| Part One The effect of alprostadil on myocardial ischemia / reperfusion injury in ratsObjective To observe the effect of alprostadil on myocardial ischemia / reperfusion injury in rats,and to explore its mechanism.Methods(1)Adult Sprague-Dawley rats were randomly divided into the following six groups:Sham operation group(sham group);I/R group;I/R+Alprostadil(4?g/kg)group;I/R+ Alprostadil(8?g/kg)group.(2)The MI/R animal model was constructed by left anterior descending coronary artery(LAD)ligation,Rats were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion.The levels of serum troponin T,creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH)were measured by NBT.The cardiac function(72 h)was measured by echocardiography.The myocardial tissue morphology was observed under light microscope and electron microscope.The apoptosis of myocardial cells was detected by TUNEL in situ labeling and DAPI staining.The contents of malondialdehyde(MDA),superoxide dismutase(SOD)Catalase(CAT)activity of myocardium were tested.Results(1)Serum troponin T,CK-MB and LDH in I/R group were higher than in Sham group(p <0.05).Compared with I / R group,serum troponin T,CK-MB and LDH were significantly lower in the high dose(p <0.05)and low dose Alprostadil group.(2)Myocardial I / R can lead to an increase in myocardial infarct size,compared with Sham group,the difference was statistically significant(p <0.01);the use of injection with alprostadil 4 or 8 ?g / kg can significantly reduce myocardial I / R caused Myocardial infarct size(p <0.01).(3)Echocardiography was performed for LVIDd,LVIDs,EF.The cardiac function of the I/R rats were decreased than Sham roup,manifested as increased LVIDd and LVIDs(p <0.01)as well as decreased EF(p < 0.05).The high dose of alprostadil could improve the cardiac function of the I/R rats,and the LVIDd and LVIDs were decreased and the EF increased,which was statistically significant compared with the I / R group(p < 0.05).(4)Myocardial histopathological morphology: The left ventricular myocardium was observed by light and electron microscopy.It was found that the rats in the alprostadil group showed mild myocardium necrosis and myocardial interstitial edema compared with the I / R group,accompanied of neutrophil infiltration.(5)By TUNEL and DAPI dyeing,the apoptosis index of I/R group is higher than Sham group(p < 0.05),.compared with the I / R group,the use of injection with alprostadil 4 or 8 ?g / kg can both significantly reduce the apoptosis index(p < 0.05).(6)Compared with the I / R group,the content of MDA in high dose Alprostadil group was significantly decreased(p < 0.05)and the activities of SOD and CAT were increased(p < 0.05).Conclusion Alprostadil can reduce the myocardial ischemia / reperfusion injury in rats.The high dose group is more effective than the low dose group,which is manifested in reducing the myocardial infarct size,reducing the level of serum myocardial enzymes,improving the cardiac function.The mechanism of Alprostadil to MIRI is related with reduce myocardial tissue oxidative stress injury and myocardial cell apoptosis.Part Two The mechanism of cardioprotection effect of alprostadil on myocardial ischemia / reperfusion injury in ratsObjective To explore the mechanism of administration of alprostadil to reduce myocardial ischemia / reperfusion injury in rats and to explore the role of PI3 K / Akt signaling pathway in the protection of alprostadil.Methods(1)Adult Sprague-Dawley rats were randomly divided into the following four groups: I/R group;Alprostadil(8?g/kg)group;Alprostadil(8?g/kg)+LY group;I/R+LY group.(2)The MI/R animal model was constructed by left anterior descending coronary artery(LAD)ligation.Rats were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion.The levels of serum troponin T,CK-MBand LDHwere measured by NBT.The cardiac function(72 h)was measured by echocardiography.The myocardial tissue morphology was observed under light microscope and electron microscope.The apoptosis of myocardial cells was detected by TUNEL in situ labeling and DAPI staining.The contents of MDA,SOD and CAT activity of myocardium were tested.The expression of cleaved caspase-3,Bcl-2 and Bax in myocardium and the expression of Akt,p-Akt,e NOS and p-e NOS were detected by Western Blot.The content of NO in left ventricular myocardium was detected by ELISA,and RT-PCR was used to detect the m RNA transcription levels of TNF-?,IL-6 and IL-1? in myocardium of rats.Results(1)The level pf serum troponin T,CK-MB and LDH in Alprostadil(8?g/kg)group were much lower than in I/R group(p <0.05).While the level of serum troponin T,CK-MB and LDH in Alprostadil(8?g/kg)+LY group were much higher than in single Alprostadil(8?g/kg)group(p <0.05).(2)Alprostadil(8?g/kg)can significantly reduce myocardial infarct size compared with I/R group(p<0.01).Myocardial infarct size of Alprostadil(8?g/kg)+LY group is small than that of I/R group,but bigger than that of Alprostadil(8?g/kg)group(p < 0.01).Alprostadil induced reduction of myocardial defense in the area of myocardial infarction after MIRI can be largely inhibited by LY294002.