The Research From The Expression Of CIRP On The Function Of Mild Hypothermia In Trauma Brain Injury Rats

Part 1 The Research from the Expression of CIRP on the Function of Mild Hypothermia in Trauma Brain Injury RatsObjective: Mild hypothermia therapy is an important treatment at the acute stage of severe trauma brain injury currently, but the regulation mechanism for the neuroprotective effect of mild hypothermia on trauma brain injury patients is still unclear. Relevant studies show that CIRP may be the temperature dependence key regulatory protein of hypothermia protection. This research took CIRP as the starting point, observed the brain cell apoptosis and the expression of CIRP in the cortex, hippocampus and hypothalamus part, and detected the CIRP protein expression and ERK pathway activation in the brain tissue at different time points, thus discussed the mechanism of the expression of CIRP during the mild hypothcrmia treatment process of trauma brain injury rats.Method: Divide randomly the rats into three groups, control group(Group 1), blank AD5-GFP(Adenovirus 5-GFP) transfection group(Group 2) and AD5-GFP-CIRP-Si RNA transfection group(Group 3), with each group subdivided into Sham group, TBI group, MH group and TBI + MH groups. After successfully modelling, transfect Group 2 and Group 3 with virus, detect viral transfection situation and the brain cell apoptosis in the cortex, hippocampus and hypothalamus part, apply RT-PCR to detect the CIRP m RNA expression in the three parts, and apply western blot to detect the CIRP, MEK, p-MEK, ERK-1/2 and p-ERK-1/2 protein expression.Results: The viral vector was effectively transfected into the corresponding brain tissue parts of Group 2 and Group 3. The apoptosis detection showed that mild hypothermia treatment could significantly reduce the brain tissue apoptosis in the cortex, hippocampus, and hypothalamus part(p <0.05), in which the anti-apoptotic effect had more significant function in the hypothalamus part. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silent expression. RT-PCR detection showed that mild hypothermia could increase the expression of CIRP in the cortex, hippocampus and hypothalamus part(p <0.05), at the same time, theexpression in the hypothalamus part significantly increased compared to that of the cortex and hippocampus(p <0.05). Western blot detection showed that mild hypothermia therapy could significantly increase the CIRP protein expression in the hypothalamic part from 6 hours(p <0.05), and these effects peaked at the 48 hour mark, showing a downward trend at the beginning of 72 hours. MEK activation detection showed that the MEK activation of the three groups of TBI, MH and TBI + MH, all showed progressive increase and peaked at 24 hours, and then showed a progressive decline. There is no correlation between the degree of MEK activation and CIRP silent expression, which indicates that CIRP protein has no effect on activation of MEK. ERK-1/2 activation detection showed that mild hypothermia therapy could lead to the antedisplacement of ERK-1/2 activation peak, and quickly reduce its activation degree, and effect disappeared during CIRP silent expression.Conclusion: During the mild hypothermia treatment of trauma brain injury, CIRP is over expressed in the cortex, hippocampus and hypothalamus part due to low temperature, in which the expression in the hypothalamus part is more obvious. CIRP action mechanism may rapidly decrease the activation degree of ERK-1/2 as time goes by directly regulating its activation, so that it may reduce the brain cell apoptosis in corresponding parts and thus play a neuro-protective effect.Part 2 The study of the expression of CIRP for Shenfu injection to the TBI in Rats treated by mild hypothermiaObjective: This study took the expression of CIRP as the key point to speculate the mechanism of the protection of Shenfu injection in the mild hypothermia treatment process of trauma brain injury rats.Method: Divide randomly the rats into three groups, control group(Group 1), blank AD5-GFP(Adenovirus 5-GFP) transfection group(Group 2) and AD5-GFP-CIRP-Si RNA transfection group(Group 3), with each group subdivided into sham group, low dose group, and high dose groups. After successfully modeled 48 h later, detect the brain cell apoptosis in the cortex, hippocampus and hypothalamus part, apply RT-PCR to detect the CIRP m RNA expression in the three parts, and apply western blot to detect the CIRP, MEK, ERK-1/2 protein expression.Results: The apoptosis detection showed that the brain tissue apoptosis in the cortex, hippocampus, and hypothalamus part have significantly reduced in low and high dose groups(p <0.05), the Shenfu injection could reduced the apoptosis of the brain cell under mild hypothermia. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silent expression. RT-PCR detection showed that the expression of CIRP have significantly risen in low and high dose groups in Group 1, 2(p <0.05), the injection could increase the expression of CIRP. Western blot detection showed that the CIRP protein expression have significantly risen in low and high dose groups in Group 1, 2(p <0.05). MEK activation detection showed no difference in Group 1, 2, 3(p >0.05). ERK-1/2 activation detection showed significantly decreased in low and high dose groups in Group 1, 2(p <0.05), however, there were no difference in Group 3(p >0.05).Conclusion: The function of the Shenfu injection using in mild hypothermia of TBI maybe adopt the way of over-expression of CIRP, and decrease the activation degree of ERK-1/2 directly, so that it may reduce the brain cell apoptosis in corresponding parts and thus play a neuro-protective effect.