| Purpose:Ischemia reperfusion injury can be seen in the heart,brain,lung,kidneys and other important organs of human body.To explore the pathological mechanism,reduce the injury as possible as we can,and improve the clinical efficacy have become one of the hottest topics on worldwide medical research field in recent years.Reflow failure or reflow not smoothly after ischemia-reperfusion may induce to arrhythmia or even reinfarction and death,which negatively influences the clinical prognosis of revascularization therapy.The mechanism of myocardial ischemia-reperfusion injury has been proved related with coronary microcirculation.Modern medical therapeutic regime is not good enough on safety and efficacy of prevention and treatment on coronary microcirculation disorder,especially on the postoperative treatment of revascularization.Traditional Chinese medicine is holding dominant position of preventing before the disease arises and preventing disease from exacerbating,which is included as two aspects,clinical curative effect is stable and more economical for patients.At present,although there had been great progress on the research of prevention and treatment by traditional Chinese medicine monomer and compound about myocardial ischemia-reperfusion injury,in-depth study about the mechanism of traditional Chinese medicine multi-target action is still inadequate.Based on the theory of "wind disturbing the veins and meridians of heart as diseases",Professor Gong proposed the treatment methods of tonifying qi and dispelling wind,nourishing blood and relieving spasm of coronary microvascular,dredging collaterals and relieving pain.The compound is summarized from her own clinical experiences and combined with the ancient prescriptions of predecessors,formed the Astragalus Compound as a tonifying qi and dispelling wind Chinese herbs prescription.The compound implied tonifying the qi and nourishing the blood and collaterals deficiency of heart,maybe effective by searching and removing the toxic wind of internal disturbance,so as to help the heart and collaterals to reconcile qi and blood,relieving spasm,and repair the damaged myocardial cells and endothelial cells due to ischemia-reperfusion injury.Professor Gong believes tonifying qi and dispelling wind can be therapeutic for myocardial ischemia-reperfusion injury without leaving pathogenic factors,and meanwhile,eliminating pathogenic factors without harming the normal tissues and organs.According to the pathological characteristics of deficiency and excess,Professor Gong's therapeutics can be treating both manifestation and root cause of disease.Taking the Sprague-Dawley rats established as model of myocardial ischemia-reperfusion injury by ligation of coronary artery,we try to explore the protective effects of tonifying qi and dispelling wind Chinese medicine compound on myocardial ischemia-reperfusion injury by observating the endothelial function,inflammatory reaction,expression of apoptosis related factors protein or gene,and other indicators of Sprague-Dawley rats,what's more,try to reveal the mechanism,enrich the theoretical connotation on "wind disturbing the veins and meridians of heart as diseases",and provide experimental basis for clinical application.Material and method:1.Establishment of myocardial ischemia-reperfusion injury model and effects of tonifying qi and dispelling wind Chinese medicine compound on endothelial function of model rats:120 SD rats were randomly divided into 6-groups,including dummy surgery group,model group,perindopril group,and tonifying qi and dispelling wind Chinese medicine compound low/medium/high dose groups,20-rats in each group.According to the daily recommended dosage for adults(calculated by weight of 60kg),the rats were given the drug after converting the dosage,once a day,and held the probe seven days,long.The rats in the dummy group and model group received normal saline solution 10 ml/Kg by probe;rats in the traditional Chinese medicine group with low/medium/high dose received the Chinese medicine 1.8 g/kg,3.6 g/kg and 7.2 g/kg by gavage;the rats in the perindopril group were given perindopril tablet 0.4 mg/kg by gavage.One hour after the end of gastric perfusion on the 7th day,the myocardial ischemia-reperfusion model was established.The operation process of the sham operation group was the same as other groups,except without ligation.ECG of rats at 30 min of ligation and 120 min of reperfusion were recorded respectively;lead ? was selected as the main observation target in the experiment,and ST segment change was taken as the marker of myocardial ischemia;successful ligation was judged by significant elevation of ST segment,and success of reperfusion was judged by 1/2 of elevation of ST segment.After the model was set up successfully,blood and heart samples were collected.The rats were fasting on the night before(the 6th day)but drinking freely.Blood was taken from abdominal aorta of rats,centrifuged after standing,and supernatant was taken for storage at-20?;after blood sampling,rats were immediately killed,and hearts were taken out under aseptic conditions,cleaned with sterile normal saline,fixed with 10%formaldehyde,and frozen at-20? for storage.The levels of TXA2,PGI2,STM,sEPCR in serum were detected by ELISA.2.Effects of tonifying qi and dispelling wind Chinese medicine compound on vascular endothelial inflammatory factors in rats with myocardial ischemia-reperfusion injury:IL-8,IL-6 and TNF-? in serum were detected by ELISA.3.Effects of tonifying qi and dispelling wind Chinese medicine compound on apoptosis of vascular endothelial cells in rats with myocardial ischemia-reperfusion injury:the heart tissues of rats in each group were collected,100 mg of left ventricular apex was weighed by electronic balance,and the tissues were ground by high-throughput tissue grinder.The ground myocardial tissues were placed in 500 UL lysate prepared in advance,centrifuged at 12000 rpm for 10 min,and then the supernatant was taken;the protein expression levels of Akt,P-Akt,Bax and Bcl-2 were determined by Western blot;the mRNA expression levels of Akt,eNOS,Bax and Bcl-2 were determined by RT-PCR.Primer design software primer premier 5.0 was used to design primers,and 2-??ct method was used to analyze the gene expression differences in each sample.The measurement data are expressed