Protective Effects And Mechanisms Of Ulinastatin On Cerebral And Myocardial Ischemia/Reperfusion Injury In Rats

BACKGROUD AND OBJECTIVES Hypoxic ischemic cardio-cerebral vascular diseases with high incidence, high morbidity, high mortality, have done serious harm to human health. Even if intensive medical and interventional therapy bring the realization of the early restoration of blood supply, the restoration of blood supply leads to reperfusion injury inevitably. So morbidity and mortality remain high. It has been accepted recently that ischemic cell injury arises from complex interactions between multiple electrophysiological, hemodynamic, and biochemical cascades, which include disturbances in energy metabolism, modifications in synaptic transmission, production of reactive oxygen species, stimulation of the inflammatory process, endothelial dysfunction, et al. All protective agents so far targeting a specific pathway in ischemic cascade failed to demonstrate clinical efficacy. Therefore, the search for efficient drug in novel mechanism and therapeutic approaches is even more critical.Ulinastatin (UTI) is one of the Kunitz-type proteaseinhibitors found in urine.It has been widely used in the systemic inflammatory syndrome, sepsis, shock, disseminated intravascular coagulation, severe acute pancreatitis, cardiopulmonary resuscitation, transplantation and ischemia reperfusion injury, etc. There is much more evidence that it can scavenge free radicals and inhibit the releasing of inflammatory mediator. But the effect and mechanism of UTI in the treatment of ischemic cardio-cerebral vascular diseases remain unknown, and the effects on learning and memory functions of neurons in the ischemic brain are not clear. This experiment is to study the effect and molecular mechanism of ulinastatin on cardiac and cerebral ischemia-reperfusion injury in rats. METHODS The first part:the effects of ulinastatin on cerebral ischemia reperfusion injury.Adult male Wistar rats were used in the experiment. Animals were randomly divided into five groups:sham operation group, ischemia-reperfusion (I/R) injured group, high dose of ulinastatin group (40OOOU/kg, ip), middle dose of ulinastatin group (20OOOU/kg, ip), low dose of ulinastatin group (10OOOU/kg, ip) Morphological examination of the CA1region of hippocampus at24h after ischemia-reperfusion injury was executed by H&E staining. Morris water maze test was done after administration for the study of neuropretive effect of UTI. The expression of NF-κB p65and ICAM-1were measured by Western-blot. The levels of tumor necrosis factor a (TNF-a) and interleukin10(IL-10) were determined by enzyme-linked immunosorbent assay. The levels of Superoxide Dismutase (SOD) and malondialdehyde (MDA) were determined by biochemical colorimetry.The second part:the effects of ulinastatin on myocardial ischemia reperfusion injury.There are ligation of the left anterior descending coronary artery for30minutes,3hour of reperfusion in rat myocardial ischemia/reperfusion injury model. Rats were randomly divided into five groups:sham operation group, ischemia-reperfusion (I/R) injured group, ischemic postconditioning group (I-postC group), ulinastatin coronary artery routes of administration group (10000U/kg), ulinastatin intravenous routes of administration group (10000U/kg). Myocardial infarct area was detected by the nitro blue tetrazolium (NBT) staining method; cardiac morphologic changes were observed by H&E staining; ECG were recorded changes at multiple time points of contrast variation; The levels of tumor necrosis factor a (TNF-a) and interleukin10(IL-10) in serum were determined by enzyme-linked immunosorbent assay. The content of superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide(NO) in serum were determined by biochemical colorimetry.RESULTS The first part:the effects of ulinastatin on cerebral ischemia reperfusion injury.Rats with focal cerebral ischemia for2h,24h after reperfusion and apparent neurological dysfunction, indicate that the model is success. The pyramidal cells of hippocampus of rats could be protected by ulinastatin and the expression of ICAM-1, MMP-9and NF-κB p65in the ischemic brain could be increased after administration of ulinastatin. In addition, ulinastatin could decrease the latency of escaping, increase the percentage of swimming distance in the platform quadrant and number of entries in the target area. Finally, under the effect of ulinastatin, the level of TNF-a in serum was decreased while IL-10was increased and the levels of MDA in brain tissue homogenate was decreased with SOD increased.The second part:the effects of ulinastatin on myocardial ischemia reperfusion injury.ECG showed ST segment, T wave of I/R group were significantly elevated, pathologic Q wave has increased significantly, compared with sham operation group with significant difference (P﹤0.05); The recovery of ST segment and T wave in ECG of the treatment groups were better than that of I/R group at each time points; pathologic Q waves appear significantly reducting. Histological examination showed that there were more regular structured myocardial fibers, less bleeding, less fracture myocardial fiber, pale cytoplasmic staining, less inflammatory cell infiltration of the treatment group compared with I/R group. The area of myocardial infarction were significantly reduced under the effect of UTI. Compared with I/R group, the level of MDA in serum was decreased together with SOD, NO increased in the treatment groups. And compared with UTI intravenous injection group and ischemic postconditioning group, the changes were more obvious in UTI coronary artery routes of administration group (P﹤0.05). Compared with I/R group,based on results of enzyme linked immunosorbent assay, the level of TNF-a in serum was decreased while IL-10was increased significantly in the treatment groups.CONCLUSIONS Our results demonstrate that ulinastatin could improve the learning and memory ability of focal brain ischemia-reperfusion injury model rats. UIT can protect the brain from ischemia-reperfusion injury in rats by improving neurological deficit and alleviating the pathological damage in hippocampus. UTI can inhibit neutrophil-mediated inflammatory response after brain ischemia reperfusion in rats by down-regulating the expression of adhesion molecules, TNF-a, MMP-9and up-regulating the level of IL-10. Besides, antioxidant effects being involved in the basic mechanism, the levels of MDA in brain tissue homogenate was decreased with SOD increased.Ulinastatin used after ischmia can produce similar protective effects as mechanical ischemic postconditioning method, the protection of cardiac muscle cell may be mediated by decreasing the level of inducible nitric oxide, inhibition of TNF-a expression, increasing the level of anti-inflammatory cytokine (IL-10), removal of peroxidation product (MDA) and elevating the level of SOD、NO.The antioxidant effect and inhibition of TNF-a expression of UTI administrated by coronary artery is sronger than intravenous administration.Ulinastatin has a protective effect of multiple links on acute cerebral and myocardial ischemia-reperfusion injury.