The Role Of ZDHHC3 In β-caryophyllene Protect Cerebral Ischemia Reperfusion Injury In Rats

Cerebral ischemia with the high incidence,high disability,and high fatality rate,seriously threatens human health.PATs,also known as the ZDHHC protein family,catalyze the post-translational palmitoylation of proteins and participate in the pathological process of various neurological diseases.However,its role in cerebral ischemia-reperfusion injury（CIRI）has been rarely studied.Objective:To investigate the effect of palmitoyltransferase 3（ZDHHC3）on cerebral ischemia-reperfusion injury and discuss the activation of ZDHHC3 in the protection ofβ-caryophyllene from cerebral ischemia-reperfusion injury in rats,it will provide a reference for finding new therapeutic targets and drugs for ischemic stroke.Methods:1.52 of male SD rats,250～270g and 6-7 weeks old,were randomly divided into two groups:Sham group and model group（MCAO）.Middle cerebral artery occlusion（MCAO）was used to establish CIRI model.The nerve function damage of MCAO rats were evaluated by neurological deficit score.The cerebral infarct volume was quantified by TTC staining and the pathological changes of hippocampus and cortex were detected by HE staining.Immunofluorescence was used to detect the localization of ZDHHC3 in rat cortical and hippocampal neurons.qRT-PCR and Western blot were used to detect the ZDHHC3 and PSD95 mRNA and protein expression in the MCAO rats hippocampus and cortex.2.PC12 cells were cultured in vitro and oxygen and glucose deprivation/reoxygenation（OGD/R）model was established by oxygen and glucose deprivation for 2h and reoxygenation for12h and 24h.In order to investigate the effects of ZDHHC3 and PSD95 on OGD/R-treated PC12cell,PC12 cells were randomly divided into 3 groups:normal group（Control）,OGD/R 12h group,OGD/R 24h group.Then,PC12 cells were randomly divided into 4 groups:normal group（Control）,OGD/R group,OGD/R+pcDNA3.1-GFP group,OGD/R+pcDNA3.1-zdhhc3 group,to observe ZDHHC3 possible protective mechanism in PC12 cells.qRT-PCR and Western blot were used to observe the ZDHHC3 mRNA and protein level in PC12 cells.ZDHHC3 overexpression plasmid was constructed and transfected into PC12 cells,qRT-PCR and Western blot were used to detect the overexpression of ZDHHC3 mRNA and protein.MTT was used to detect PC12 cell survival;Flow cytometry was used to observe PC12 cell apoptosis;The protein expression of ZDHHC3 and PSD95,AMPAR2,Bax,and Bcl2 were detect by Western blot.3.36 of male SD rats,250～270g and 6-7 weeks old,were randomly separated into three groups:Control group（Sham）,Model group（MCAO）,BCP treated group（BCP）.Rats cerebral ischemia reperfusion injury（CIRI）model established by Middle cerebral artery occlusion（MCAO）.The rats were given BCP intragastrically for 7 days(BCP,306mg.kg^-1)before operation.The neurological deficit score was used to assessed the neurological damage of MCAO rats.TTC staining was used to quantify the cerebral infarct volume of MCAO rats and the pathological changes of hippocampus and cortex were dctected by HE staining.Western blot was used to detect the AMPAR2 and PSD95 protein expression.4.PC12 cells were cultured and established damage model induced by OGD/R.In order to investigate the effect of BCP,PC12 cells randomly divided into three groups:Control group,OGD/R group,Control+BCP group,which cells were treated by 2h of oxygen and glucose deprivation and 24h of reperfusion.The Control+BCP group was treated with BCP（2.5μM）before OGD/R.Then,PC12 cells were divided into six groups:Control group,OGD/R group,OGD/R+SiRNA-NC group,OGD/R+ZDHHC3-SiRNAgroup,OGD/R+ZDHHC3-SiRNA+BCPgroup,OGD/R+BCP group.Cell proliferation were detected by MTT,and apoptosis was detected by flow cytometry.qRT-PCR was used to investigate the efficiency of SiRNA and Western blot was used to detect the expression of PSD95,AMPAR2.Results:1.The effect of ZDHHC3 on MCAO/R-treated ratsCompared with the sham group,the MCAO group had significantly increased neurological deficit scores,increased cerebral infarction volume（P<0.01）,increased nuclei deep staining and pyknosis after HE staining.Immunofluorescence results showed that ZDHHC3 expression was identified on cytomembranes and in the cytoplasm of neurons in the hippocampus and cortex of rats,but not in astrocytes.Compared with the sham group,the qRT-PCR and Western blot tests showed that decreased mRNA and protein expression of ZDHHC3 and PSD95 in cortex and hippocampus of MCAO rats（P<0.01）.2.The effect of ZDHHC3 onthe OGD/R-Treated PC12 CellsCompared with control group,the mRNA and protein expression of PSD95 and ZDHHC3 decreased significantly（P<0.05）,and the expression decrease at 24h was more than at 12h in OGD/R group.The apoptosis rate of PC12 cells increased（P<0.01）and the proliferation rate（P<0.01）decreased in OGD/R group.Compared with the OGD/R group and OGD/R+pcDNA3.1-GFP group,the expression of synaptic related protein PSD95 and AMPA receptor subunit（GLUR2）of pcDNA3.1-zdhhc3 group were significantly increased（P<0.05）and there was no significant difference in the OGD/R+PCDNA3.1-GFP group.Compared with the OGD/R group and pcDNA3.1-GFP group,increased cells proliferation（P<0.01）,decreased Bax expression,as well as increased expression of Bcl-2in OGD/R+pcDNA3.1-zdhhc3 group（P<0.05）.3.The effect of BCP on MCAO rats and OGD/R-treated PC12 cells and its relationship with ZDHHC3.Compared with Sham group,BCP group decreased the neurobehavioral score and alleviated the cerebral infarcation（P<0.01）.Compared with MCAO group,the expression level of PSD95,AMPAR2protein in cortex of BCP group was significantly increased（P<0.01）.Compared with the Control group,the proliferation of PC12 cells was significantly decreased after OGD/R（P<0.01）.Compared with OGD/R group,the apoptosis of PC12 cells was significantly decreased and increased cells survivial in BCP group（P<0.05）.Compared with OGD/R+DHHC3-SiRNA group,the protein expression levels of PSD95and AMPAR2 were increased in OGD/R+DHHC3-SiRNA+BCP.Conclusion:1.Rats have neurological impairment and pathological changes,and the expression of synapse-related protein PSD95 and ZDHHC3mRNA and protein also decreases accordingly after cerebral ischemia.Combined with bioinformatics analysis,suggested that ZDHHC3 might interact with PSD95 to participate in cerebral ischemia reperfusion injury.2.The expression of PSD95 and ZDHHC3 mRNA and protein of PC12 cells in OGD/R group decreased significantly,and the apoptosis rate and proliferationin OGD/R group increased significantly.Overpression of ZDHHC3 may play protective roles by regulating AMPA receptors and PSD95 to participate in synaptic plasticity and alleviate the apoptosis of oxygen-glucose deprivation/reoxygenation PC12 cells.3.BCP has a protective effect on cerebral ischemia/reperfusion injury,which effect may be related to decreased cell apoptosis and alleviate OGD/R injury in PC12 cells by increasing the expression of ZDHHC3 and synaptic related proteins PSD95 and AMPAR.