| ObjectivesThe myocardial ischemia-reperfusion injury is very common in our daily clinical work, such as thrombolysis therapy, coronary angioplasty, aorta blockage, post-cardiopulmonary bypass, etc. It not only caused regional damage, but also spread to the distant non-ischemia organ, which lead to arrhythmia myocardial stunning cardiac insufficiency, or even fatal multiple organ dysfunction syndrome. These phenomena displayed as descending blood pressure after the reperfusion, no-reflow or slow-flow phenomenon when the blood flow was rebuilt in patients with the acute myocardial infarction , so it could not implement myocardial reperfusion, what's more the incidence and death rate of re-infarction malignant arrhythmia and heart failure, which seriously affected the prognosis of patients. Therefore, how to mitigate the reperfusion injury and safeguard the ischemic myocardium utmost have been paid more and more attention.It's Murry who reported the ischemic preconditioning opened up one original study domain for the prever(?)on of myocardial ischemia-reperfusion in 1986.Its original definition was that a kind of transient post-ischemic evoked quick adaptation reaction would be able to reduce the cellular necrosis in the sequent long ischemia. At present, people gradually realize that IPC is a widespread existing biological phenomena , IPC can effectively diminish the myocardial damage at ischemia-reperfusion and protect the myocardium. The animal experiment and clinical observation have confirmed that IPC is an effective myocardial protection method. Some investigations proved remote non-myocardial ischemic preconditioning such as renal artery mesenteric artery and femoral artery etc can also provide protection for the myocardium. The intervention of IPC is before the myocardial ischemia, but the patients' ischemia is impossibly predicted, so IPC is confined in the clinical application. Clinically, we can't enforced preconditioning on the health adults in order to mitigate the myocardium damage which is brought out by the PCI, In addition, because of short of medical consciousness, the patients often visit the doctor when the myocardium is serious ischemic or infarct. Therefore, to search a non-traumatic myocardial protection behind the myocardial ischemia events will have very significant practical value and clinical sense.Recently, domestic and overseas scholars proposed the ischemic post-conditioning and carry out related investigation. Compared with preconditioning, they discovered it has similar heart protection. Its mechanism may be different , ischemic post-conditioning will have better clinical manipulation and application. Ischemic post-conditioning was original raised by Zhao, he carried out several studies. When the coronary artery was reperfused, repeated temporal coronary artery open/close would be able to mitigate ischemia-reperfusion damage and produce notable protection, which was named ischemic post-conditioning. Experimental result indicated: ischemic post-conditioning can reduce infarct size, protect endothelial function, reduce the concentration of neutrophil leucocyte in the ischemic infarct domain, accordingly produce protection. This kind of protection is similar to IPC to some degree, at the first minutes of reperfusion , ischemic post-conditioning can specifically mitigate ischemia-reperfusion damage.At present, Ischemic post-conditioning has been confirmed by different animal model (including dog hare and rat) and isolated heart, It can produce similar heart protection to ischemic precondition. Galagudza who came from Russia federal Medical College discovered: ischemic post-conditioning can notably reduce the ventricular tachycardia which is produced by the continuing reperfusion on the isolated rat heart perfusion model, it is a new method to prevent reperfusion damage. It is also reported that non-traumatic both pelvic limbs ischemic post-conditioning can protect the ischemia-reperfusion myocardium. People discovered : the post-conditioning of non-myocardium tissue such as kidney intestine and skeletal muscle not only mitigated local reperfusion damage , but also protected the remote heart, we called the phenomenon remote post-conditioning.Even though IPC has been already recognized by us, its exactly mechanism, especially intra-cellular signal transduction mechanism initiated by IPC, was still incompletely clear. People have carried out considerable studies and gradually realized