Effect Of TRPV1 Gene Silence On Cell Apoptosis Induced By Lidocaine And Its Mechanism

Objective:The aim is to construct a recombinant retroviral vector expressing sh RNA targeting to human TRPV1 gene and observe its down-regulation effect in the U87-MG cell line.By silencing TRPV1 gene,the effect on status and apoptosis was observed in U87-MG cell line after treated with lidocaine,thus to explore whether local anesthetic producing neurotoxicity by TRPV1.Methods:The oligonucleotides designed by Ambion on-line CAD software targeting to TRPV1 were cloned into the p SUPERretro RNAi plasmid.The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis.The p SUPERretro-puro TRPV1 sh RNA retrovirus was prepared by calcium phosphate method and transfected into U87-MG cells.After the screening by purinase,the expression levels of TRPV1 m RNA and protein were detected by fluorescent quantitation PCR and Western blotting.U87-MG cells were divided into control group,gene silencing group and empty vector group.For cells in each group,intracellular calcium ion concentration and mitochondrial membrane potential at different time point were study from the perspective of cell using flow cytometry after treated with lidocaine.Meanwhile,the cell vitality and cell apoptosis of U87-MG was assayed by MTT and flow cytometry.Results:The expression vector p SUPERretro-puro TRPV1 sh RNA was successfully constructed,which was confirmed by the DNA sequencing and the enzyme digestion analysis.The p SUPERretro-puro TRPV1 sh RNA retrovirus can down-regulate expression of TRPV1 effectively after transfection in U87-MG cells.The cell vitality decreased in pace with the increase of the concentration of lidocaine.After treated with lidocaine,the calcium ion concentration increased along with time in the cell of each group.The calcium ion concentration in TRPV1 gene silencing group was significantly lower than that in other groups at each time point(P<0.05).After treated wiht lidocaine,the mitochondrial membrane potential of U87-MG cell in control significantly decreased.The mitochondrial membrane potential in TRPV1 gene silencing group was significantly higher(P<0.05).Flow cytometry result showed that cell apoptosis in each group increased after treated with lidocaine.Cell apoptosis in TRPV1 gene silencing group was lower than that in control group(P<0.05).Conclusion:The p SUPERretro-puro TRPV1 expression vector was successfully constructed.The expression of TRPV1 gene was down-regulated effectively in U87-MG cells transfacted with p SUPERretro-puro TRPV1.It was proved that lidocaine could induce the increase of intracellular calcium ion concentration,the decrease of mitochondrial membrane potential and the increase of cell apoptosis.Meanwhile,the silence of TRPV1 attenuated cytotoxicity of lidocaine,which revealed the correlations between TRPV1 and the neurotoxicity injury mechanism of lidocaine.Lidocaine might have increased intracellular calcium ion concentration by activating TRPV1 channel proteins and induced cell apoptosis by decreasing mitochondrial membrane potential.