Exploring The Neuroprotective Mechanism Of Naotai Fang Against Cerebral Ischemia-reperfusion Injury Based On Endoplasmic Reticulum Stress-mediated Cell Autophag

Objective The intervention mechanism of Naotai Fang on neuronal cell injury in the ischemic penumbra region was investigated based on endoplasmic reticulum stress.MethodIn vivo study: 75 male SD rats were randomly divided into a sham surgery group of 15 rats,a model group of 15 rats,a low-dose Naotai Fang group of 15 rats,a medium dose Naotai Fang group of 15 rats,and a high-dose Naotai Fang group of 15 rats.Then,the model group and Naotai Fang groups of rats underwent surgical modeling of middle cerebral artery occlusion reperfusion(MCAO/R).After modeling,the corresponding drugs were given by gavage for feeding.Zea longa scoring method and balance beam scoring method were used to evaluate the neuromuscular function and vestibular motor function of rats,TTC staining was used to observe the infarct area,HE staining was used to observe the tissue morphology of cerebral ischemic penumbra,immunofluorescence double staining and Western blot were used to detect the expression of endoplasmic reticulum stress and autophagy related marker proteins in brain tissue and ischemic penumbra area.In vitro study: PC12 cells were used to perform hypoxia glucose deprivation / reoxygenation to mimic cerebral ischemia-reperfusion injury in vitro,to set up the normal group,the model group,the 4-PBA(endoplasmic reticulum stress inhibitor)group,and Naotai Fang low(10%),medium(15%),and high(20%)groups.Cell viability was assessed by CCK8 assay,LDH activity in each group was detected by lactate dehydrogenase(LDH)assay kit,the nuclear morphology was observed by Hoechst 33258 staining,the apoptotic rate was detected by TUNEL staining,and the ER stress and positive expression of autophagy pathway related proteins in injured PC12 cells were detected by immunofluorescence and protein immunoblotting.ResultIn vivo study:(1)Compared with the rats before treatment,the scores of Zea longa and balance beam of rats in each Naotai Fang dose group were significantly reduced after treatment(P<0.05,P<0.01).Compared with the model,the scores of Zea longa and balance beam of rats in Naotai Fang middle and high dose groups were significantly reduced after treatment(P<0.05,P<0.01);(2)The infarct volume of rats in the model group significantly increased(P<0.01),while the infarct volume of rats in each dose group of Naotai Fang decreased to varying degrees(P<0.05,P<0.01)The neural cells of the model group were significantly damaged,while the pathological changes of the neural cells in the Naotai Fang group were relatively mild,with significant improvement compared to the model group.The arrangement of neural cells was more orderly and the number was larger;(4)The positive expression of Beclin-1,CHOP,GRP78,PERK,and LC3 II in the ischemic penumbra of the model group rats increased significantly(P<0.05,P<0.01),while the positive expression of Beclin-1,CHOP,GRP78,PERK,and LC3 II in the ischemic penumbra of the rats in each dose group of Naotai Fang decreased to varying degrees(P<0.05,P<0.01);Compared with the low-dose and high-dose groups of Naotai Fang,the positive expressions of Beclin-1,CHOP,PERK,and LC3 II in the ischemic penumbra of rats in the low-dose and high-dose groups of Naotai Fang increased to varying degrees(P<0.05,P<0.01).The positive expression rate of GRP78 in the ischemic penumbra of rats in the low-dose group of Naotai Fang significantly increased(P<0.01).In vitro study:(1)The survival rate of PC12 cells in the model group decreased,LDH activity increased(P<0.01),while the survival rate of cells in the medium and high concentration groups of Naotai Fang increased,and LDH levels decreased(P<0.05,P<0.01);(2)The model group cells showed typical apoptotic characteristics,with a decrease in the number of apoptotic cells and a reduction in nuclear shrinkage in the Naotai Fang medium and high concentration groups The apoptosis rate of PC12 cells in the model group significantly increased(P<0.01),while the apoptosis rate of PC12 cells in the Naotai Fang medium and high concentration groups decreased to varying degrees(P<0.05,P<0.01)The expression of GRP78,PERK,CHOP,LC3 II,and Beclin-1 protein was significantly increased in the model group,while the expression of LC3 I protein was significantly reduced(P<0.05,P<0.01).The expression of GRP78,PERK,CHOP,LC3 II,and Beclin-1 protein was significantly reduced in the medium and high concentration groups of Naotai Fang,while the expression of LC3 I was increased(P<0.05,P<0.01).Conclusion(1)Cerebral ischemia-reperfusion can lead to endoplasmic reticulum stress in neurons in penumbra;Endoplasmic reticulum stress mediates apoptosis caused by autophagy of damaged nerve cells,which is the pathological mechanism of cerebral ischemia-reperfusion injury.(2)By inhibiting endoplasmic reticulum stress and reducing the expression of endoplasmic reticulum stress-related proteins GRP78,PERK and CHOP,the autophagy of cells can be alleviated,the expression of LC3Ⅱ and Beclin1 protein can be increased and decreased,and finally the apoptosis of injured nerve cells can be reduced,thus playing a certain neuroprotective role;(3)Naotai Fang can regulate the expression of ER stress and cell autophagy related proteins,thereby alleviating the state of ER stress and its mediated excessive autophagy in injured neuronal cells in the penumbra area,increasing cell survival rate,reducing cerebral infarction volume,improving neurological function,and thus playing a role in the prevention and reperfusion injury of cerebral ischemia.