| ObjectiveMyocardial ischemic reperfusion injury(MIRI)is a frequent tissue and organ injury in clinical practice,but the exact mechanisms of MIRI is not very clear at present.The previous study about MIRI consider that is an acute inflammatory reaction, Tumor Necrosis Factor-alpha(TNF-α)is one of the important reason that induced cardiac function impairment and myocardial cell necrosis increased while MIRI, however,inhibiting of TNF-αis beneficial to the recovery of myocardial function.For the past few years,the prevention of MIRI have already acquired much progression by using of the traditional Chinese medicine.Xue Bi Jing Injection is abstracted from 36 prescriptions of traditional Chinese medicine,having the properties of anti-bacterial toxin,decreasing the level of endotoxin,regulating immunity and mediators of inflammation,improving microcirculation,protecting vascular endothelial cells etc. The main task of this research is to observe the protection of Xue Bi Jing Injection to MIRI and to approach the possible mechanisms of this effects by using of the isolated Langendorff perfusion model.Material and MethodsThirty adult Wistar rats(body weight:250±50g)were used in this study with no restricting paroecious.All animals were randomly divided into test groups(Xue Bi Jing Injection)and control groups respectively(n=15 per group).After anesthesia induction(10%ChloralHydrate,intraperitoneal injection)and heparinization (heparin-sodium intraperitoneal injection 500U for each Wistar rats),hearts were excised rapidly,rinsed thoroughly in chilled 4℃Krebs-Henseleit buffer(K-H buffer), connecting the aortic cannulation to the the isolated Langendorff perfusion model,and then perfused retrogradely in the isolated Langendorff model,at a constant perfusion pressure of 70 mmHg,with 37℃oxygenated K-H buffer.After 15 minutes equilibration,the hearts were perfused retmgradely with cardioplegia(Test group:implicated Xue Bi Jing Injection in St ThomasⅡsolution(4ml/kg);control group:St ThomasⅡsolution only).All these hearts received 30 minutes of global ischemia and reperfused with K-H solution at 37℃until 120 minutes.The samples of myocardium of left ventricle were collected after the experiment is completed,and then be fixed in 10%formaldehyde for 12-20h.The tissue was then dehydrated,embedded in paraffin,and sectioned(5p,m sections)in a standard manner,the other sample of myocardium was excised and then frozen rapidly and stored at-70℃.we collected and measured the cornory flow at pre-ischemia and 30,60,90,120 minutes of reperfusion respectively(ml/min),and detected CK of the collected effluent(3ml)with automatic biochemistries' detecting instrument and Viewing the morphology changes of myocardium by optical microscope.Then detecting TNF-αwith ELISA.Statistical analysis:All measurement data is expressed as(?)±s,and statistical significance was evaluated by multiple comparison of T test and ANOVA.A P value of 0.05 or less was considered to indicate statistical significance.All statistical processes were completed via SPSS(V13.0).Results1.Changes of cornory flow:Compared with control group,the CF was significant raised in test group(Xue Bi Jing Injection)at 30,60,90,120 minutes after reperfusion(P＜0.05).2.Changes of CK in the collected cornory effluent:Compared with control group,the decreases of CK were significant in test group(Xue Bi Jing Injection)at 30,60,90,120 minutes after reperfusion(P＜0.05).3.Myocardial morphology changes:The endocardium in control group was found oedema,hemangiectasis and hyperaemia in some parts of myocardium and inflammatory cell infiltration in myocardium.However,the cadiocyte are normal on the whole and the oedema is not very obvious comparatively.4.Concentration of TNF-αin myocardium:Compared with control group,the concentration of TNF-αwas significantly decreased in test group(Xue Bi Jing Injection) after reperfusion(P＜0.05). DiscussionTNF-αis an inflammatory cell cytokine,with the properties of autocrine, produced by cadiocyte at the early age of MIRI,inducing myocardial function abnormal and cadiocyte necrosis in MIRI,pyaemia,chronic cardiac insufficiency,viral myocarditis and the homograft rejection of heart.When reperfusion begins, macrophage and cadiocyte will secrete TNF-αby stimulus,the effects on leukocyte by TNF-αis related to two different ways,which will induce inflammation and tissue impaired:(1)The direct action:About 10 minutes after reperfusion,the chemotactic effect and accumulation of leukocyte will induce the release of chemical materials and then reinjury.(2)TNF-αeffects on endotheliocyte in the primarily injury site,which will activate the inert adherence factor on the surface of leukocyte,and then the leukocyte will move to the primarily injury site adhering to the injuryed tissue cells to release a series of chemical materials inducing tissue reinjury.The process of the above usually slowly,it will take about 4 hours the serious tissue reinjury development. Besides the significant increase of TNF-αin blood plasma at 1 and 4 hours after reperfusion,myeloperoxidase in the ischemic myocardium is also obviously increased, which reflects the infiltration of neutrophil.With optical microscope,it is found that the myocardium is infiltrated by generous of neutrophil,The aggregation of neutrophil in myocardium is closely correlated with the release of TNF-α,malonaldehyde,reflecting oxyradical injury,is significant increased in the ischemic myocardium.These consequence are:When MIRI happens,the level of TNF-αis increased,meanwhile, the infiltration of neutrophil and myocardial damage are also aggravated.Accordingly, TNF-αplays an important role in the MIRI.For myocardial preservation under MIRI, inhibiting of TNF-αmust be critical.The effective constituent of Xue Bi Jing Injection are safflor yellow A,Ligustrazine,tanshinol,ferulic acid,peoniflorin and protocatechualdehyde,mainly extracted from Safflower,Red Peony Root,Szechwan Lovage Rhizome,Danshen Root and Chinese Angelica.The proceed investigations have manifested that Xue Bi Jing Injection is an effective antagon for endotoxin,besides,it has the ability of down regulation pro-inflammatory mediators and accommodation of immunological reaction, finally,Xue Bi Jing Injection also provide the function of protection endothelium, improving microcirculation,avoidance tissue damage induced by endotoxin.Some other experiments confirmed Xue Bi Jing Injection can also obviously inhibit the level of TNF-αin liver and lung tissue and make it in normal range.Many constituents of traditional Chinese medicine contained in Xue Bi Jing Injection were confirmed has the ability of inhibitory action in injury of capillary vessel,release of neutrophil and apoptosis.The animal model of MIRI made in this experiment is successful,compared with control group,CF was significantly raised in test group(Xue Bi Jing Injection)at each time point after reperfusion,while the level of CK is significant decreased; inflammatory reaction and oedema are also decreased obviously under optical microscope.The concentration of TNF-αin myocardium was obviously decreased. From the mechanisms of MIRI and the produce mechanism of TNF-α,Xue Bi Jing Injection can reducing the production of oxyradical,protect cellular membrane,decrease lipid peroxidation,relieving calcium overload,mitigation inflammatory reaction and reducing attack of endotoxin,restrain the level of TNF-α.by this means, maybe to provid myocardial preservation after MIRI.Conclusion1.Xue Bi Jing Injection could increase CF,inhibit the release of CK,relieve the injury of cadiocyte,protect cellular membrane while MIRI.2.Xue Bi Jing Injection could obviously improve myocardial ultrastructure after MIRI,therefore,it has the ability of myocardial preservation.3.Xue Bi Jing Injection could significantly decreased TNF-αin myocardium.And maybe to provid myocardial preservation after MIRI by this way.
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