| Cerebral ischemic stroke（CIS）is currently treated by intravenous thrombolysis,which may lead to cerebral ischemia-reperfusion injury and further aggravate the damage of brain tissue.Glucose regulated protein 78（GRP78）,an important molecular chaperone in the endoplasmic reticulum（ER）,can aggravate the inflammatory response in the brain by regulating the nuclear factor-κB（NF-κB）inflammatory pathway.it is hypothesized that the regulation of GRP78 or other important molecules to further affect endoplasmic reticulum stress（ERS）may improve the occurrence and development of inflammation and apoptosis during cerebral ischemia-reperfusion injury.After cerebral ischemia-reperfusion,cerebroprotein hydrolysate（CH）can decrease the release of pro-inflammatory factors and inhibit oxidative stress by regulating Ca2+and oxygen free radicals.It can also up-regulate brain-derived neurotrophic factor,increase synaptic den-sity,and restore white matter integrity and axonal plasticity.In this study,firstly,the related target genes between CIS and ERS are screened out,and the enrichment analysis and prediction are carried out according to the obtained target genes.Then the model of cerebral ischemia-reperfusion injury in mice is established by the method of thread embolism,and the related target proteins are detected by immunohistochemistry and Western blot to explore the possible mechanism related to ERS in the neuro pro-tective effect of cerebroprotein hydrolysate.Firstly,Gene Cards and OMIM databases were used to screen out the targets related to ischemic stroke and ERS in the experiment,and Venny2.1.0 was used to draw Venn’s diagram to obtain the intersecting genes of the two.Then String database was used to obtain the Protein-protein Interaction（PPI）diagram.Cytoscape was used for visual analysis,and cyto Hubba plug-in was used to screen out the key genes and conduct enrichment analysis.In animal experiments,a mouse model of ischemic stroke induced by ischemia-reperfusion injury of middle cerebral artery was established by thread embolism method.According to the Longa"5-point scale"scoring method,a score of 1-3 points was considered as successful modeling and included in the follow-up experiments.Mice with successful modeling were randomly divided into sham group,model group,cerebroprotein hydrolysate 5 mg·kg-1(5 mg·kg-1CH)group,cerebroprotein hydrolysate 10 mg·kg-1(10 mg·kg-1CH)group,cerebroprotein hydrolysate5 mg·kg-1combined with edaravone 5 mg·kg-1(5 mg·kg-1CH+5 mg·kg-1EDA),cerebroprotein hydrolysate 10 mg·kg-1combined with edaravone 5mg·kg-1(10 mg·kg-1CH+5 mg·kg-1EDA)and edaravone 10 mg·kg-1(10mg·kg-1EDA)positive drug control group according to neurological function score with 11 mice in each group.Intraperitoneal injection was performed for 14 days.The second neurological deficit score was made according to Longa method,and the changes of scores in each group were observed after the administration.2,3,5-triphenyltetrazole chloride（TTC）was used to detect the changes of cerebral infarction area in mice.The expressions of Caspase-3,Akt,CREB and p-CREB in brain tissue of mice were detected by immunohistochemistry.Western blot was used to detect the expressions of Akt,p-Akt,GRP78,Caspase-12 and CHOP in the cortex and hippocampus of mice to preliminarily explore the effect of ERS on the expression of PI3K/Akt pathway-related proteins at the molecular level,and how cerebroprotein hydrolysate regulates ERS to play a neuroprotective role.The results of network pharmacology show that there are 3744 targets related to CIS and 8675 targets related to ERS,and 41 common targets between them.There are 37 nodes and 390 edges in PPI network,and there are 37 interacting proteins and 390 interacting relationships.The top 10genes in MCC algorithm are IL-6,ALB,INS,TNF,AKT1,CASP3,MAPK3,TP53,SIRT1 and VEGF respectively.Biological processes were highly related to reaction to oxidative stress,the regulation of neuron death,and negative regulation of cell differentiation,etc.;cellular components were highly related to the membrane raft,smooth endoplasmic reticulum,endoplasmic reticulum lumen and other components;molecular function aspects were related to signalling receptor activator activity,chaperone binding and protease binding.Enrichment analysis of pathway revealed that the intersecting targets were involved in PI3K/Akt and protein processing in endoplasmic reticulum,etc.The neurological score again showed that the neurological score in the sham group was 0 after 14 days of administration.Compared with the mice in the sham group,the neurological deficit score of the model group was significantly increased（P<0.001）.Compared with the model group,the scores of mice in each dosage group of cerebroprotein hydrolysate,the combination treatment group and the edaravone positive control group were significantly reduced,and the neurological impairment was signi-ficantly improved（P<0.05）.TTC results showed that the cerebral infarct area of mice in the sham group was 0.the other groups of mice showed different infarct areas.Compared with the sham group,the cerebral infarc-tion area in the model group was significantly increased（P<0.001）.Compared with the model group,the cerebral infarct area was reduced to different extents in each administration group（P<0.05）.The results of immunohistochemical showed that compared with the sham group,the content of Caspase-3 in the model group was significantly increased（P<0.001）,and the expression levels of Akt,CREB and p-CREB were significantly decreased（P<0.001）.Compared with the model group,the content of Caspase-3 in mice of each administration group was decreased（P<0.05 or P<0.01 or P<0.001）;the levels of Akt and CREB were both increased（P<0.05 or P<0.01 or P<0.001）;the contents of p-CREB in 5mg·kg-1CH+5 mg·kg-1EDA,10 mg·kg-1CH+5 mg·kg-1EDA and EDA control group were significantly increased（P<0.05 or P<0.01）,and expression of p-CREB was not enhanced in the CH two dose groups（P>0.05）.Compared with the 10 mg·kg-1CH+5 mg·kg-1EDA group,the contents of Akt and p-CREB in the 5 mg·kg-1CH group were significantly reduced（P<0.05 or P<0.01）;the expressions of Caspase-3,Akt and p-CREB in the 10 mg·kg-1CH group were significantly reduced（P<0.05）;the expression of Akt in the EDA control group was reduced（P<0.05）.The results of Western Blot showed that p-Akt/Akt ratios in the cortex and hippocampus were significantly decreased（P<0.01）and the expressions of GRP78,CHOP and Caspase-12 were significantly increased in the model group（P<0.01 or P<0.001）compared with the sham operation group.Compared with the model group,each administration group increased the p-Akt/Akt ratio in the cortex and hippocampus（P<0.05 or P<0.01）;GRP78,Caspase-12,and CHOP were significantly decreased in both cortex and hippocampus（P<0.05 or P<0.01 or P<0.001）.Compared with 5 mg·kg-1CH+5 mg·kg-1EDA,the expression of GRP78 in the 10 mg·kg-1CH group was significantly reduced（P<0.05）,as well as the expression levels of GRP78 and CHOP in the 10 mg·kg-1EDA group（P<0.05）.In summary,it is speculated that the main action targets that are closely related to the ERS mechanism of CIS are Akt and Caspase-3,involving PI3K/Akt and protein processing in endoplasmic reticulum according to network pharmacology analysis.Animal experiments showed that the cerebroprotein hydrolysate could inhibit the expression of endoplasmic reticulum stress-related proteins such as GRP78,CHOP and Caspase-12 by increasing the ratio of p Akt/Akt and the expression of CREB.Its neuroprotective mechanism may be related to the activation of PI3K/Akt pathway and the inhibition of GRP78-induced apoptosis pathway. |