that IPC was a cardiac self-protect mechanism, it triggered the heart to release some endogenous active compound in the earlier period of ischemic, regulating the heart function through intra-cellular signal transduction system, accordingly it raised the toleration of myocardium to sequent longer ischemia and provided earlier and later protection for the myocardium. Today, we considered three basic component elements involved in the cellular signal transduction: triggering materials (endogenous active materials), mesomerism materials (protein kinase) and effecter substances (ion channel and protector protein) .At present, IPC's mechanism was that temporal ischemia-reperfusion could release considerable endogenous substances such as adenosine bradykinin meconium etc, these substances could affect correlative receptor, induced the activation of protein kinase C, then activated other kinases, eventually these enzymes affected mitochondrial ATP sensitive channels and protected the heart. PKC is one of important ingredient in myocardial cell monophosphoinositide signal transduction system, PKC s role has been proved in the ischemic preconditioning. Recently, findings indicated: IPC can also protect the heart through affecting the reconstitution of myocardial cellular membrane, reducing and inhibiting myocardial cell apoptosis.At present, we studied little about ischemic post-conditioning, especially remote organic ischemic post-conditioning, it should be studied whether or not its mechanism was similar to that of ischemic preconditioning . Someone guessed the protection of renal ischemic post-conditioning was possibly related to protein kinase C, it activate reperfusion injury salvage enzyme pathway through activating protein kinase C, at last it made effective apparatus phosphorylation and induced myocardial preservation, those effective apparatus was mainly mitochondrial ATP sensitive channels.The aim of this experiment was to approach whether or not renal ischemic post-conditioning could mitigate ischemic-reperfusion injury through animal experiment whether or not protein kinase C played an important role in renal ischemic post-conditioning through interfering with protein kinase C inhibitor-GF109203X.Materials and Methods1 Experimental animalsHealthy New Zealand rabbits 64 (offered by Henan Medical College animal experiment center), female and male are not restrained, their quality are about 2.0-2.5kg.2 Main agentsNitrotetrazolium blue chloride (Lingjin fine chemicals limited company, Shanghai), SOD kits MDA kits(Jiancheng biotechnology research institute, Nanjing); Creatine kinase isozyme kits (Dong' ou biotechnology limited company, Zhejiang), Cardiac troponin I quick quantitation diagnostic kits (Jidan biotechnology limited company, Nanjing); Condensing DAB kits Sticking tablets (polylysine) (Zhongshan jinqiao biotechnology limited company, Beijing), Tunel kits (Roche company, Germany); Anti-Bax Anti-Bcl-2 (Boster biotechnology limited company, Wuhan),β-actin Goat anti-Rb IgG (Boisynthesis biotechnology limited company, Beijing), PVDF membrane Glycocine Tris base SDS Methanol (Fulin biotechnology limited company, Qingdao), SDS-PAGE Gel Parparation Kit Prestained Protein Molecular Weight Marker BCA Protein Assay Kit SDS-PAGE protein loading buffer (5×) Ponceau S Staining Solution Commassie Blue Fast Staining Solution RIPA Lysis Buffer PMSF (Beyotime Institute of Biotechnology, Haimen) .3 MethodsAll rabbits were subjected to a total of 60 minutes of left anterior descending coronary artery occlusion(LADO)and 6 hours of reperfusion. The rabbits are randomly distributed (n=8 in each group) : (1)Ischemia-reperfusion(IR): LADO and reperfusion only with no other intervention; (2)Ischemic preconditioning(IPC):Three cycles of myocardial ischemia(5min) and reperfusion(10min) preceded the 60 minutes of left anterior descending coronary artery occlusion (LADO) and 6 hours of reperfusion; (3) Renal ischemic postconditioning(RI-Post): This group is divided into two subgroups: the first subgroup (RI-PostF) : after 60 minutes of LADO, the left renal artery was occluded 30 seconds and released 30 seconds for three cycles, then the coronary artery was perfused 6 hours; the second subgroup (RI-PostS) : after 10 minutes of reperfusion, the left renal artery was occluded 30 seconds and released 30 seconds for three cycles, then the coronary artery was perfused 6 hours ; (4)Ischemic preconditioning+ the first subgroup of Renal ischemic postconditioning(IPC+RI-PostF) : as the basic of IPC , executing the manipulation of RI-PostF group, then the coronary artery was perfused 6 hours ; (5)Medicine intervention(MI): after 50 minutes of LADO rabbits were received 0.05 mg/kg IV of the PKC antagonist GF109203X,then the left renal artery was occluded 30 seconds and released 30 seconds for three cycles at coronary artery reperfusion.4 . Animal experimental surveying index(1) Hemodynamics indexsEach group was recorded heart rate and blood pressure at different time, and analyzed the degree of ST raising.