| Objective:1.To observe the effects of HSYA combination with paeoniflorin on brain infarct size,the neurological deficit score and pathological by establishing the model of acute focal cerebral ischemia-reperfusion injury in rats,and to explore the synergistic effect of neuroprotection on cerebral ischemia-reperfusion injury;2.To explore the synergistic inhibitory effec of HSYA combination with Paeoniflorin on platelet activation by observing the changes of CD62 P in whole blood;3.To explore the synergistic effect of HSYA combination with paeoniflorin on PI3K/Akt signal pathway By observing the expression of PI3 K m RNA(ρ110),Akt m RNA(Akt1)gene in PI3K/Akt signaling pathway in hippocampus,and the expression of p-Akt(Akt1)protein in brain tissue;4.Integrated the above indicators,to explore the possible mechanism of the synergistic protective effect of HSYA combination with paeoniflorin on PI3K/Akt signaling pathway in cerebral ischemia-reperfusion injury.Methods:1.SPF male SD rats(120 total)were randomly divided into 6 groups(20 in each group):HSYA,paeoniflorin,HSYA combination with paeoniflorin(combination group),ginkgolide,model and sham groups.The modified thread bolt method of middle cerebral artery occlusion(MCAO)was used as model to induce an acute focal ischemic cerebral ischemia-reperfusion injury in rats.The neurological defici scoret was 1h of ischemia and6 h of reperfusion,the rats were removed who rated as 0 points and died less than the scheduled time.The rats were treated by tail vein injection for 7 days,once a day.All rats were daily obsered hair luster,mental state,drinking,eating,weight change and so on.Dosage: HSYA(5mg·kg-1)group,paeoniflorin(5mg·kg-1)group,HSYA combination with paeoniflorin(HSYA5mg·kg-1 + paeoniflorin 5mg·kg-1)group and gingkolides(5mg·kg-1)group,model group and sham group received normal saline.The rats were rescored at the 2h after the last injection.2.The area ratio of cerebral infarction in each group were detected by TTC staining,to observe the effect of HSYA combination with paeoniflorin on the area of cerebral infarction.3.The effect of HSYA combination with paeoniflorin on the pathological changes of brain tissue were observed by HE staining.4.The degree of activation of platelet activation marker CD62 P in whole blood of rats were detected by flow cytometry(FCM),to observe the effect of HSYA combination with paeoniflorin on platelet activation function.5.The expression of p-Akt protein in rat brain were detected by immunohistochemistry(IHC),to observe the effect of HSYA combination with paeoniflorin on the expression of Akt protein in the PI3K/Akt signaingl pathway.6.The expression of PI3 K m RNA and Akt mRNA genes in rat hippocampus were detected by qRT-PCR method,to observe the effect of HSYA combination with paeoniflorin on the gene expression in PI3K/Akt signaling pathway.Results:1.Model group showed significant reduce on the weight of within 24 h in rats after the operation compared with the sham group(P<0.01);Combination group showed ignificant increase on the weight of within 24 h after the operation compared with the model group(P<0.01);HSYA group and paeoniflorin group had significantly lower weightwithin 24 h after the operation compared with the combination group(P<0.05).2.Model group showed significant increase on neurological function both before and after treatment(the difference of pre-treatment / post-treatment rate scores)compared with sham group(P<0.01);HSYA,Paeoniflorin,HSYA+Paeoniflorin and Ginkgolide rats showed significant improvement in on neurological function both before and after treatment compared with model group(P<0.05,P<0.01);There was no significant difference between combination group,HSYA group and paeoniflorin group(P>0.05).3.Model group showed significant increase on the cerebral infarction area compared with sham group(P<0.01);HSYA group,combination group and ginkgolide group showed significant reduce on the cerebral infarction area compared with model group(P<0.05,P<0.01);There was no significant difference between combination group,HSYA group and paeoniflorin group(P>0.05).4.Sham rats revealed a right hemisphere structural integrity,clear and complete structure of cortex and hippocampus,with no obvious damages or disruption.On the other hand,cerebral tissue in model rats exhibited liquefactive necrosis,non-clear structure,neuronal degeneration and necrosis.And cortical damage seems to be more severe than hippocampal damage.Rats treated with ginkgolide,HSYA and paeoniflorin showed partial cell arrangement disorder in hippocampal CA1 region,cell shrinkage,smaller in size,isolation from surrounding cells,eosinophilic changes and apoptosis.On the other hand,rats treated with HSYA combination with paeoniflorin exhibited a normal amount of CA1 hippocampal neurons,normally arranged,with no obvious abnormalities.5.The Model group showed significant increase on the activation degree of CD62 P in whole blood compared with the sham group(P<0.01).The combination group and ginkgolide group exhibited significant reduction on the activation degree of CD62 P in whole blood compared with model group(P<0.05).The combination group reduction was greater than the paeoniflorin group(P<0.01),but there was no significant difference between the combination group and the HSYA group(P>0.05).6.The expression of p-Akt in cortex was significantly higher than that in hippocampus(P<0.01).The Model groupshowed significant increased on the expression of p-Akt in braintissue compared with sham group(P<0.01).HSYA group,paeoniflorin group,ginkgolide group and combination group showed significant increase on the expression of p-Akt compared with the model group(P<0.01).HSYA group and paeoniflorin group showed significant reduce on the expression of p-Akt compared with the combination group(P<0.01).7.The Model group showed significant decrease on PI3 K mRNA genes expression in hippocampus compared to sham group(P<0.01).The HSYA,paeoniflorin,ginkgolide and combination groups had all significantly downregulated PI3 K m RNA pathological overexpression in comparison to model group(P<0.01,P<0.05).Howwere,the HSYA and ginkgolide groups have siginificantly groups higher PI3 K mRNA expression in comparison to combination group(P<0.05).There was no significantly difference between the combination group and paeoniflorin group(P>0.05).The differences observed in Akt mRNA expression in the hippocampus of these groups were not statistically significant(P>0.05).Conclusions:1.HSYA combination with Paeoniflorin has a synergistic protective effect on acute cerebral ischemia-reperfusion injury in rats,which may be related to significant reduce cerebral infarct size and neurological deficit scores,inhibit postoperative 24 h weight loss,improve the pathological damage of brain tissu and obviously reduce the content of CD62 P in whole blood.2.HSYA combination with Paeoniflorin has a synergistic protective effect on acute cerebral ischemia-reperfusion injury by acting on the PI3 K /Akt signaling pathway in rats,which by inhibiting the pathological overexpression of PI3 K mRNA,Akt mRNA gene and upregulating the expression of Akt phosphorylation to play a role in the brain protection. |
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