Effects Of Huazhuo Jiedu Huoxue Tongluo Prescription On Expression Of ZO-1 And Occludin In Rats Of Cerebral Ischemia Reperfusion Injury

Objective:To investigate the effects of Huazhuo Jiedu Huoxue Tongluo prescription on Blood Brain Barrier and expression of ZO-1 and occludin in rats of Cerebral Ischemia Reperfusion Injury,and to reveal its neuroprotectiive mechanism from the protein of tight junction in Blood Brain Barrier.Methods:We established the mice model of cerebral ischemia reperfusion injury(CIRI)by using Zea-Longa method.Fifty-four healthy male SD mice were randomly divided into six groups: the sham operation group,the model group,the nimodipine group,the high dose of Huazhuo Jiedu Huoxue Tongluo prescription(the high dose group for short),the middle dose of Huazhuo Jiedu Huoxue Tongluo prescription(the middle dose group for short)and the low dose of Huazhuo Jiedu Huoxue Tongluo prescription(the low dose group for short),nine mice in each group.All mice were returned to their cages after the operation,ate food and water freely after models were successfully established,and the drugs were given by gastric perfusion the next day.The drugs were given by gastric perfusion the next day.Physiological saline were given to the mice in the sham operation group and the model group.The nimodipine group were given the same amount of nimodipine solution and the respective dose of Huazhuo Jiedu Huoxue Tongluo prescription were given to the high dose group,the middle group and the low dose group.The drugs were given once a day and for 7 days continuously.The animals were neurologically assessed on a 5 Longa point scale 24 hours atter operation and 7days after the tyeatment,and then beheaded.Each group of rats' right cerebral hemisphere by HE staining,we observedunder the shape variation of ischemic brain tissue,and the expression of ZO-1 and occludin were detected by the immunohistochemical and RT-PCR method.Results:1 The Neurologic deficits symptoms score of each group: 24 hours after operation :There were no obvious Neurologic deficits in the sham operation group.The Neurologic deficits symptomsd in the model group and other isposed groups were in varying degrees,but there were no significant differences in these gyoups(P>0.05).7 days after tyeatment:Compared with the sham operation group,the scores of the model group increase obviously(P<0.05);compared with the model group,the scores of the nimodipine group,the high dose group decreased significantiy(P<0.05),but there was no significant difference between the two groups(P>0.05);the middle dose group and low dose group had decreased compared with the model group(P<0.05);but there was no significant difference between the two groups(P>0.05);2 The pathological morphological changes of the right cerebral tissues in mice: Tissues on the right side of the cerebral cortex in the sham operation group were arranged regularly and tightly,the form of cells were normal,the cytoplasm were full,homogeneously dyed in purple and The nucleus were in blue.there had little dead cells.The ischemic regions were obviously on the cerebral cortex of the model group,tissues were arranged irregularly,the extra cellular space of cells was broadening significantly,a large number of necrotic neuron cells existed;That pathologic change also existed in other disposed groups.Ischemic necrosis area of middle dose group and low dose group decreased compared with the model group.Ischemic necrosis area of nimodipine group and high dose group decreased significantly compared with the model group.3 The expression of ZO-1 by immunohistochemistry:Under optical microscope,most of the positive cells could be seen around ischemia areain infarct side,and the positive cells immunohistochemisty staining were sandy,the nucleus of the positive endothelial cells were round or oval.By the count of the ZO-1 in each group,we can see that:The ZO-1 in the sham operation group expressed Continously.The ZO-1 in the model group was obviously decreased compared with the sham operation group(P<0.05),and they were expressed scatteredly in a small amount;The ZO-1 in the nimodipine group?the high dose group were higher than the model group(P<0.05),Continuous expression and Discontinuous express coexisted;but there was no significant difference between the two groups(P>0.05);The ZO-1 in the he middle dose group,the low dose group were higher than the model group(P<0.05);but there was no significant difference between the two groups(P>0.05),they were in Discontinuous express.4 The expression of occludin by immunohistochemistry :Under optical microscope,most of the positive cells could be seen around ischemia area in infarct side,and the positive cells immunohistochemisty staining were sandy,the nucleus of the positive endothelial cells were round or oval.By the count of the occludin in each group,we can see that:The occludin in the sham operation group expressed Continously.The occludin in the model group was obviously decreased compared with the sham operation group(P<0.05),and they were expressed scatteredly in a small amount;The occludin in the nimodipine group ? the high dose group were higher than the model group(P<0.05),Continuous expression and Discontinuous express coexisted;but there was no significant difference between the two groups(P>0.05);The occludin in the middle dose group,the low dose group were higher than the model group(P<0.05),the middle dose group increased more significantly than the low dose group(P<0.05).5 The expression of ZO-1 by RT-PCR:.The ZO-1 in the model group was obviously decreased compared with the sham operation group(P<0.05);The ZO-1 in the nimodipine group,the high dose groupwere higher than the model group(P<0.05);but there was no significant difference between the two groups(P>0.05);The ZO-1 in the he middle dose group,the low dose group were higher than the model group(P<0.05);but there was no significant difference between the two groups(P>0.05).6 The expression of occludin by RT-PCR:The occludin in the model group was obviously decreased compared with the sham operation group(P<0.05);The occludin in the nimodipine group ? the high dose group were higher than the model group(P<0.05);but there was no significant difference between the two groups(P>0.05);The occludin in the he middle dose group,the low dose group were higher than the model group(P<0.05);but there was no significant difference between the two groups(P>0.05).Conclusion:1 Huazhuo Jiedu Huoxue Tongluo prescription can significantly reduce the neurologic deficits symptoms score in CIRI model rats,and improve the pathological morphological damage in the ischemic regions,promote the recovery of neural function.2 Huazhuo Jiedu Huoxue Tongluo prescription has some protective effects on blood brain barrier in Ischemic brain tissue of in CIRI model rats,the mechanism might iovolved increasing the expression of tight junction ZO-1 and occludin.