(2) Biochemical indicatorsEach group was determined the density of serum superoxide dismutases (SOD ) and malonaldehyde (MDA) at different time.(3) Myocardium enzymes detectingEach group was determined the density of serum creatine kinase isoenzyme and cardiac troponin I at different time.(4) Creatinine and Blood urea nitrogen detectingIn renal ischemic postconditioning group, Cr and Bun were detected before and after the left renal artery was occluded.(5) Morphology indexs When the experiment was finished, each group was quickly taken the heart and dyed by NBT, thus we determined the myocardium infarct size.(6) Tunel detecting apoptosisAccording to the immune histochemistry principle, the apoptosis information of each group was observed.( 7) Spectroscopy observingWhen the reperfusion was finished, we observed the myocardium morphology changing by HE dying ischemic necrosis myocardium. We also observed the left renal tissue before and after the left renal artery was occluded.( 8) Electron microscope observingWhen the reperfusion was finished , we could observe myocardial cell ultrastructure changing by undertaking ultrathin section.(9) The expression of Bcl -2 Bax protein kinase CWe detected the expression of myocardium apoptosis related gene bcl-2 bax by western blot technology. We undertook quantitative analysis by gel analysis software--Quantity one and undertook single-factor analysis of variance by software-Origin 75.We detected the expression of protein kinase C in the medicine intervention group and renal ischemic postconditioning group.5 Data statistical analysisExperimental data was expressed with x±s and analyzed by statistics software-SPSS 12.0, data was compared with single-factor analysis of variance in the samegroup and compared with q test in two groups.P <0.05 was defined a statistical significance.Experimental results1 The protection of renal ischemic post-conditioning to rabbit myocardium ischemic reperfusionWhen the myocardium was reperfused, the renal ischemic post-conditioning could produce the same protection as ischemic preconditioning: (1)Improving hemodynamics indexs strikingly, inhibiting the decreasing of heart rate and blood pressure; (2) Improving the changing of serum biochemical indicators, inhibiting the decreasing of SOD and the increasing of MDA; (3)Inhibiting the releasing of myocardium enzymes, lowering the increasing degree of CK-MB and cTNI; (4)Contracting the infarct size. On the base of ischemic preconditioning, however, renal ischemic post-conditioning did not improve those above detection index.(5)Cre and Bun were no statistical difference in the serum level before and after the left renal artery was occluded.2 The mechanism of the protection to rabbit myocardium ischemicreperfusion by renal ischemic post-conditioningCompared with ischemic reperfusion group, renal ischemic post-conditioning, which could reduce the apoptosis, inhibit the ultrastructural changing of myocardium and increase the expression of bcl-2, reduce the expression of bax, would protect the myocardium eventually. Protein kinase C inhibitor GF109203X could reduce the expression of protein C and abolish the myocardium protection by renal ischemic post-conditioning.Conclusions1 The animal experiment indicates: in the rabbit ischemic reperfusion model, at the earlier period of reperfusion, repeated many times blocking and opening renal artery (ischemic post-conditioning) can produce the same protection as the ischemic preconditioning ; but delayed ischemic post-conditioning can not protect the myocardium.2 When the ischemic myocardium is reperfused , renal ischemic post-conditioning can not produce supernumerary protection on the base of ischemic preconditioning.3 The mechanism of the protection of renal ischemic post-conditioning is possible related to activation protein kinase C.
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