| Introduction:Coronary heart disease has become one of the main diseases of high incidence and mortality.The main cause of this disease is acute myocardial infarction.With the development of therapy in acute myocardial infarction,such as percutaneous coronary intervention,thrombolysis,angioplasty and coronary bypass surgery,injury of myocardium due to ischemial reperfusion includes cardiac contractile dysfunction,myocardial stunning,arrhythmias and irreversible myocytes damage has become an important problem for clinical research.Several studies show that the use of drugs and myocardial protective agent in ischemia/reperfusion or before reperfusion could reduce ischemia/reperfusion injury and the myocardial infarction area.It is great significance for preventing myocardial ischemia/reperfusion injury.At present,it is still a lack of effective strategies and drugs to treat myocardial ischemia/reperfusion injury.How to alleviate ischemia/reperfusion injury and improve postoperative survival rate of patients with acute myocardial infarction is still a problem urgently needs to solve.Triptolide(TPL)is a diterpene lactone extracted from the root of tripterygium wilfordii.It has anti-inflammatory,antioxidant and anti-cancer effects.Its anti-inflammatory effect could promote ventricular function recovery in diabetic cardiomyopathy rats,alleviate myocardial fibrosis and protect heart.In addition,TPL also plays a protective role in organ or tissue injury due to ischemia/reperfusion.TPL inhibits oxidative stress and the expression of inflammatory factor,reduces neutrophil infiltration,alleviates ischemia/reperfusion injury in liver or lung.Oxidative stress and inflammation is closely related to myocardial ischemia/reperfusion injury.It is not known whether TPL could prevent myocardial ischemia/reperfusion injury caused by oxygen free radicals and inflammation.Thus,our study established the model of myocardial ischemia/reperfusion injury in rats.After intraperitoneal injection of TPL solution,the protective effects of TPL on myocardial ischemia/reperfusion injury and its mechanism were investigated.This will provide the theoretical and experimental basis of ischemia/reperfusion injury clinical treatment.Objective:1.Myocardial ischemia/reperfusion model in rat was established to explore whether TPL has protective effect on ischemia/reperfusion.2.The effects and mechanism of TPL on ischemia/reperfusion injury inflammation in rat myocardial tissues.3.The effects and mechanism of TPL on ischemia/reperfusion injury oxidative stress in rat myocardial tissues.Methods:1.TPL has protective effect on ischemia/reperfusion.(1)Myocardial ischemia/reperfusion model in rat was established.The male health Wistar rats were randomly divided into six groups:(1)the sham group;(2)the sham with high-dose TPL group(sham+H-TPL group);(3)Ischemia/reperfusion model group(I/R group);(4)I/R model with low-dose TPL group(I/R+L-TPL group);(5)I/R model with middle-dose TPL group(I/R+M-TPL group);(6)I/R model with high-dose TPL group(I/R+H-TPL group).(2)Intraperitoneal injection of different doses(25 ?g/kg,50 ?g/kg,100 ?g/kg)TPL solution.Philips IE33 ultrasound hemodynamic data acquisition system was used to evaluated hemodynamic data,including left ventricular end systolic pressure(LVESP),left ventricular end diastolic pressure(LVEDP)and fractional shortening(FS).(3)The rat blood samples were collected.The Creatin kinase(CK),Lactate Dehydrogenase(LDH)and Glutamic oxalacetic transaminase(GOT)activities in the serum were measured by Detection Kits.(4)Myocardial sections were stained with hematoxylin and eosin(H&E)and the myocardial tissue morphology were examined with optical microscope.(5)Immunohistochemistry was conducted to observe macrophages infiltration.(6)Myocardial infarct size was detected using the double staining with 2,3,5-triphenyl-2H-tetrazolium chloride(TTC).2.The effects and mechanism of TPL on ischemia/reperfusion injury inflammation in rat myocardial tissues.(1)Myocardial ischemia/reperfusion model in rat was established.Intraperitoneal injection of different doses(25 ?g/kg,50 ?g/kg,100 ?g/kg)TPL solution.Real-time PCR was performed to determine transcript levels of TNF-?,IL-1?,IL-6 in rat myocardial tissues.