The Effect And Mechanism Of Resveratrol On Neurovascular Units In Rats With Acute Cerebral Ischemia

1.Part Ⅰ Objective: To investigate the protective effects of different doses of resveratrol on rats with cerebral ischemia and reperfusion.Methods: Focal cerebral ischemia was induced by the right MCAO with a nylon monofilament suture.The rats were randomly divided into sham group,I/R model group,low-dose group(Res 10mg/kg/d),middle-dose group(Res 20mg/kg/d)and high-dose group(Res 40mg/kg/d).Neurological deficit score was carried out at 6h,1d2 d,3d,5d,respectively.TTC staining and HE staining were performed at 2d after MCAO.Results:(1)Neurological function score: the score of high-dose group was significantly lower than that of the model group on 1d.Both the high-dose and middle-dose group were significantly lower than that of model group on 3d.All resveratrol groups were significantly lower than the model group on 5d.(2)TTC results: Cerebral infarction volume was 0% in the sham group,(35.33±3.14)% in the model group,and(29.83±2.04)% in the high-dose group,respectively,and the differences among them were statistically significant(P<0.05).(3)Resveratrol improved the pathological changes of HE staining induced by I/R injury,with the most remarkable improvement in the high-dose group.Conclusions: Different doses of resveratrol showed protective effects against acute cerebral ischemia-reperfusion in rats.In the short term(1 day),the high-dose group of resveratrol has a significant protective effect With the extension of time,the beneficial actions were demonstrated in all 3 doses of resveratrol treatment,with the greatest protection in high-dose group.In this experiment,the optimal therapeutic dose of resveratrol was 40 mg/kg/d.2.Part Ⅱ Objective: To investigate the protective effect and mechanism of resveratrol on BBB injury induced by cerebral ischemia/reperfusion.Methods: Dry-wet weight method was used to detect the degree of brain edema,Evans blue staining was used to detect the permeability of BBB,electron microscopy was used to observe the ultrastructure of BBB,immunohistochemistry and Western blot were used to observe the expression of ZO-1,claudin-5 and occludin in brain tissue.Results:(1)The water content of brain tissue in I/R group reached the highest value on the 3rd day,while that in the Res group was significantly lower than that in the I/R group(P<0.05).(2)Evans blue content in brain tissue of I/R rats had two peaks at 6h and 3d,respectively,with the higher content at 3d.The values of Evans blue content were 0.87±0.23,8.60±2.31,and 6.45±1.11 in sham group,I/R model group and resveratrol group,respectively,and the differences among 3 groups were statistically significant(P<0.05).(3)Resveratrol improved the integrity of BBB ultrastructure examined under electron microscopy.(4)The number of positive-staining cells for ZO-1,Claudin-5 and Occludin in the I/R model group decreased gradually with time,while the resveratrol treatment inhibited these decreases when compared with the model group,and the difference were statistically significant.The levels of ZO-1,Claudin-5,and Occludin is lower in the model group and resveratrol group.The results of Western blotting showed that the levels of ZO-1,Claudin-5 and Occludin in the Res group were significantly lower than those in the sham operation group but higher than those in the I/R model group,and the difference was statistically significant.Conclusions: Resveratrol reduced I/R induced brain edema.BBB leakage and ultrastructure destruction.The protective effect of Resveratrol may be related to the reversal the decrease of ZO-1,Claudin-5 and Occludin during cerebral ischemia and reperfusion.3.Part Ⅲ Objective: To investigate the protective effect and mechanism of resveratrol on neurovascular units in acute cerebral ischemia reperfusion.Methods: The ultrastructural changes of neurovascular unit were observed by electron microscopy.The expressions of Neu N,GFAP and LN were observed by immunohistochemistry and Western blot.The apoptosis of neurons was detected TUNEL assay.The expression of XIAP,Smac and Caspase-9 proteins was observed by Western blot.The levels of inflammatory cytokines IL-1β,IL-6 and TNF-αwere determined by ELISA.Results:(1)The resveratrol treatment inhibited the ultrastructural damage of neurovascular units as shown by electron microscopy.(2)The differences of Neu N,GFAP and LN in three groups were statistically significant(P<0.05).The differences of XIAP,Smac,Caspase-9 and Cx43 in three groups were statistically significant.The levels of IL-1β,IL-6 and TNF-αin model group and the resveratrol group were up-regulated significantly when compared with those in sham group.However,the levels in the resveratrol group were remarkable lower than those in model group.Conclusions: Neurons,astrocytes and endothelial cells in neurovascular units are damaged after cerebral ischemia reperfusion,which was inhibited by the intervention of resveratrol in a time-dependent manner.The protective effect of resveratrol on neurovascular units may be related to the up-regulation of the expression of Neu N and LN,and the inhibition of GFAP expression.Resveratrol reduced the occurrence of neuronal apoptosis after cerebral ischemia and reperfusion in rats,which may be related to the up-regulation of XIAP protein expression and down-regulation of Smac and Caspase-9 expression.The inhibition of resveratrol on the GFAP may be related to the inhibition of Cx43 protein expression.Resveratrol also inhibited the expression of inflammatory cytokines after ischemia reperfusion.