| Objective:Cerebral ischemic disease is a common disease, serverely harmful to the human’s health and life quality. Recently research found that the normal apoptosis plays an important role in ischemia and reperfusion, while too much apoptosis will increase brain ischemia and reperfusion injury. Otherwise, to inhibit the apoptosis is critical for the protection against ischemia and reperfusion (I/R) injury in brain. The process of apoptosis has a critical role for the judge theI/R injury of prognosis. Association between the cerebral ischemic injury and apoptosis has become increasingly researched topic, especially discovery of the basic and highly conserved the mechanisms of neurons apoptosis and protection against the ischemic.As we all know, ischemia and reperfusion (I/R) injury is when the body organ suffered an interruption of blood flow for a certain amount of time, the blood reperfusion and organ tissues’injury increased. Ischemia and reperfusion injury is a quick cascade of reaction, this cascade include energy failure, inflammatory response, excitotoxicity, altered calcium homeostasis, free radical production and activation of apoptosis gene,ect. This reciprocal causation, overlapping and interrelated, a vicious, eventually leading to apoptosis or necrosis.Excitatory amino acid after focal cerebral ischemia plays an important role in the process of neuronal cell death. Gamma-aminobutyric acid (GABA) is recognized as the most important of the inhibitory neurotransmitter within the central nervous system. GABA can protect the neurons of ischemia-induced against overexcitation. GABA needs to through its receptor, like gamma-aminobutyric acid (GABA) receptor which is distribute very widely, play an dominant role of the neuroprotoction in ischemic brain injury.Xingnaojing injection is refine by the country’s classical prescriptions angongniuhuang pill, which is good at restoring consciousness and inducing resuscitation that is widely used in vascular lesions of the brain. Xingnaojing injection can reduce the brain neurons damage and cerebral infarction after ischemic injury, but the mechanism of how does its part in protecting nerve cell is not very clear. Therefore, the purpose of the experiment is to select the binding of the receptor GABAA s antagonist bicuculline confrontation of xingnaojing injection and GABAA receptor, to investigate the mechanisms of xingnaojing injection on cerebral ischemia’s protection whether through the GABAA receptor as a target or not.Methods:(A) to explore the effect of xingnaojing injection on cell apoptosis and its mechanism of action.1. PC12cells cultured in vitro using cell culture techniques, MTT colorimetric method for detecting effect of xingnaojing injection on cell activity.2. The use of flow cytometry for further verification role of xingnaojing injection against cell apoptosis, and then select the GABAA receptor antagonist bicuculline (bicuculline, BIC)to against the combination of xingnaojing injection and the GABAA receptor, to investigate the mechanisms of xingnaojing injection on cerebral ischemia’s protection against cell apoptosis whether through the GABAA receptor as a target or not.3. Extract the cell protein and use Western blot analysis to detect the xingnaojing injection on expression of GABAA receptors, further investigate the mechanisms of xingnaojing injection protection against cell apoptosis whether through the GABAA receptor as a target or not.(B) To research the effect of xingnaojing injection in SDrats with cerebral ischemia-repcrfusion model.1. Use the method of plug wire in the internal carotid artery to make the SD rats with cerebral ischemia-reporfusion model. Neurobuhavioral scores, TTC staining and Tunel staining method are use to test the effect of xingnaojing injection in MACO (Middle cerebral artery occlusion) SD rats2. Western blot analysis to detect the xingnaojing injection on expression of GABAa receptorsResults:1. MTT results revealed that xingnaojing injection can improve the activity of PC12cells.2. Annexin V/PI double staining flow cytometry results found, xingnaojing injection is effective in inhibiting apoptosis of PC12cells, after xingnaojing injection combined with GABAA receptor antagonist bicuculline (bicuculline, BIC), its anti-apoptotic role is weaker than xingnaojing group, it means the effect of ati-apoptosis by xingnaojing injection is dependent on GABAA receptor.3. Western blot analysis prove that xingnaojing injection can increase the damage cells expression of GABAA receptors, the expression of GABAA is significantly decrease by the combination of xingnaojing injection and GABAA receptor antagonist bicuculline,it further explain the effect of ati-apoptosis by xingnaojing injection is dependent on GABAA receptor.4. In the method of plug wire in the internal carotid artery to make the SD rats with cerebral ischemia-reperfusion model. The results of Neurobehavioral scores, the infarct size and cell apoptosis of positive rates significantly lower than the model group, shows that the protection of xingnaojing is obviously.5. Western blot analyses reveal that xingnaojing injection can increase the expression of GABAA receptors, further express that the protection of xingnaojing is dependtent on GABAA receptor.Conclusion:in summary, we demonstrated that xingnaojing injections have the effect of anti-apoptosis, but its effection would significantly decrease via the GABAa antagonist bicuculline antagonist in vitro. It proves that mechanisms of xingnaojing injection protection against cell apoptosis are related to the anti-apoptosis effect of GABAA receptor. In addition, the present study showed that xingnaojing injection can significantly reduced the brain infarct size with cerebral ischemia injury, and enhance the expression of GABAA receptors in vivo. These results indicate that xingnaojing injection may go through the GABAA receptors to play the role of protection. |
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