The Possible Mechanism Of Naotaifang In Preventing Cerebral Injury After Focal Cerebral Ischemia Based On Endoplasmic Reticulum Stress

Objective: To investigate the the protective effect and the mechanisms of Naotaifang,a formula benefiting qi and activating blood circulation,on focal cerebral ischemia based on endoplasmic reticulum stress damage in order to provide scientific evidence for its further clinical application.Methods:1.Animal experiment1.1 Animal experiment: SPF grade SD male rats were randomly divided into sham-operated group,model group(normal saline 10 m L/kg.weight),low dose Naotaifang group(4.5g/kg.weight),medium dose Naotaifang group(9g/kg.weight),high dose Naotaifang group(18g/kg.weight)and nimodipine group(32mg/kg.weight).Except for normal control group,the rest rats were prepared for middle cerebral artery occlusion(MCAO)using suture-occluded method.The rats received continuous lavage 7 days before operation once daily according to above-mentioned dose and their neurobehavioral score were graded according to Longa method 6 hours after MCAO operation.The brains were taken 24 hours after MCAO operation,and the area of cerebral infarction was detected and the area percentage was calculated using TCC staining method.The survival of ischemic brain tissue neurons was observed using Nissl staining method 72 hours after operation.1.2 Then the expressions of protein GRP78,p-PERK and CHOP related to hippocampal endoplasmic reticulum stress at different time points after MCAO was detected using immunohistochemical method.The protein expressions of p-PERK,GRP78 and CHOP were detected using western-blot and the gene expressions of GRP78 and CHOP at different time points were detected using RT-q PCR.2.Cell experiment2.1 Artificially isolated and cultured rat(fetal rat)hippocampal neurons and HT22 mice hippocampal neurons were randomly assigned into 4 groups: hippocampal neurons with control plasma group,hippocampal neurons,control plasma and hypoxia-hypoglycemia group,hippocampal neurons with Naotaifang plasma group,hippocampal neurons,Naotaifang plasma and hypoxia-hypoglycemia group.Build models using OGD method and conduct MTT test(10000/well for rat and 8000/well for mice)at 6h,12 h,24h,48 h,and 72 h respectively after the models succeeded.Meanwhile,observe the survival and growth of neurons.2.2 detect the protein expressions of ATF4 and c ATF6 related to endoplasmic reticulum stress in HT22 mice hippocampal neurons using western-blot at corresponding time points.3.Data analysis The experimental data was statistically analyzed with SPSS18.0 and the measurement data was expressed by mean ± standard deviation(`X ± S).Use one-way ANOVA in the comparison of the groups and P<0.05 means significant difference.Results:1.Animal experiment1.1 Compared with control group,the neurobehavioral score results showed that Naotaifang could reduce the neurobehavioral score 12 hours after MCAO(P<0.05),especially the high dose Naotaifang(P<0.05).1.2 For morphological observation,TTC staining indicated that Naotaifang could significantly reduce the area percentage of infarction after MCAO(P<0.05)and Nissl staining indicated that Naotaifang could obviously increase the survival amount of hippocampal neurons per unit area after MCAO(P<0.05)espeicially the high does Naotaifang(P<0.05).1.3 Immunohistochemical results showed that GRP78 protein expression level in model group gradually increased since 6 hours after operation,reached the peak at 24 hours and significantly decreased after 72 hours.After using Naotaifang intervention,GRP78 expression obviously increased after operation(P<0.05).While in model group,p-PERK protein expression was quite high at 6 hours and then began to decrease after operation(P<0.05).But after using Naotaifang intervention,p-PERK protein expression was significantly higher than that in control group and remained at a high level since 24 hoursafter operation(P<0.05).Meanwhile,in model group,CHOP protein expression gradually increased along with time(P<0.05)and Naotaifang began to significantly inhibit CHOP protein expression 12 hours after operation(P<0.05).1.4 Western-blot results showed that compared with model group,Naotaifang could upregulate GRP78 protein expression after operation(P<0.05)and began to promote p-PERK expression 12 hours after operation(P<0.05),while CHOP expression was obviously downregulated(P<0.05).1.5 RT-q PCR results suggested that in the model group GRP78 gene expression increased,reached to the peak at 24 hours after operation,and then decreased,while Naotaifang began to promote GRP78 gene expression since 12 hours(P<0.05).In model group,CHOP gene expression gradually increased along with time and Naotaifang began to significantly inhibit CHOP gene expression in hippocampus 24 hours later(P<0.05).2.Cell experiment2.1 MTT test results showed that the MTT value of fetal rat hippocampal neurons graduallydecreased with the extension of inoculation time since 12 h after OGD and compared with OGD model group,the MTT value of hippocampal neurons after given Naotaifang plasma began to significantly increase 12 h after OGD.The MTT value of HT22 mice hippocampal neurons gradually decreased with the extension of inoculation time since 24 h after OGD and compared with OGD model group,the MTT value of HT22 mice hippocampal neurons after given Naotaifang plasma was significantly increased 24 h later after OGD.2.2 Western-blot results showed that ATF4 protein expression of the hippocampal neurons 6 hours after OGD had no significant difference with normal cultured neurons,but gradually increased 12 hours after OGD compared with normal cultured neurons 12 hours after operation(P<0.05).After using Naotaifang plasma intervention,ATF4 expression obviously decreased 12 hours later compared with OGD model group 12 hours after operation(P<0.05).The expression of c ATF6 protein in cultured HT22 mice in 6 hours after OGD had no significant difference with normal cultured neurons,but significantly increased 24 hours after OGD,while decreased a bit 72 hours later,which was still slightly higher than that of normal group(P<0.05).After using Naotaifang plasma intervention,c ATF6 protein expression was higher than that of OGD model group within 24 hours after OGD(P<0.05).Conclusions:Naotaifang carried out its protective function through the regulation of PERK and ATF6 endoplasmic reticulum stress pathways and the downregulation of CHOP after focal cerebral ischemia.