Molecular Mechanism Of HtrA2/Omi Dysfunction And DA Neuron Apoptosis In Parkinson’s Disease

Objective:This study mainly discussed the molecular mechanism of the correlation between HtrA2/Omi dysfunction and endoplasmic reticulum stress(ERS),apoptosis and Wnt signaling pathway in models of PD,and the neuroprotective effects of intervening of molecular targets such as HtrA2/Omi,Wnt and GSK3 were evaluated..Methods:1.6 hydroxydopamine(6-OHDA)was used to intervene PC12 cells,and the damage model of PD cells was constructed.CCK8 method was used to detect the cell viability and to screen the optimum concentration.The expression of tyrosine hydroxylase(TH)was detected by immunohistochemistry.The apoptosis rate was determined by flow cytometry.the levels of ERS and apoptosis-related proteins such as α-syn,CHOP,Grp78,active caspase3,XIAP and TH were detected by Western blotting(WB).The siRNA was transfected by Lentivirus to silence the HtrA2/Omi gene.The silencing efficiency of HtrA2/Omi gene was detected by real-time fluorescent quantitative(RT-PCR).WB was used to detect proteins about endoplasmic reticulum stress,apoptotic and Wnt pathway.2.The optimal concentration of Ucf101(a specific inhibitor of HtrA2/Omi)was screened by CCK8.WB was used to detect the effect of Ucf101 on ERS in PD model cells.The PD rat model was constructed by 6-OHDA injection of right substantia nigra compact(SNc)and Midbrain ventral tegmental area(VTA).APO was intraperitoneally injected at 2,3 and 4 weeks postoperatively to induce rotation behavior.WB assay was used to detect the expression of proteins such as HtrA2/Omi,activate Caspase3,α-syn,ERS and apoptosis-related proteins.Ucf101 continued to intervene in rats for 4 weeks,observe thebehavior of rats,and detect the expression of TH in midbrain tissues by immunohistochemistry.WB was used to detect the expression of HtrA2/Omi,TH,ERS,apoptosis-related proteins and Wnt pathway proteins in rat midbrain tissues.3.WB was used to analyze the expression of Wnt pathway protein in PD cell model;The optimal concentration of Wnt1 protein was screened by CCK8,the effect of Wnt1 protein on apoptosis of PD model cells was detected by flow cytometry,and the expression of ERS-related proteins was detected by WB.The best concentration of LiCL(specific inhibitor of GSK3β)was screened by CCK8 method,and the expression of TH and Wnt1 in cells was detected by immunofluorescence method.Results:1.The cell survival rate and 6-OHDA showed a time dose-dependent decrease trend,and the cell survival rate at 60μM was 51.37±2.83%.The expression of HtrA2/Omi in PD model cells increased from 0 to 24 h.Compared with the control group,the expression ofα-syn,Bip/Grp78,CHOP and active caspase3 were up-regulated(P < 0.05).The expression of Xlinked apoptotic inhibitory protein(XIAP)was down-regulated,P<0.05,and the apoptosis rate of the model group was significantly increased.Silence of HtrA2/Omi gene,the expression of HtrA2/Omi mRNA decreased significantly,P <0.05.Compared with the model cell group,the apoptosis rate of HtrA2/Omi gene silencing group was increased,and the expression of α-syn,Bip/Grp78,CHOP,and active caspase3 were up-regulated,P<0.05.The expression of Xlinked apoptotic inhibitory protein(XIAP)was down-regulated(P < 0.05),which aggravated the ERS damage induced by6-OHDA.The GSK3β phosphorylated molecule tyr216/Ser9 ratio increased,and the Wnt pathway was inhibited.2.The intervention concentration of Ucf101 significantly increased the cell viability by 2.5uM,while both 10 uM and 20 uM decreased the cell viability of the model(P <0.05).2.5Um of Ucf101 could reduce the apoptosis rate of model cells,and 10 uM Ucf101could accelerate the apoptosis of model cells.2.5uM Ucf101 pretreated PD model cells,expression of ERS marker molecules Bip/Grp78 and CHOP decreased,P<0.05.Ucf101 can improve the APO-induced rotation behavior in PD rats,and the expression of TH in the midbrain increased.Ucf101 reduced the accumulation of α-syn in the midbrain of PD rats,reduced ERS level,down-regulated caspase3,up-regulated TH and XIAP,and protected DA neurons.The ratio of p-GSK3β(ser9)/p-GSK3β(tyr216)in PD rats was decreased when Wnt-3α and β-catenin were down-regulated.Ucf101 up-regulated Wnt-3α,β-catenin and p-GSK3β(ser9)and down-regulated p-GSK3β(tyr216)in PD rats.Ucf101 activates the Wnt pathway in DA neurons of PD rats,reduces the activity of GSK3β and protects DA neurons.3.The expression of p-GSK3β(ser9),β-catenin and p-Akt in PD model cells within24 hours showed a decreasing trend,while the expression of p-GSK3β(Tyr216)showed an increasing trend.6-OHDA inhibited activation of the Wnt pathway and increased activity of GSK3β.The optimal concentration of Wnt1 was 100ng/mL by CCK8.Wnt1 significantly reduced the expression of Bip/Grp78 and CHOP in PD model cells.Wnt1 could anesis 6-OHDA induced ERS and apoptosis.The optimal concentration of LiCL is2 mM,LiCL can increase the survival rate of model cells,up-regulate the expression of Wnt and TH,activate the Wnt signaling pathway,and reduce the cell damage caused by6-OHDA.Conclusion:1.6-OHDA up-regulated α-syn,HtrA2/Omi,ERS and apoptosis-related proteins,in vivo and vitro PD models to inhibited Wnt pathway,activated GSK3β,and promoted DA neuron apoptosis.2.The silencing of HtrA2/Omi gene can aggravate ERS and apoptosis of PD model cells,further inhibit Wnt pathway,activate GSK3β,and aggravate cell damage.3.Mild inhibition of HtrA2/Omi can alleviate 6-OHDA induced ERS,reduce the apoptosis of DA neurons,activate Wnt pathway,and inhibit GSK3β,which has certain neuroprotective value.4.nt1 alleviated ERS of PD model cells and reduces apoptosis;LiCl down-regulated ERS of model cells,reduces cell apoptosis,inhibits GSK3β,and activated Wnt pathway,which has certain nerve repair value.5.HtrA2/Omi dysfunction and ERS may play an important role in the pathogenesis of PD,and Wnt pathway and GSK3β,Wnt and other molecules may play key roles in the cascade reaction of cell apoptosis,which may be used as a target for PD research and treatment.