Study On The Effect Of Musk On Immune Inflammatory Response Induced By Ischemic Stroke Based On HMGB1

ObjectiveTaking the successfully established rat cerebral ischemia-reperfusion injury model as the object,the intervention effects of musk on cerebral ischemia-reperfusion injury was studied.Using Q-PCR,immunohistochemistry,Western Blot and immunofluorescence double-labeling method,etc.,we have verified the hypothesis of cerebral protection resulting from inhibiting the inflammation in the brain tissue of the ischemic area from the perspective of the regulation of HMGB1 by musk.MethodsThe experimental rats were randomly divided into Control group,Sham group,MCAO group and MCAO+Musk group.The MCAO(middle cerebral artery occlusion)model of rats was replicated by suture method,and the neurological deficit scores of each group were observed.The rats were sacrificed after reperfusion of 24 hours to obtain the samples.the cerebral infarction volume was evaluated by TTC staining;Q-PCR technology was used to detect the expression levels of brain tissue IL-1β and IL-6;the expression level of brain tissue MMP-9 was detected by immunohistochemistry;the HMGB1/NeuN immunofluorescence double-labeling method was used to observe the translocation of neuron HMGB1(Highmobility group box 1 protein)of brain tissues;Western Blot was used to detect the expression of HMGB 1 in brain tissue.ResultsNeurological function score:After the establishment of the local cerebral ischemia reperfusion model,the neurological function score of the musk treatment group was significantly reduced(P<0.001)compared with the model group.The blank group and the sham operation group showed no symptoms of neurological impairment;Cerebral infarction volume:TTC staining showed that there was no infarction in the brain tissue of the blank group and the normal group.There were infarctions in the brain tissue of the MCAO group and the MCAO+Musk group and the cerebral infarction range of the MCAO group was larger.The range of infarction decreased in the MCAO+Musk group compared with the MCAO group.The difference between the two groups was statistically significant(P<0.001);Expression of inflammation in cerebral infarction:IL-1β:Compared with the MCAO group,the expression level of IL-1β mRNA in the MCAO+Musk group significantly was reduced and the difference was statistically significant(P<0.01).IL-6:Compared with the MCAO group,the expression level of IL-1β mRNA in the MCAO+Musk group decreased and the difference was statistically significant(P<0.05).MMP-9:Compared with the MCAO group,the expression level of MMP-9 in the MCAO+Musk group declined and the difference was statistically significant(P<0.05).HMGB1 displacement:The percentage of the positive expression of HMGB1 in the cytoplasm and nucleus of neurons in the brain tissue of ischemia-reperfusion rats as a percentage of the total number of DAPI was detected by immunofluorescence doublelabeling method.Ratio of double staining of the HMGB1 and neuronal cell nuclei:The blank group was 31.24%±3.36%,the sham operation group was 32.89%±10.56%,the MCAO group was 4.79%±2.21%,and the MCAO+Musk group was 23.92%±4.75%.There were statistical differences in the overall mean of each group.The differences between the groups were compared:no statistical difference between the blank group and the sham operation group(P=0.676>0.05).Compared with the blank group and the sham operation group,the ratio of double-stained nuclei in the MCAO group was significantly reduced,which had a statistical difference(P<0.001,P<0.001)Compared with the MCAO group,the ratio of double-stained nuclei of the MCAO+Musk group rise and there were statistical differences between the two groups of data(P<0.001).The double staining of HMGB 1 and neuron cell cytoplasm was 9.83%±2.29%in the blank group,8.79%±5.72%in the sham operation group,63.04%±9.04%in the MCAO group,and 27.22%±4.77%in the MCAO+Musk group.There was a statistical difference in the overall mean of each group.The differences between the groups were compared.There was no statistical difference between the blank group and the sham operation group(P=0.999>0.05).Compared with the blank group and the sham operation group,the ratio of double-stained cytoplasm of the MCAO group was significantly increased(P<0.001,P<0.001)Compared with the MCAO group,the ratio of doublestained cytoplasm of the MCAO+Musk group decreased(P<0.01).The expression of HMGB 1 in brain tissue:Western Blot was used to detect the expression of HMGB1 in brain tissue.There was no statistical difference between the blank group and the sham operation group(P=0.221>0.05),Compared with the blank group and the sham operation group,the expression of HMGB1 in the MCAO group increased(P=0.009<0.01,P=0.001<0.01);compared with the MCAO group,the expression of HMGB1 in the MCAO+Musk group decreased(P=0.016<0.05).ConclusionsMusk treatment helps to reduce the brain tissue damage volume and neurological deficit symptoms in the infarcted area of MCAO rats.The protective mechanism may be closely related to reducing the inflammatory response after the cerebral ischemia-reperfusion injury by inhibiting the translocation of HMGB1 from neuronal cell nucleus to cytoplasm and inhibiting the expression of HMGB 1 in brain tissue to reduce the expression of IL-1βmRNA IL-6 mRNA,and MMP-9.