Research On The Regulation Of MJS On TRP Channel

Objective: To study the primary cultured dorsal root ganglion(DRG)neurons in C57BL/6 mice.MJS for histamine(Hist),serotonin(5-hydroxytryptamine,5-HT),endothelin Inhibition of-1(Endothelin-1,ET-1)and Chloroquine(Chlo);Regulation of TRP channels by MJS in DRG neurons;HEK293T cells transfected with plasmids of TRPV1,TRPV4 and TRPA1 The regulation of MJS on the TRP channel.Method and result:1.Inhibition of Hist,5-HT,ET-1 and Chlo by MJS in primary cultured DRG neurons Rapid separation and collection of C57BL/6 male mice L2-L6(Lumbar two-Lumbar six)DRG for 4-6 weeks,cultured for 16-18 h,in calcium imaging system(RC-26 type amplifier,MP-285 type 1550 B digital-to-analog converter,BT100-2J The microscope and DG-4 ion imaging light source were used to study the inhibitory effect of MJS on Hist,5-HT,ET-1 and Chlo by fluorescent probe Fura-2-AM.Intracellular calcium concentration assay of Fura-2-AM showed that 20 ?M MJS had no significant effect on intracellular calcium concentration of DRG neurons;100?M Hist,100 ?M 5-HT,100 ?M ET-1 and 100 ?M Chlo Both can cause a significant increase in calcium ion concentration;20 ?M MJS can significantly inhibit the increase of calcium concentration caused by 100 ?M Hist,100 ?M 5-HT,100 ?M ET-1 and 100 ?M Chlo.2.Effects of different TRP channel specific agonists on calcium ion concentration in primary cultured DRG neurons and regulation of MJS The isolated and cultured DRG neurons were assayed for intracellular calcium concentration by Fura-2-AM to study TRPV1 specific agonist Capsaicin(Cap),TRPV4 specific agonist GSK1016790A(GSK101)and TRPA1 specific agonist.The effect of AITC on calcium ion concentration and the regulation of MJS.Fura-2-AM calcium imaging results showed that 20 ?M MJS had no significant effect on calcium ion concentration in DRG neurons;1 ?M Cap,100 n M GSK101 and100 ?M AITC could cause a significant increase in calcium ion concentration;20 ?M MJS It can significantly inhibit the increase of calcium ion concentration caused by100 n M Cap and 50 n M GSK101;however,it has no significant effect on the increase of calcium ion concentration induced by 100 ?M AITC.3.Effects of different TRP channel specific agonists on calcium ion concentration of plasmid transfected TRPV1,TRPV4 and TRPA1 protein HEK293 T cells and regulation of MJS HEK293 T cells were cultured in DMEM high glucose medium and transfected with TRPV1,TRPV4 and TRPA1 proteins respectively.The transfection reagent was LIPOFECTAMINE 2000.The RFP red protein in the plasmid was used as the reporter gene.After transfection for 48 hours,the red fluorescent cells with representative visual field were counted under the fluorescence microscope to calculate the transfection rate.Western blotting(WB)and real-time fluorescent quantitative PCR(Q-PCR)were used.The transfection results were detected by the Q-PCR method.The effects of Cap,GSK101 and AITC on the calcium ion concentration of HEK293 T cells transfected with TRPV1,TRPV4 and TRPA1 proteins and the regulation of MJS were observed by Fura-2-AM.Transfection of TRP protein N-terminal fusion Red fluorescent protein(RFP)red fluorescent protein,fluorescence results showed red fluorescent protein expression in HEK293 T cells,indicating that the recombinant plasmid was successfully transferred into host cells and expressed,the transfection rate was more than 60%;WB The expression of positive protein and m RNA was detected by Q-PCR results,indicating that the plasmid transfected TRP protein into HEK293 T cells was successfully expressed.Fura-2-AM calcium imaging showed that 20 ?M MJS had no significant effect on the calcium concentration of HEK293 T cells transfected with TRPV1,TRPV4 and TRPA1 proteins;100 n M Cap,50 n M GSK101 and 100 ?M AITC could all lead to corresponding plasmid transformation.The calcium concentration of HEK293 T cells was significantly increased;20 ?M MJS significantly inhibited the increase of calcium ion concentration in HEK293 T cells transfected with plasmids transfected with TRPV1 and TRPV4 by 100 n M Cap and 50 n M GSK101,and induced calcium ions at 100 ?M AITC.There was no significant effect on the increase in concentration.Conclusion: MJS significantly inhibits the excitatory effects of Hist,5-HT,ET-1 and Chlo in primary cultured DRG neurons;TRPV1,TRPV4 and TRPA1 channels are involved in the excitability of HEK293 T cells transfected with DRG neurons and plasmid transfected TRP proteins Sex,MJS can inhibit the activation of TRPV1 and TRPV4 channels,and has no effect on TRPA1 channel activation.