滋肾活血方通过GRP78/PERK/ATF4信号通路减轻2-VO模型大鼠海马神经元内质网应激的保护机制OA

Objective:To investigate the mechanism by which the Zishen Huoxue prescription(ZSHXP)ameliorates cognitive dysfunction in rats with vascular dementia(VD)induced by the bilateral common carotid artery ligation(2-VO model rats)through regulating the glucose-regulated protein 78(GRP78)/protein kinase R-like endoplasmic reticulum kinase(PERK)/activating transcription factor 4(ATF4)signaling pathway.Methods:A VD rat model was established via the 2-VO method.A total of 72 male Sprague-Dawley(SD)rats were randomly divided into six groups:Sham group,Model group,donepezil hydrochloride group(0.45 mg·kg-1),and ZSHXP groups at low(8.90 g·kg-1),medium(17.80 g·kg-1),and high(35.60 g·kg-1)doses,with 12 rats in each group.The Morris Water Maze test was utilized to assess spatial learning and memory abilities of rats,and the Novel Object Recognition test was used to evaluate cognitive performance.Hematoxylin-eosin(HE)and Nissl staining were applied to observe the histological and morphological changes in hippocampal tissues.Transmission electron microscopy(TEM)was used to observe the morphological changes of endoplasmic reticulum in rat hippocampal neurons.Immunofluorescence staining was adopted to detect the colocalization of neuronal nuclei antigen(NeuN)with GRP78 and β Ⅲ Tubulin with gasdermin D(GSDMD)in hippocampal neurons.Western blot was used to detect the expression levels of endoplasmic reticulum stress(ERS)-related proteins including GRP78,PERK,ATF4,phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK),C/EBP homologous protein(CHOP),NOD-like receptor protein 3(NLRP3),Caspase-1 and GSDMD.Results:Compared with the sham operation group,the model group showed a significantly prolonged escape latency(P＜0.01),a significant decrease in the number of platform crossings and the residence time in the target quadrant(P＜0.01),and a markedly reduced recognition index(P＜0.01).Histological observations revealed that the hippocampal neurons in the model group were disorderly arranged with reduced quantity,deformed and shrunken cell bodies,and pyknotic and hyperchromatic nuclei.The number of Nissl bodies decreased significantly.The number of endoplasmic reticula reduced obviously,accompanied by abnormal dilation and swelling,and the loss of normal folding structure.The fluorescence colocalization of NeuN with GRP78 and β ⅢTubulin with GSDMD in the hippocampus was significantly increased in the model group.The protein expression levels of GRP78,p-PERK/PERK,ATF4,CHOP,NLRP3,GSDMD and Caspase-1 in the model group were significantly elevated(P＜0.01).Compared with the model group,the donepezil hydrochloride group and the ZSHXP medium-and high-dose groups had a significantly shortened escape latency(P＜0.01)and an increased number of platform crossings(P＜0.05,P＜0.01).The residence time in the target quadrant was increased in the donepezil hydrochloride group and all ZSHXP groups(P＜0.05,P＜0.01),with a significantly improved recognition index(P＜0.01).In the donepezil hydrochloride group and all ZSHXP groups,the number of hippocampal neurons increased with a more compact arrangement and reduced nuclear hyperchromasia.The number of Nissl bodies increased with morphological structures tending to be normal.In the ZSHXP high-dose group,the number of endoplasmic reticula increased and the folding structure was restored.The fluorescence colocalization of NeuN with GRP78 and β Ⅲ Tubulin with GSDMD in the hippocampus was significantly weakened in the treatment groups.In the donepezil hydrochloride group,the protein expressions of GRP78,ATF4 and CHOP were increased(P＜0.01),while the expression of p-PERK/PERK was decreased(P＜0.05).In the ZSHXP low-dose group,the expressions of GRP78,p-PERK/PERK and CHOP were elevated(P＜0.05,P＜0.01).The ZSHXP medium-and high-dose groups showed a significant decrease in the protein expressions of p-PERK/PERK,ATF4 and CHOP(P＜0.01),and the high-dose group had a markedly reduced GRP78 protein expression(P＜0.01).In the donepezil hydrochloride group,the Caspase-1 protein expression was increased(P＜0.01)and the NLRP3 protein expression was decreased(P＜0.01).In the ZSHXP low-dose group,the GSDMD expression was elevated(P＜0.01)while the NLRP3 protein expression was reduced(P＜0.01).After treatment with medium and high doses of ZSHXP,the protein expression levels of NLRP3,GSDMD and Caspase-1 were significantly decreased(P＜0.01).Conclusion:The ameliorative effect of ZSHXP on cognitive function in 2-VO model rats may be associated with its regulation of the GRP78/PERK/ATF4 signaling pathway,which ameliorates ERS and inhibits neuronal pyroptosis.