| Objectives The blood supply of brain were restored by various means and methods after being subjected to a certain time of ischemia, but at this point it could easily lead to cerebral ischemia reperfusion injury flow recanalization. in recent years, ischemic cerebral vascular disease incidence rate has been continued to rise, when cerebral ischemia improved, reperfusion injury may also occur, research in this field has become hot spot in recent years, mechanism of cerebral ischemia reperfusion injury is complicated, there are almost no targeted therapies and drugs. The purpose of this study is to explore the mechanism of the damage of nerve vascular unit after cerebral ischemia reperfusion injury, and the protective effects of diammonium glycyrrhizinate on neurovascular unit.Methods 160 male sprague-dawley rats weight 250-300 g were randomly divided into normal group(group A), sham operation group(group B), ischemia-perfusion with normal saline control group(group C), ischemia-perfusion with Diammonium Glycyrrhizinate(DG) treatment group(group D).rats model of cerebral ischemia reperfusion injury were established by simulating ischemia-reperfusion with reversible middle cerebral artery occlusion(MCAO), sham operation group give the same operation, do not plug line, model group pull-out suture at 2 hours after operation. Ras in DG treatment group were treated with DG by intraperitoneal injection, model control group, sham operation group and the normal group were injected with equal volume of saline, administered once a day, a total of three days. Rats neural function scores were taken in postoperative rats at 3 hours after anesthesia and three days later. The rats were all killed at 3 days after operation. The volume of infarction were detected by the technique of TTC staining. Brain water content were detected by dry wet weight method. The number of neurons in cerebral cortex of rats were detected by HE staining. The number of nissl bodies in cerebral cortex of rats were detected by Nissl staining. The apoptosis of neurons in cerebral cortex of rats were detected by TUNEL staining. The expression of serine/threonine protein kinase(Akt) and cysteine proteinase 3(Caspase-3) in cerebral cortex of rats were detected by immunohistochemical staining. The expression of phosphorylation of Akt(p-Akt) in cerebral cortex of rats were detected by Western blotting. The expression of aquaporin-4(AQP4) and tight junction related occludin protein in cerebral cortex of rats were detected by immunofluorescence staining.Results Rat model of ischemia reperfusion injury was successfully established. Zealonga score of all rats preoperative is 0, 2 hours after the operation, Zea-longa score of rats in A,B,C,Dgroup were 0, 0.30±0.48, 2.80±0.92, 2.90±1.20,respectively.There was no significant difference between group A and B(P>0.05),There was significant difference between group C,D and A,B(P<0.05). Three days after the operatiion, the score of group D(1.90±0.74) was lower than group C(2.70±0.48), the difference is statistically significant(P<0.05). 3 days after operatiion, infarct volume calculated by TTC staining was0, 0,(42.48±5.81)%,(31.70±6.57)%, respectively.There was no significant difference between group A and B(P>0.05), the infarct volume in C and D groups were higher than that in group A and B, the group C was higher than that in group D, The difference was statistically significant(P<0.05).Brain water content was(63.11±6.83)%,(65.34±7.49)%,(87.51±5.75)%,(72.23±7.21)%, respectively. There was no significant difference between group A and B(P>0.05), the brain tissue water content in group C and D were higher than that in group A and B, the group C was higher than that in group D, the difference was statistically significant(P<0.05). Number of neurons with HE staining were 233.10±32.47,230.30±41.61,60.20±16.25,116.30±21.45, respectively. There was no significant difference between group A and B(P>0.05), the number of neurons in group C and D were lower than that in group A and B, the group C was lower than that in group D, the difference was statistically significant(P<0.05). Number of nissl bodies were 137.20±27.55,138.90±33.89,50.80±11.10,78.80±16.35, respectively.There was no significant difference between group A and B(P>0.05), the number of nissl bodies in group C and D were less than that in group A and B, the number of group C was less than that of the group D, the difference was statistically significant(P<0.05).TUNEL staining positive cells were 8.90±3.81,7.50±3.66,65.40±8.40,44.80±7.69, respectively. There was no significant difference between group A and B(P>0.05), the number of apoptosis cells in group C and D were more than that in group A and B, the number of group C were more than that in group D,the difference was statistically significant(P<0.05).The numberof caspase-3 positive cells were 10.20±6.01, 11.60±5.99, 99.00±14.85, 56.00±14.90, respectively. There was no significant difference between group A and B(P>0.05), the number of Caspase-3 positive cells in group C and D were more than that in group A and B, the number of group C were more than that in group D, The difference was statistically significant(P<0.05).The number of Akt positive cell were 86.20±11.41,83.30±10.32,16.60±5.36,47.00±7.06, respectively. There was no significant difference between group A and B(P>0.05), the number of Akt positive cells in group C and D were more than that in group A and B, the number of Akt positive cells in group C were more than that in group D, the difference was statistically significant(P<0.05). pAkt expression levels were 0.83±0.08,0.81±0.11,0.30±0.07,0.62±0.07, respectively. There was no significant difference between group A and B(P>0.05), the p-Akt expression levels in group C and D were lower than that in group A and B, p-Akt expression levels in group C were lower than that in group D, the difference was statistically significant(P<0.05).Average optical density of AQP-4 were 0.020±0.003,0.019±0.002,0.044±0.004,0.029±0.005,respectively.There was nosignificant difference between group A and B(P>0.05), the average optical density of AQP-4 in group C and D were higher than that in the group A and B, the average optical density of AQP-4 in group C were higher than that in group D, the difference was statistically significant(P<0.05).The average optical density of occludin were 0.228±0.021, 0.216±0.024, 0.060±0.013, 0.129±0.017,respectively.There was no significant difference between group A and B(P>0.05), the average optical density of occludin in group C and D were lower than that in the group A and B, the average optical density of occludin in group C were lower than that in group D, the difference was statistically significant(P<0.05).Conclusions This study successfully established rat model of cerebral ischemia reperfusion injury.and confirmed the important role of neurovascular unit injury during ischemia reperfusion, that the expression of apoptosis related Caspase-3 protein was significantly increased and expression of anti-apoptosis related Akt protein decreased after the occurrence of ischemic reperfusion, resulting in apoptosis increased significantly in the ischemia reperfusion brain tissue. The expression of blood brain barrier water regulating metabolism related AQP-4 protein was elevated, the expression of occludin protein,one of the major proteins of tight junction was decreased, which leads to the destruction of the blood brain barrier. This study also confirmed diammonium glycyrrhizinate relieve the ischemia reperfusion injury apoptosis level by increasing the expression of Akt and reduce the expression level of caspase 3. It also relieve the ischemia reperfusion injury of blood brain barrier damage by reducing the expression of AQP-4 and increased the expression of occludin protein. So diammonium glycyrrhizinate play a big role in the overall protective effect of the nerve vascular unit in rats after ischemia reperfusion injury in rats. |
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