Effect And Mechanism Of Lentivirus Mediated Pannexin-1 Gene On Spinal Cord Injury In Rats

Objectives1.To establish an animal model of spinal cord injury(SCI)in rats and observe the expression changes of Pannexin-1(Panx-1)after SCI.2.To culture,identify the primary spinal neurocytes and construct the oxidative stress injury model of spinal neurocytes in rat in vitro.3.To construct the overexpression vector LV5-Panx-1 and interference vector sh-Panx-1 and transfect into rat primary spinal neurocytes in vitro.4.To investigate the effect of regulating the expression of Panx-1 on primary spinal neurocytes of oxidative stress injury in rat.5.To explore the mechanism of Panx-1 aggravating SCI in rat in vitro.Methods1.The spinal cord injury model(Allen's model)of rat was constructed by NYU Impactor-?.And the hind limb motor function of rats was observed by BBB score.The expression of Panx-1 was detected by Real-time PCR and Western Blot.2.Rat embryos of 15 days were used to isolate spinal cord tissues and culture primary spinal neurocytes with modified trypsin digestion combined mechanical separation method.Immunofluorescence staining(Neu N/?tubulin-?/Hoechst 33258)was used to identify spinal neurocytes,and hydrogen peroxide(H₂O₂)was used to construct the oxidative stress injury model of spinal neurocytes in rat in vitro.3.Panx-1 overexpressed lentivirus vector(LV5-Panx-1)and lentivirus interference vector(sh-Panx-1)were constructed.Lentiviral titer was detected by finite dilution method.The above viruses infected primary spinal neurocytes respectively,and multiplicity of infection(MOI)suitable for fluorescence expression determination was observed.Western Blot was used to detect the expression of Panx-1 protein in rat spinal neurocytess after transfection.4.According to different treatment factors,the cells were divided into normal control group(NC group),simple injury group(Vehicle group),empty plasmid transfection group(LV-con group:LV5-con group,sh-con group),LV5-Panx-1 group and sh-Panx-1 group.Cell proliferation-toxicity detection kit(CCK-8)was used to observe the cell activity and proliferation rate in different groups.The immunofluorescence staining(?tubulin-?/Hoechst33258)and flow cytometry were used for detecting apoptosis of each group.5.The intracellular Ca²⁺concentration of each groups was detected by laser confocal microscopy.Real-time PCR and Western Blot were used to determine the mRNA and protein expressions of Bcl-2,Bax,Caspase-3(cleaved-Caspase-3)and PARP-1(cleaved-PARP-1)in each group to investigate the mechanism of Panx-1 aggravating oxidative stress injury of spinal neurocytes in rat in vitro.Results1.The rat spinal cord injury model(Allen's model)was successfully established by NYU Impactor-?.And the BBB score showed that the score gradually recovered after injury,but the score significantly decreased at D 2.The expression of Panx-1 in the SCI group detected by Real-time PCR and Western Blot was significantly increased with time compared with the normal control group and the Sham group,and reached a peak at D 2 after SCI.Moreover,the expression of Panx-1 protein was negatively correlated with the BBB score of hind limb motor function of rats at D 1,D 2,D 3 and D 5 after SCI.2.Primary spinal neurocytes were isolated and cultured from 15-day pregnant rat embryos by modified trypsin digestion and mechanical separation.Fetal rat spinal cord was easy to peel off and had strong survival ability in vitro.Keep the whole operation at low temperature,strictly control the trypsin concentration and digestion time,add an appropriate amount of DNA enzyme and MgCl₂ before gentle resuspending the cell to prevent the aggregation of cells,use serum-free NPC culture medium can effectively improve the purity and production of primary spinal neurocytes,and keep the cells in good viability.Specific Neu N and?tubulin-? staining were observed by immunofluorescence staining,which was consistent with the characteristics of spinal neurocytes.The purity of spinal neurocytes was identified to be 90%.The cell model of SCI in vitro was constructed by H₂O₂ intervention of primary spinal neurocytes in rats,and the concentration of H₂O₂ intervention was 700?M and the time was 3 h by CCK-8 method.3.Panx-1 overexpressing lentivirus