as(?).One-way ANOVA was used to compare the data between different groups,LSD test was used to compare the two groups.P<0.05 means the statistical difference make sence,P<0.01 means significant difference,or P>0.05 means the statistical difference make no sence.Results:1.Establishment of myocardial ischemia-reperfusion injury model and effects of tonifying qi and dispelling wind Chinese medicine compound on endothelial function of model rats:1.1 Rat myocardial ischemia-reperfusion injury model:the survival of rats in each group during the model establishment:1 rat died after anesthesia;37 rats died due to respiratory arrest,massive hemorrhage and ventricular fibrillation during tracheal intubation,ligation and reperfusion,including 5 rats in sham operation group and 32 rats in other models.The number of surviving rats in each group met the needs of follow-up experiments.Modeling:ECG monitoring was used to observe the changes of lead ?ECG during ligation and after reperfusion.Eight rats that did not meet the ST segment change standard were screened out,and the total number of successful modeling was 59.The number of successful modeling rats in each group met the requirements of index detection.1.2 serum TXA2,PGI2 levels and TXA2/PGI2 ratio:compared with sham operation group,TXA2 level in model group was significantly increased,PGI2 level was significantly decreased,and TXA2/PGI2 ratio was significantly increased(P<0.01);Compared with the model group,the TXA2 content of perindopril group and each dose of Yiqi soufeng Huangqi compound were significantly decreased,the PGI2 content was significantly increased,and the TXA2/PGI2 ratio was significantly decreased(P<0.01);Compared with perindopril group,the contents of TXA2 and PGI2 and TXA2/PGI2 in Yiqi soufeng middle dose group had no significant difference(P>0.05).1.3 the levels of serum STM and sEPCR in the model group were significantly higher than those in the sham operation group(P<0.01);Compared with the model group,the contents of STM and EPCR in perindopril group and Yiqi soufeng Decoction groups were significantly decreased(P<0.01);Compared with perindopril group,the contents of STM and EPCR in each dose group were increased(P<0.05).2.Effects of Yiqi soufeng herbs on vascular endothelial inflammatory factors in myocardial ischemia-reperfusion injury model rats2.1 the levels of serum IL-6 and IL-8 in the model group were significantly higher than those in the sham operation group(P<0.01);Compared with the model group,the contents of IL-6 and IL-8 in perindopril group and each dose group were significantly decreased(P<0.01);Compared with perindopril group,the contents of IL-6 and IL-8 in middle dose group had no significant difference(P>0.05).2.2 the level of serum TNF-a in the model group was significantly higher than that in the sham operation group(P<0.01);Compared with the model group,the content of TNF-a in perindopril group and each dose of Chinese medicine group were significantly decreased(P<0.01);Compared with perindopril group,there was no significant difference in TNF-a content in middle dose group(P>0.05).3.Effect of Yiqi soufeng herbs on apoptosis of vascular endothelial cells in myocardial ischemia-reperfusion injury model rats3.1 expression of Akt and eNOS mRNA in each group:compared with sham operation group,the expression of Akt and eNOS mRNA in model group was significantly lower(P<0.01);Compared with the model group,the expressions of Akt mRNA and eNOS mRNA in perindopril group and Yiqi suofeng Decoction groups were increased(P<0.05);Compared with perindopril group,the expression of Akt mRNA and eNOS mRNA in Yiqi soufeng middle dose group had no significant difference(P>0.05).3.2 protein expression of Akt and p-Akt and p-Akt/Akt ratio:compared with sham operation group,protein expression of Akt and p-Akt in model group decreased significantly(P<0.01);Compared with the model group,the protein expressions of Akt and p-Akt were significantly increased in perindopril group and Yiqi soufeng Decoction groups(P<0.01);Compared with perindopril group,there was no significant difference in Akt protein expression and p-Akt/Akt ratio between middle dose group and high dose group(P>0.05).3.3 the expression of Bcl-2 and Bax mRNA in each group:compared with the sham operation group,the expression of Bcl-2 and Bax mRNA in the model group increased(P<0.05);Compared with the model group,the expression of bcl-2 mRNA in perindopril group and Yiqi suofeng group was increased,while the expression of Bax mRNA was decreased(P<0.05);Compared with perindopril group,the expression of bcl-2 mRNA and Bax mRNA in Yiqi soufeng middle dose group had no significant difference(P>0.05).3.4 the expression of Bcl-2 and Bax protein and the ratio of Bcl-2/Bax in each group:compared with the sham operation group,the expression of Bcl-2 and Bax protein in the model group were significantly increased,and the ratio of Bcl-2/Bax was significantly decreased(P<0.01);Compared with the model group,the expression of Bcl-2 protein and the ratio of Bcl-2/Bax in perindopril group and Yiqi soufeng middle dose group were significantly increased,while the expression of Bax protein was significantly decreased(P<0.01);Compared with perindopril group,there was no significant difference in the expression of Bcl-2 protein in Yiqi soufeng middle dose group(P>0.05).Conclusion:1.Astragalus membranaceus compound can regulate the expression of vascular endothelial function injury factors,suggesting that it may have protective effect on vascular endothelial function in Miri rats.2.Yiqi soufeng prescription can down regulate IL-8,IL-6 and TNF in rats-? These results suggest that the protective effect of Yiqi soufeng compound on myocardium of Miri rats may be related to the inhibition of inflammatory reaction.3.Yiqi soufeng herbal compound can up regulate the expression of Akt and its downstream eNOS mRNA in PI3K/Akt signaling pathway;Intervention of Akt and p-Akt protein expression level,regulation of p-Akt/Akt balance;At the same time,it regulates the expression of Bcl-2 and Bax genes and proteins.It is suggested that the mechanism of Yiqi soufeng Chinese herbal compound in improving vascular endothelial function of Miri may be related to activation of PI3K/Akt signaling pathway and regulation of cell apoptosis. |
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