A Study On Mechanisms Of MPST's Inhibition On Neural Apoptosis Induced By Sodium Arsenite Through GGRP78/CHOP Signaling Pathways

Part one Construction of the Lentiviral vector carrying MPST and its expression in SH-SY5 Y cellsObjective: To construct the lentiviral vector of MPST gene and obtain SH-SY5 Y cell line stably expressing the exogenous MPST gene.Methods: The target gene of MPST was amplified by polymerase chain reaction(PCR)and cloned into the lentiviral expression vector p EB-GFP(T2A)PURO.The recombinant lentiviral expression vector and lentivirus packaging plasmid system(p LP/VSVG,p LP1,p LP2)were co-transfected into 293 T cells,SH-SY5 Y cells were infected with the recombinant lentivirus solution,and puromycin was employed to screen the cell stably expressing the exogenous MPST gene(named SH-MPST cells).Real-time PCR,Western blot and ELISA were used to identify the expression and function of MPST protein in the SH-MPST cells.Results: Compared with the SH-PEB group,the expression of MPST m RNA and protein in the SH-MPST group was significantly increased(p<0.01),and the MPST activity,content,and relative levels of hydrogen sulfide were significantly increased(p<0.01).Conclusion: The lentiviral vector of MPST gene was constructed successfully and the SH-SY5 Y cell line stably expressing exogenous MPST gene was also obtained,which would lay the foundation for the further study of endogenous H2 S function.Part two Protection of MPST on SH-SY5 Y cells injured by arsenateObjective: To investigate the effect of over-expression of MPST gene on nerve injuries induced by sodium arsenite(Na As O2)in SH-SY5 Y cells.Methods: The empty vector group(SH-PEB)and over-expressed group(SH-MPST)cells were transfected and exposed to Na As O2 to induce injuries.MTT assay and colorimetric assay were used to detect the cell viability and Caspase-3 activity.Western blot was used to observe expressions of GRP78,CHOP,Bax,and Bcl-2 proteins related with endoplasmic reticulum stress(ERS)and apoptosis.Results: After treatment with 50 ?M Na As O2 for 24 h,the cell viability and Bcl-2 protein expression in SH-PEB group were significantly decreased(p<0.01),while the Caspase-3 activity and protein expression of GRP78,CHOP,and Bax were significantly increased(p<0.01).Compared with the SH-PEB group,the Bcl-2 expression was up-regulated(p<0.01)whereas the expression of GRP78 and CHOP was down-regulated in SH-MPST group.There were no difference in the cell viability,Bax expression and Caspase-3 activity between two groups(p>0.05).Conclusion: Overexpression of MPST gene in SH-SY5 Y cells can protect the neural cells from Na As O2-induced injuries.Part three ERS participates in the effects of MPST on the GRP78/ CHOP pathwayObjective: To investigate whether GRP78/CHOP signaling pathway involve in the protective mechanism of H2 S on arsenic-induced injuries in SH-SY5 Y cells.Methods: Pretreated with ERS blocker Sodium tauroursodeoxycholate(TUDCA),cells were exposed to 50 ?M Na As O2 for 24 h.Then,MTT assay,colorimetric assay and western blot were used to detect the cell viability,Caspase-3 activity and the expression of GRP78,CHOP,Bax and Bcl-2 proteins related with endoplasmic reticulum stress and apoptosis,respectively.Results: 1.The decrease of cell viability,the increase of Caspase-3 activity,the down-regulation of Bcl-2 expression,and the up-regulation of GRP78,CHOP and Bax proteins induced by Na As O2 in the SH-PEB group were reversed by TUDCA pretreatment.2.In the SH-MPST group,the up-regulation of Bcl-2 and down-regulation of GRP78 and CHOP proteins induced by Na As O2 were reversed by TUDCA pretreatment.However,Na As O2 and TUDCA had no effects on cell viability,Caspase-3 activity,and Bax expression(p>0.05).Conclusions: 1.Endoplasmic reticulum is the critical mechanism of arsenate-induced injuries in neural cells.2.GRP78/CHOP pathway plays an important role in MPST's neuroprotection.