Effects Of Tongxinluo Supermicropowder On NF-κB,iNOS Expression In Artery Of Rabbits After Coronary Spasm Induced By ET-1

ObjectiveTo observe the effects of Tongxinluo Supermicropowder on nuclear factor-kappaB (NF-κB),inducible nitric oxide synthase(iNOS)in the artery of rabbits after coronary spasm induced by endothelin-1(ET-1)injection.Methods1.Healthy male Japanese rabbits were randomly divided into 5 groups(n=6 each)(1)control group(normal saline group,10/ml/day),(2)Tongxinluo Supermicropowder low-dose group(150mg/kg/day),(3)middle-dose group(300mg/kg/day),(4)High-dose group(600mg/kg/day),(5)diltiazem group(10mg/kg/day)Rabbits were fed with these drugs 5 days.2.Coronary spasm modelAll animals stop eating 18 hours before experiment.Feed rabbits with cousulting drugs 30 minutes befer endothelin-1 injection.Wulatan(1000mg/Kg/10ml) intraperitoneal injection 15 minutes befor ET-1 injecton.Using multichannel physiologic recorder to record electrocardiogram by standard limb leadⅡcontinuously.3.Blood samples Achieve rabbit blood samples before and after 2,6 hours of ET-1 injection.Wait until the blood samples become solid.After centrifugation,3000rpm,5 minutes, seperate serum to EP tubes。4.Tissue samplesExecute rabbits after 6 hours of ET-1 injection.Heparin(300IU/Kg)was injected intravenous 15minutes befor ET-1 injection.Bloodletting from carotid 2 minutes after heparin injection.Hearts were taken out of the chest immediately,and seperate the coronary artery in 4℃heparin saline.Put the tissue samples in liquid nitrogen until detection.5.Indicators detction(1)Using multichannel physiologic recorder to record electrocardiogram 30 minutes by standard limb leadⅡcontinuously,and compare T-wave height in each group at 3,5,10,15,30 minutes.(2)Using Rabbit serum TnI detcting kit(96-well plate,ELLISA method)to analyse serum treponin I level.(3)Using reverse transcription PCR(RT-PCR)to detect inducible nitric oxide synthase,nuclear factor-kappaB expression in the coronary artery.Usingβ-actin as the relative control,compare the relative gray values of goal straps in each group.(4)Using NF-κB activation assay kit(96-well plate,ELLISA method)to detect NF-κB activity in coronary artery.6.Statistical AnalysisValues were implicated by Mean±Standard((?)±s).Multiple values were compared by One-way ANOVA method.Between-group values were compared by LSD test or Dunnett's T3 test.Two sample meas were compared by t test.P value was set at 0.05. All deta were analysed by SPSS 13.0.Results1.The height of T wave on ECG leadⅡwas most large at 5 minutes after ET-1 injection.Either at 5 or 10 minutes after ET-1 injection,T wave changes in Tongxinluo Supermicropowder middle-dose group,High-dose group and diltiazem group were significant compared with control group(P＜0.05),but Tongxinluo Supermicropowder low-dose group didn't reach significance(P＞0.05).At 5 minutes after ET-1 injection, T wave changes in Tongxinluo Supermicropowder high-dose group was larger than diltiazem group(P＜0.05),but Tongxinluo Supermicropowder middle-dose group didn't reach significance(P＞0.05).2.Serum treponin I level raised at 2 hours after ET-1 injection.At this time Tongxinluo Supermicropowder middle-dose group and high-dose group didn't reach significance compared with baseline(P＞0.05),but Tongxinluo Supermicropowder low-dose group and diltiazem group reached significance compared with baseline(P＜0.05).Serum treponin I level significantly raised at 6 hours after ET-1 injection.At this time only Tongxinluo Supermicropowder high-dose group didn't reach significance compared with baseline(P＞0.05),Tongxinluo Supermicropowder middle-dose group and diltiazem group reached significance compared with baseline(P＜0.05).Not only that,at this time,Tongxinluo Supermicropowder middle-dose group,High-dose group and diltiazem group reached significance compared with control group(P＜0.05),but Tongxinluo Supermicropowder low-dose group didn't reach significance(P＞0.05).3.NF-κB RT-PCR results of thoracic aorta showed that the straps of control group were the brightest,secondarily Tongxinluo Supermicropowder low-dose group and diltiazem group,and then Tongxinluo Supermicropowder middle-dose group and high-dose group.4.iNOS RT-PCR results of thoracic aorta showed that the straps of control group were the brightest,secondarily Tongxinluo Supermicropowder low-dose group and diltiazem group,and then Tongxinluo Supermicropowder middle-dose group and high-dose group.5.NF-κB activity of thoracic aorta in Tongxinluo Supermicropowder High-dose group reached significance compared with control group(P＜0.05),but other groups didn't reach significance(P＞0.05).Conclusions1.Coronary artery spasm model can be astablished sucessfully by ET-1 injecton.2.Tongxinluo Supermicropowder can abviously reduce T wave elevation at the time of coronary spasm,and decrease serun TnI level when myocardium in ischemia. Tongxinluo Supermicropowder can reliease coronary spasm and myocardium injury after that.3.Tongxinluo Supermicropowdercan inhibit NF-κB,iNOS expression and reduce NF-κB activation in the coronary artery.4.Tongxinluo Supermicropowder may improve endothelial ruction and relieve coronary spasm to prevent mycarial injury by inhibitting expression of NF-κB,iNOS and reducing NF-κB activation in the artery.