(3)The high dose of alprostadil could improve the cardiac function of the I/R rats,and the LVIDd and LVIDs were decreased and the EF increased,which was statistically significant compared with the I / R group(p< 0.05).The cardiac function of Alprostadil(8?g/kg)+LY group is a little better than that of I/R group,but much worse than that of Alprostadil(8?g/kg)group(p< 0.05).It was shown that LY294002 could inhibit the protective effect of alprostadil on cardiac function in myocardial I / R rats.(4)Myocardial histopathological morphology: It was found that the rats in the Alprostadil(8?g/kg)group showed mild myocardium necrosis and myocardial interstitial edema compared with the I / R group,accompanied of neutrophil infiltration.Alprostadil(8?g/kg)+LY group showed more myocardium necrosis and myocardial interstitial edema compared with Alprostadil(8?g/kg)group.(5)From TUNEL and DAPI dyeing,the apoptosis index of Alprostadil(8?g/kg)group is decreased than I/R group(p<0.01).The apoptosis index of Alprostadil(8?g/kg)+LY group is higher than that of Alprostadil(8?g/kg)(p< 0.05).(6)Compared with the I/R+ Alprostadil(8?g/kg)group,the content of MDA was high while the activities of SOD and CAT were decreased in I/R+ Alprostadil(8?g/kg)+LY group(p < 0.05).Indicating that alprostadil of high dose can anti-oxidative stress,this cardioprotective effect can be partly blocked by LY294002.(7)High dose of alprostadil(8?g/kg)could promote the expression of Bcl-2 in myocardium of myocardium I/R rats and inhibit the expression of Bax and cleaved caspase-3(p < 0.01).This effect could be mostly blocked by PI3 K inhibitor LY294002.(8)High dose of alprostadil(8?g / kg)can promote the phosphorylation of Akt and e NOS in myocardium of myocardial I / R rats(p < 0.01)and promote the release of NO in ischemic myocardium(p < 0.01).The above effects can be largely inhibited by PI3 K inhibitor LY294002.(9)High dose of alprostadil(8?g/kg)down-regulates the m RNA levels of TNF-?,IL-6 and IL-1? in myocardium of myocardium I / R rats(p < 0.01).This effect can be largely blocked by PI3 K inhibitor LY294002.Conclusions(1)Alprostadil plays an anti-apoptotic effect,by increasing the expression of Bcl-2,down-regulating cleaved caspase-3 and Bax protein.Alprostadil can also upregulate phosphorylated Akt and phosphorylated e NOS levels,and promoted NO expression.PI3 K / Akt / e NOS / NO signal transduction pathway is involved in the mechanism of action of alprostadil against cell apoptosis.(2)Alprostadil decreases the transcription of protein m RNA of TNF-?,IL-6 and IL-1? induced by MIRI.Thus alprostadil plays a protective effect of myocardial ischemia / reperfusion injury by anti-inflammatory reaction.It can also reduce the expression of TNF-? protein,by inhibiting the death receptor dependent on the TNF receptor family,plays an anti-apoptotic effect to achieve its mitigation of myocardial ischemia / reperfusion injury in the heart protection.Part Three The effect of ginsenoside Rg3 on myocardial ischemia / reperfusion injury in rats and its mechanismObjective To evaluate the effect of ginsenoside Rg3 on cardiac function impairment induced by myocardial I/R inrats.Methods(1)Adult Sprague-Dawley rats were randomly divided into the following four groups(n =18-20):Sham operation group(sham group);I/R group;ginsenoside Rg3(5mg/kg)group,and ginsenoside Rg3(20mg/kg)group.(2)The MI/R animal model was constructed by left anterior descending coronary artery(LAD)ligation,Rats were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion.(3)Electrocardiogram of reperfusion 24 h,echocardiogram,hemodynamics,blood serum for Tnt,CK-MB,LDH were tested.The apoptosis of cardiac myocyte detected by terminal deoxynucleotidy 1 transferase-mediated d UTP-biotin nick end labeling(TUNEL)method.The expression of p53,Bcl-2,Bax and cleaved caspase-3 were extracted from the myocardium after 24 h of reperfusion by Western Blot.The protein content of TNF-? and IL-1? in left ventricular myocardium were determined by ELISA.Results(1)Ginsenoside Rg3 increased a left ventricular fractional shortening and left ventricular ejection fraction.Treatmentwith ginsenoside Rg3 also alleviated increases of left ventricular enddiastolic pressure and decreases of left ventricular systolic pressure and ądp/dt in myocardial I/R-rats.(2)Ginsenoside Rg3 decreased apoptosis cells through inhibiting the activation of caspase-3.(3)Ginsenoside Rg3 also caused significant reductions of the contents of TNF-?and IL-1? in left ventricles of myocardial I/R-rats.Conclusion The findings suggested that ginsenoside Rg3 possessed the effect of improving myocardial I/R-induced cardiac function impairment and that the mechanism of pharmacological action of ginsenoside Rg3 was related to its properties of antiapoptosis and anti-inflammation. |
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