾抗衰片也明显降低了家兔p38 MAPK水平;p38 MAPK mRNA,iNOS mRNA基因表达明显下降,辛伐他汀也有类似的抑制硝基化效应。结论:动脉粥样硬化时,组织蛋白质酪氨酸发生硝基化损伤,中药复方制剂补肾抗衰片在AS病变中具有重要的抗硝基化作用,其保护机制可能是通过调控p38 MAPK mRNA,iNOS mRNA基因的表达以及影响iNOS/NO-COX-2通路中相关酶的活性,同时补肾抗衰片可能通过对抗硝基化反应稳定动脉粥样硬化斑块。

Objective：This study was to determine the effect of the Bushen Kangshuai tablet on nitric xidesynthase （NOS） /nitric oxide （NO）-cyclooxygenase-2 （COX-2） pathway and its associated protein nitrative modification influence in atherosclerotic rabbits.Method：Fifty-six rabbits were randomly divided into normal group,model group,Bushen Kangshuai tablet therapeutic group （1 g· kg-1· d^-1） and simvastatin therapeutic group （5 mg·kg-1·d^-1）.The blood sample of all animals were collected before administration,after supplementing with the 8 weeks,12 weeks and 16 weeks,the animals were sacrificed under aseptic conditions.Aortic iNOS mRNA,p38-mitogen activated protein kinase （p38 MAPK） mRNA expression was measured by QPCR method,the serum COX-2 activity and NO,3-nitrotyrosine （3-NT） levels were measured by ELISA,and eNOS protein level was detected by western blot analysis.Result：iNOS expression and protein tyrosine nitration could be detected in different periods of atherosclerosis,model group showed intimal thickening,thin fibrous cap,a large lipid plaque deposition.Significant ieduction of serum 3-NT levels was found when atherosclerosis occured,but Bushen Kangshuai tablets could decreased the expression of serum 3-NT levels,NO levels were significantly increased,while COX-2 activity did not change significantly.Bushen Kangshuai tablets significantly reduced the rabbit p38 MAPK levels.The model group showed a significant increase in iNOS and p38 MAPK gene expression.The simvastatin had a similar inhibitory effect of nitration.Conclusion：Bushen Kangshuai tablets in atherosclerotic lesions plays an important role in anti-nitration,the protective mechanism may regulate p38 MAPK mRNA,iNOS mRNA gene expression and iNOS/NO-COX-2 pathway related enzymes possibly through anti-nitration reaction to stabilize of the atherosclerotic plaques.