vector(LV5-Panx-1)and lentivirus interfering vector(sh-Panx-1)were successfully constructed,and the above viruses were respectively infected to rat spinal neurocytes.The appropriate MOI was determined to be 50 by observing fluorescence staining.Western Blot was used to detect the expression of Panx-1 protein after transfection,which proved that the transfected rat spinal neurocytes could continuously express Panx-1 protein at a high level or silence it.4.Observation of the cells in the NC group,the Vehicle group,the LV-con group,the LV5-Panx-1 group and the sh-Panx-1 group showed that:the cell swelling,shrinkage,floating and death in the LV5-Panx-1 group were significantly increased compared with the sh-Panx-1 group,while the level of LV-con group and the Vehicle group were in the middle.CCK-8 assay showed that the viability of sh-Panx-1 group was significantly higher than that of LV-con group and LV5-Panx-1 group.Immunofluorescence staining and flow cytometry were used to detect cell apoptosis.It was found that the apoptotic cells and dead cells in LV5-Panx-1 group were significantly increased at 24 h,48 h and72 h compared with LV-con group,Vehicle group and NC group,while the apoptotic cells and dead cells in sh-Panx-1 group were significantly reduced compared with LV-con group,Vehicle group and NC group.The detection of intracellular Ca²⁺in each group by laser confocal microscopy showed that the intracellular Ca²⁺concentration in LV5-Panx-1 group was significantly higher than that in LV-con group and Vehicle group at 24,48 and 72 h,while that in sh-Panx-1 group was significantly lower than that in LV-con group and Vehicle group.Real-time PCR detection showed that the expressions of Bax and Caspase-3 in LV5-Panx-1 group were significantly increased,and the expressions of Bcl-2 and PARP-1 were significantly decreased,compared with Vehicle group and LV-con group at 24 h,48 h and 72 h.However,the expressions of Bax and Caspase-3 in sh-Panx-1 group were significantly decreased,and the expressions of Bcl-2 and PARP-1 were significantly increased,compared with Vehicle group and sh-con group at 24 h,48 h and 72 h.Western Blot analysis showed that the expression of Bax,cleaved-Caspase-3 and cleaved-PARP-1 in LV5-Panx-1 group was significantly higher than that in Vehicle group and LV-con group,while the expression of Bcl-2 was significantly lower at 24 h,48 h and 72 h.However,the expression of Bax,cleaved-Caspase-3 and cleaved-PARP-1 in sh-Panx-1 group was significantly lower than that in Vehicle group and LV-con group,while the expression of Bcl-2 was significantly higher at 24 h,48 h and 72 h.In addition,the above indicators were basically consistent with the changes in gene and protein expression levels,and the expression level gradually increased or decreased with time,reaching a peak or trough at 48 h.Conclusion1.The SCI model(Allen's model)of rat established by NYU Impactor-? has high reliability and good stability,and can perform spinal cord impact with controllable repeatability standard on rats.The expression of Panx-1 after SCI was negatively correlated with BBB score.2.The primary spinal neurocytes of rat were isolated and cultured by modified trypsin digestion combined with mechanical separation.H₂O₂ interfered with the primary spinal neurocytes to construct the oxidative stress injury model of spinal neurocytes in rat in vitro with high reliability and good repeatability.3.Lentivirus-mediated LV5-Panx-1 and sh-Panx-1 were successfully constructed and transfected into spinal neurocytes of rat to achieve stable and efficient expression.4.Up-regulation the expression of Panx-1 in vitro can aggravate oxidative stress injury of spinal neurocytes,inhibit cell viability,and promote apoptosis of spinal neurocytes.However,silencing the expression of Panx-1 can significantly improve the oxidative stress injury of spinal neurocytes,restore cell viability,and inhibit the apoptosis of spinal neurocytes.5.As a membrane half channel,Panx-1 may lead to intracellular Ca²⁺overload by accelerating extracellular Ca²⁺influx and Ca²⁺release of the endoplasmic reticulum,which leads to endoplasmic reticulum stress(ERS)and promotes the apoptosis of spinal neurocytes through the endoplasmic reticulum pathway,thus aggravating the secondary injury of SCI.