(2)The expression of TNF-?,IL-1?,IL-6 in rat myocardial tissues was detected by ELISA.(3)Western blot was conducted to detect the expression of P38 MAPK and p-P38 MAPK.3.The effects and mechanism of TPL on ischemia/reperfusion injury oxidative stress in rat myocardial tissues.(1)Myocardial ischemia/reperfusion model in rat was established.Intraperitoneal injection of different doses(25 ?g/kg,50 ?g/kg,100 ?g/kg)TPL solution.The Superoxide Dismutase(SOD),Heme Oxygenase-1(HO-1),Glutathione Peroxidase(GSH-Px)activities and Malondialdehyde(MDA),Glutathione(GSH)content in rat myocardial tissues were measured by Detection Kits.(2)The expression of Nrf2 in cytoplasm and nuclei was detected by Western blot.4.Statistical analysis.SPSS16.0 software was used for statistical analysis.Data are expressed as the mean ± standard deviation(SD).Comparisons within each group for a given parameter used t tests,and p<0.05 was considered statistically significant.Results:1.Hemodynamic data showed that high-dose TPL significantly reduced LVEDP,increased LVESP and FS(p<0.05 vs.I/R).2.Serum enzyme activity test results showed that high-dose TPL treatment(100 ?g/kg,1 h before surgery)markedly decreased CK,LDH and GOT activities compared with I/R group.3.The HE staining revealed that high-dose TPL(100 ?g/kg)I/R rat presented little necrotic area,the myocardial fibers arranged neatly,only a small amount of inflammatory cells infiltration,the structure was clear,the pathological changes was obviously better than that of the I/R group.4.Immunohistochemical analysis showed that the indices of macrophages were significantly decreased in TPL group.5.Myocardial infarct size was detected by TTC method.Intraperitoneal injection of high-dose TPL(100 ?g/kg)I/R rat drastically decreased I/R induced myocardial infarct size(p<0.01 vs.I/R).6.The real-time PCR results indicated that m RNA expression of TNF-?in middle and high-dose TPL was significantly lower than that in I/R groups(p<0.01).Different dose of TPL could markedly decreased the expression of IL-1?and IL-6(p<0.01).7.ELISA results showed that intraperitoneal injection of different doses(25 ?g/kg,50 ?g/kg,100 ?g/kg)TPL could significantly decreased the expression of TNF-?,IL-1? and IL-6(p<0.05,p<0.01 vs.I/R).8.Western blot analysis demonstrated that different dose of TPL could drastically elevated the expression of p-P38(p<0.01 vs.I/R).9.Intraperitoneal injection of middle-dose TPL(50 ?g/kg)drastically elevated SOD,HO-1 and GSH-PX activities in rat myocardial tissues(p<0.05,p<0.01 vs.I/R),but the level of MDA and GSH had no significant differences.The activities of SOD,HO-1 and GSH-PX were obviously elevated,the level of GSH was obviously increased and the production of MDA markedly reduced in the high-dose TPL(100 ?g/kg)I/R rat compared with the I/R group(p<0.01).10.Western blot analysis demonstrated that intraperitoneal injection of different doses(25 ?g/kg,50 ?g/kg,100 ?g/kg)TPL significantly up-regulated the expression level of nucleus Nrf2 in the rat myocardial tissues(p<0.01 vs.I/R).Conclusions : 1.High-dose TPL(100 ?g/kg)improved the haemodynamic parameters,enhanced myocardial contraction and promoted myocardial functional recovery after ischemia/reperfusion injury.2.High-dose TPL markedly decreased CK,LDH and GOT activities,reduced myocardial injury,markedly ameliorated pathological changes,significantly reduced macrophages infiltration,drastically decreased I/R induced myocardial infarct size.3.TPL(25 ?g/kg,50 ?g/kg,100 ?g/kg)could significantly decreased the expression of TNF-?,IL-1?and IL-6 in I/R rat myocardial tissues,markedly attenuated myocardial ischemia/reperfusion induced inflammation.The activation of P38 MAPK signal pathway by TPL was closely related to ischemia/reperfusion induced inflammation.4.High-dose TPL(100 ?g/kg)enhanced the I/R rats antioxidant effect,obviously elevated SOD,HO-1 and GSH-PX activities,increased the level of GSH and markedly reduced the production of MDA.5.TPL could activated Nrf2 antioxidant signal pathway.TPL played anti-oxidation myocardial ischemia/reperfusion by activating Nrf2 signal pathway. |
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