The Interaction Between TRPV1 And NR1 In Lidocaine-induced DRG Neurons Neurotoxicity In Vitro

Objective:To investigate the interaction between TRPV1 and NR1 in lidocaine-induced neurotoxicity on dorsal root ganglion (DRG) neurons in vitro.Methods:(1) DRG neurons harvested from 3-day neonatal rats were cultured and purified in vitro. The purity (the percentage of purified DRG neurons) were calculated under immunocytochemical stain.(2) The purified DRG neurons were incubated with 0,0.5,1,2,4 and 8mM lidocaine for 30 min, respectively. Their viabilities were examined using Cell Counting Kit (CCK-8) assay, and the lethal concentration 50 (LC50) of lidocaine on DRG neurons was calculated.(3) Then, the variation of DRG cell viability induced by LC50 lidocaine together with different concentrations of TRPV1 agonist capsacin (0,1,10 and 100?M), TRPV1 antagonist capsazepine (0,1,10 and 100?M), NMDA receptor agonist NMDA (0,100,200 and 400?M), and NMDA receptor antagonist AP-5 (0,1,10 and 100?M), respectively, were detected with CCK-8 assay to identify the best reactive concentrations, respectively.(4) And, the protein levels and their phosphorylation levels of DRG TRPV1 and NR1 were measured with western blot after DRG neurons were incubated for 30 min with LC50 lidocaine together with the best reactive concentrations of capsacin (100 uM), capsazepine (10 uM), NMDA (200 uM), and AP-5 (100 uM), respectively.Results:(1) The isolated and cultured DRG neurons survived and grew in good condition in vitro, and the purity was as high as 91%.(2) Lidocaine decreased cell viability of DRG neurons in a dose-dependent manner, and the LC50 is about 1.56mM at 30 min.(3) Compared with the LC50 group:? capsacin decreased cell viability of DRG neurons induced by lidocaine at LC50, with 100?M capsacin hit the maximal effect that decreased the cell viability from 50% to 40%;? capsazepine improved the viability of LC50 lidocaine-induced DRG neurons, 10?M capsazepine hit the maximal effect that increased the viability from 50% to 77%; ? NMDA together with LC50 Lidocaine decreased cell viability of DRG neurons, showing a dose-dependent trend, with 200?M hit the maximal effect that decreased the cell viability from 50% to 25%;? AP-5 improved the viability of DRG neurons induced by lidocaine at LC50, with 100 ?M AP-5 hit the maximal effect that increased the viability from 50% to 68%.(4) Compared with the LC50 group: ? 100?M capsacin added to LC50 lidocaine increased the phosphorylation level of TRPV1 (P<0.05) and NR1 (P<0.05), but did not change the expressions of TRPV1 and NR1; ? 10?M capsazepine added to LC50 lidocaine down regulated the phosphorylation levels of TRPV1 (P<0.05) and NR1 (P<0.05), but did not change the expressions of TRPV1 and NR1; ?200?M NMDA added to LC50 lidocaine increased the phosphorylation level of NR1 (P<0.05), but did not change the expressions of TRPV1 and NR1, or the phosphorylation level of TRPV1;? 100?M AP-5 added to LC50 lidocaine down regulated the phosphorylation level of NR1 (P<0.05), but did not change the expression of NR1 and TRPV1, or the phosphorylation level of TRPV1.Conclusion:(1) The novel method in this experiment is very effective to obtain good DRG neurons;(2) Lidocaine has neurotoxicity on DRG neurons in vitro and the LC50 of lidocaine on DRG neurons is about 1.56mM at 30 min.(3) The phosphorylations of TRPV1 and NR1 are both involved in lidocaine-induced DRG neurotoxicity in vitro. Activation and depression of TRPV1 could affect the phosphorylation level of NR1, whereas activation or depression of NR1 did not show any effect on the phosphorylation level of TRPV1.