Effects Of Ulinastatin On Expression Of Tumor Necrosis Factor-α,Mitogen-activated Protein Kinase P38and JNK In Cerebral Ischemia Reperfusion Rats

Background and ObjectiveCerebral infarction is very common in China now, morbidity and disability of which are frighteningly high. Especially, the "aging" society our country has entered, the fast pace of life and a poor diet increase the incidence of cerebral infraction. Thrombolytic therapy can recover cerebral perfusion in the ischemic tissue, but it also can cause reperfusion injury in brain tissue. Free radicals, calcium overload, excitatory amino acids are the factors related with ischemia reperfusion(I/R) injury. The latest research think that the inflammatory response plays an important role in the process of ischemia-reperfusion injury, and can promote the conversion of the ischemic penumbra to the infarct zone. Tumor necrosis factor alpha (TNF-a) is considered to be a cytokine which initiate inflammatory response in early stage of cerebral ischemia and the increase of TNF-a is adverse to cerebral I/R injury, which can measure the severity of the inflammatory response.Ulinastatin, a human urinary trypsin inhibitor (UTI), is a serine-type protease inhibitor, Which can inhibit a variety of enzyme activity especially the trypsin. Some studies reported that UTI reduces oxidative injury and cell apoptosis. Recently, it has been reported that UTI offers a cytoprotective effect against I/R injury in many organs like liver, heart, lung and pancrea. There was no research focused on anti-inflammatory mechanism of UTI in cerebral tissue. Mitogen-activated protein kinase in the P38and JNK pathways plays an important role in stress response and apoptosis. Whether ulinastatin can influence JNK and P38signaling pathways to accommodate inflammatory responses still needs further research.In this study, we aimed to explore the influence of UTI on inflammation in rats with I/R injury, so we produced the cerebral ischemia reperfusion injury model with SD rats, compared mRNA expression of TNF-a, JNK and P38of cerebral tissue in rats with I/R injury with that in rats with the administration of UTI. The correlations between TNF-a and P38, TNF-a and JNK, were also analyzed to investigate the role of TNF-a in inflammation response of rats with cerebral I/R injury.Materials and methodsWe randomly allocated45Sprague-Dawley rats into3groups:sham operation group, I/R injury group (control group) and UTI group. All rats were handled according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The rats model with I/R injury was successfully produced according to Zea-Longa’s report. The middle cerebral artery occlusion (MCAO) last120minutes.UTI group were immediately given ulinastatin (10U/g) intraperitoneal injection, and the other two groups of rats are given1ml physiological saline intraperitoneal injection. All the rats were sacrificed1day after I/R injury. Reverse transcription-polymerase chain reaction (RT-PCR) technology was used to measure mRNA expressions of TNF-a, P38and JNK in the brain tissues of rats.All data are recorded as mean±standard deviation (SD) of seven measurements and analyzed with the SPSS17.0statistical software package. The statistical significance of the current data and parameters was determined with analysis of variance (ANOVA). When the results of ANOVA were significant, the Bonferroni post hoc test was applied to determine specific group differences. Statistical analysis of the correlation between TNF-a and P38, TNF-a and JNK, was performed using Pearson’s correlation coefficient. A value of P<0.05was considered to be statistically significant. ResultsThe relative quantity of TNF-a mRNA in the sham operation group, the control group and the UTI group is0.074±0.019,0.328±0.043and0.165±0.031. The relative quantity of P38mRNA in the sham operation group, the control group and the UTI group is0.125±0.02,0.386±0.065and0.217±0.038. The relative quantity of JNK mRNA in the sham operation group, the control group and the UTI group is0.335±0.057,1.147±0.136and0.217±0.038. The control group and the UTI group rat brain tissue TNF-a, P38, JNK, mRNA expression were significantly higher than sham operation group (P<0.001); TNF-a, P38, JNK, mRNA expression was significantly lower in the UTI group than in the control group (P<0.01).We observed a significant correlation between TNF-a and P38mRNA expression (R=0.901, P<0.01) and between TNF-a and JNK mRNA expression (R=0.904, P<0.01).Conclusion1. The mRNA levels of TNF-a, JNK and P38increased significantly in the control group, suggesting that the TNF-a, JNK, and P38involved in the pathological process of cerebral ischemia and reperfusion injury.2. Ulinastatin can mitigate inflammatory responses through decreasing the mRNA levels of TNF-a, JNK and P38. The reduction of TNF-a expression may be associated with inhibition of expression of P38and JNK of UTI.