| Background: Diabetes mellitus(DM)is an independent risk factor for increased incidence and mortality in ischaemic stroke.In patients with acute cerebral infarction(ACI),the area of cerebral infarction is larger,the clinical prognosis is worse,the mortality rate increases,but the specific mechanism is not fully understood.Endoplasmic reticulum stress(ER stress)is one of the important mechanisms of cerebral ischemia reperfusion injury(IRI)and an important participant in the pathogenesis of diabetes mellitus and its complications.RTN1-C is a ER-associated protein,which mediates ER stress-induced apoptosis and regulates cellular susceptibility to different apoptosis pathways.Studies have shown that overexpression of RTN1-C can aggravates cerebral IRI.Objective: In this study,we aimed to investigate the role of RTN1-C in high glucose-aggravated IRI in N2 a cells and its mechanism.Methods: Mouse neuroblastoma N2 a cells were cultured in DMEM:F12 culture medium supplemented with 10% fetal bovine serum.The cells were seeded in 96-well(100 ?l/well)or 24-well(1 ml/well)or 6-well(2 ml/well)plates in the density of 105/m L.To simulate high glucose damage model,cells were incubated in DMEM containing 30 mmol/L glucose(high glucose)for 48 h in normal glucose groups.Then the cells were randomly divided into 10 goups : group normal glucose control(group NG-CON),group normal glucose oxygen-glucose deprivation/reoxygenation(group NG-OGD/R),group normal glucose 4-PBA preconditioning(group NG-OGD/R+4PBA),group normal glucose transfected with RTN1-C sh RNA(group NG-sh RTN1-C),group normal glucose transfected with scrambled sequence of sh RNA(group NG-sh NC),group high glucose control(group HG-CON),group high glucose oxygen-glucose deprivation/reoxygenation(group HG-OGD/R),group high glucose 4-PBA preconditioning(group HG-OGD/R+4PBA),group high glucose transfected with RTN1-C sh RNA(group HG-sh RTN1-C),group high glucose transfected with scrambled sequence of sh RNA(group HG-sh NC).In NG-OGD/R,NG-OGD/R+4-PBA,NG-sh NC,NG-sh RTN1-C,HG-OGD/R,HG-OGD/R+4-PBA,HG-sh NC,HG-sh RTN1-C groups,after changing the culture medium for glucose-free serum-free culture medium,the cells were exposed to 95% N2-5% CO2 in an incubator at 37 for 3 h.Subsequently,the culture medium was changed to DMEM culture medium ?suppemented with 10% fetal bovine serum and glucose at the corresponding concentration,and the cells were incubated in the normal oxygenation condition for 2 h.In group NG-OGD/R+4-PBA and group HG-OGD/R+4-PBA,the cells were incubated in the DMEM culture medium supplemented with 4-PBA at the final concentration of 2 mmol/L for 2 h before OGD/R injury.In NG-sh NC,NG-sh RTN1-C,HG-sh NC,HG-sh RTN1-C groups,the cells were respectively transfected with scrambled sequence of sh RNA or RTN1-C sh RNA for 24 h before OGD/R injury.At 2 h of reoxygenation,the cells were selected to measure the cell viability by CCK-8 assay,apoptosis rate by PE Annexin V/7-AAD flow cytometry,and the expression of RTN1-C,GRP78,cleaved-caspase-12,CHOP,cleaved-caspase-3 was analyzed by Western blot.Results: 1.Cell viability Compared to group NG-CON,the cell viability was reduced in group NG-OGD/R(P<0.05).Compared to group NG-OGD/R,the cell viability was increased in group NG-OGD/R+4-PBA(P<0.05).Compared to group HG-CON,the cell viability was reduced in group HG-OGD/R(P<0.05).Compared to group HG-OGD/R,the cell viability was increased in group HG-OGD/R+4-PBA(P<0.05).Compared to group NG-OGD/R,the cell viability was reduced in group HG-OGD/R(P<0.05).There was no obvious change of the cell viability,between groups of NG-OGD/R+4-PBA and HG-OGD/R+4-PBA(P>0.05).Compared to group NG-sh NC,the cell viability was reduced in group HG-sh NC(P<0.05).There was no obvious change of the cell viability,between groups of NG-sh RTN1-C and HG-sh RTN1-C(P>0.05).2.Cell apoptosis rate Compared to group NG-CON,the cell apoptosis rate was increased in group NG-OGD/R(P<0.05).Compared to group NG-OGD/R,the cell apoptosis rate was reduced in group NG-OGD/R+4-PBA(P<0.05).Compared to group HG-CON,the cell apoptosis rate was increased in group HG-OGD/R(P<0.05).Compared to group HG-OGD/R,the cell apoptosis rate was reduced in group HG-OGD/R+4-PBA(P<0.05).Compared to group NG-OGD/R,the cell apoptosis rate was increased in group HG-OGD/R(P<0.05).There was no obvious change of the cell apoptosis rate,between groups of NG-OGD/R+4-PBA and HG-OGD/R+4-PBA(P>0.05).Compared to group NG-sh NC,the cell apoptosis rate was increased in group HG-sh NC(P<0.05).There was no obvious change of the cell apoptosis rate,between groups of NG-sh RTN1-C and HG-sh RTN1-C(P>0.05).3.The expression of protein Compared to group NG-CON,the protein expressions of RTN1-C,GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3 were increased in group NG-OGD/R(P<0.05).Compared to group NG-OGD/R,the protein expressions of GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3 were decreased in group NG-OGD/R+4-PBA(P<0.05).Compared to group HG-CON,the protein expressions of RTN1-C,GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3 were increased in group HG-OGD/R(P<0.05).Compared to group HG-OGD/R,the protein expressions of GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3 were decreased in group HG-OGD/R+4-PBA(P<0.05).Compared to group NG-OGD/R,the protein expressions of GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3 were increased in group HG-OGD/R(P<0.05).There were no obvious change of protein expressions of GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3,between groups of NG-OGD/R+4-PBA and HG-OGD/R+4-PBA(P>0.05).Compared to group NG-sh NC,the protein expressions of GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3 were increased in group HG-sh NC(P<0.05).There were no obvious change of the protein expressions of GRP78,cleaved-caspase-12,CHOP and cleaved-caspase-3,between groups of NG-sh RTN1-C and HG-sh RTN1-C(P>0.05).Conclusions: High glucose significantly increases the expression of RTN1-C in OGD/R treated cells.RTN1-C is involved in high glucose-aggravated OGD/R injury by exacerbating ER stress. |
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