| Objective The middle cerebral artery occlusion(MCAO)method was used to establish a stable cerebral ischemia model in rats,and the neuroprotection of alpha 2adrenoceptor agonist clonidine to cerebral ischemia 2 h and reperfusion 4 h Role,and further explore its specific molecular mechanism from the apoptotic pathway.Methods(1)Healthy adult male SD rats were randomly divided into 6 groups:sham operation group,MCAO model group,low-dose clonidine(20 ug/kg)group,high-dose clonidine(40 μg/kg)group,yohimbine(5 mg/kg)+ High-dose clonidine(40 μg/kg)group and positive drug control MK801(0.5 mg/kg)group,pre-administered according to body weight for 1 week,sham operation and model group were given the same dose of normal saline,and then used thread plug MCAO model was prepared,2 hours after ischemia and 4hours after reperfusion,the brains were sacrificed and scored by TTC staining after the neurological deficit score,and the cerebral ischemic injury was evaluated according to the neurological deficit score and cerebral infarction volume;(2)Observe the cortical and hippocampal neuron morphology of each group by HE staining,and evaluate the neuron damage;(3)Further evaluate the apoptosis of rat cortex and hippocampus by fluorescent TUNEL staining;(4)Immunoblot was used to detect the expression of ischemic cortex and hippocampal apoptosis-related proteins in each group of rats;(5)Real-time quantitative PCR was used to detect the expression of ischemic cortex and hippocampal apoptosis-related protein mRNA in each group of rats.Results(1)The statistical results of neurological deficit scores showed that compared with the sham operation group,the neurological deficit scores in the model group were significantly increased(P <0.01);compared with the model group,the clonidine group scores were all reduced(P <0.05),The MK801 group was also significantly reduced(P<0.01);compared with the low-dose clonidine group,the high-dose clonidine group score was significantly reduced(P <0.01);compared with the high-dose clonidine group,yohimbine + high-dose cola The score in the fixed group increased significantly(P <0.01),but there was no significant difference in the MK801 group.(2)The statistical results of TTC-stained cerebral infarction volume ratio showed that compared with the sham operation group,the cerebral infarction volume ratio in the model group was significantly increased(P <0.01);compared with the model group,the clonidine group had a lower proportion(P <0.05),the MK801 group was also significantly reduced(P <0.01);compared with the low-dose clonidine group,the proportion of the high-dose clonidine group was significantly reduced(P <0.01);The proportion of the Yohimbine + high-dose clonidine group was significantly increased(P <0.01),but there was no significant difference in the MK801 group.(3)Cortical and hippocampal HE staining results in each group of rats showed that the cortical and hippocampal neurons in the sham operation group were arranged tightly,the cells were large and round,and the nucleus was clearly visible;compared with the sham operation group,the hippocampal neurons in the model group were more obvious Atrophy and deformation,disorderly arrangement,deep condensed nucleus,and cell atrophy was significantly improved after clonidine pretreatment,and the damaged neurons were significantly reduced(P <0.01).(4)Cortical and hippocampal TUNEL staining statistics of rats in each group showed that there were very few apoptotic positive cells in the cortex and hippocampus in the sham operation group,which were scattered sporadically.Compared with the sham operation group,the apoptotic cells in the cortex and hippocampus in the model group were significantly increased.The positive rate of apoptosis was significantly increased(P <0.01);compared with the model group,after clonidine pretreatment,apoptotic cells were significantly reduced,and the positive rate of apoptosis was significantly reduced(P <0.01);compared with high-dose clonidine Compared with the group,apoptotic cells increased significantly in the yohimbine + high-dose clonidine group,and the apoptosis rate increased significantly(P <0.01).(5)The results of immunoblotting detection of apoptosis-related proteins in the ischemic cortex and hippocampus of each group of rats showed that:1).Compared with the sham operation group,the expressions of pro-apoptotic proteins AIF,Caspase3,P53,p-P53,and Bax were increased in the model group(P <0.05);compared with the model group,each of the pro-apoptotic proteins in the high-dose clonidine group The expressions were reduced;compared with the high-dose clonidine group,the expressions of all pro-apoptotic proteins in the yohimbine + high-dose clonidine group were increased;2).There was no significant difference in expression of Bcl2 and p-Bcl2 between the ischemic cortex and hippocampus.(6)Real-time fluorescent quantitative PCR detection of ischemic cortex and hippocampal apoptosis-related mRNA in each group of rats showed that:1)a.Compared with the sham operation group,the expressions of cortical proapoptotic proteins AIF,Caspase3,P53,and Bax in the model group were significantly increased(P <0.05).Compared with the model group,the mRNA expressions of the high-dose clonidine group were significantly reduced.(P <0.01);compared with the high-dose clonidine group,the mRNA expression of yohimbine +high-dose clonidine group was increased;b.The expression of apoptotic protein Bcl2 mRNA in the ischemic cortex was between the groups There are no significant differences;2)There was no significant difference in the expression of various apoptosis-related protein mRNAs in the hippocampus of ischemic side.Conclusion(1)α2 adrenoceptor agonist clonidine can improve neurological deficit scores in acute focal rats,reduce cerebral infarction volume,reduce neuronal damage,reduce apoptosis,and have significant effects on acute focal cerebral ischemia rats Neuroprotective effect;(2)Clonidine’s neuroprotective effect on acute focal cerebral ischemia in rats is closely related to apoptosis,which may be through inhibiting the expression of pro-apoptotic related proteins AIF,Caspase3,P53,p-P53,Bax and their mRNAAnd has nothing to do with the expression of Bcl2. |
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