利多卡因对发育期海马神经细胞生长的抑制作用及相关机制研究

Inhibitory effect of lidocaine on the growth of hippocampal neurons in developing period and related mechanisms

目的研究利多卡因对发育期海马神经细胞生长的影响及相关分子机制。方法通过做浓度依赖实验确定利多卡因实验浓度,依据不同浓度利多卡因分为3组,其中对照组给予0.9%生理盐水处理,不加利多卡因,A组加入1μM利多卡因,B组10μM利多卡因。采用四唑盐(MTT)法检测利多卡因对发育期海马神经细胞增殖能力的影响;采用PI单染法检测利多卡因对发育期海马神经细胞的细胞周期影响;采用Annexin V-FITC双染法检测利多卡因对发育期海马神经细胞凋亡水平的影响;采用RT-PCR实验检测mTOR、CyclinD1及Cleaved caspase3基因水平。采用Western blot法检测利多卡因对发育期海马神经细胞mTOR、CyclinD1、Cleaved caspase3蛋白表达的影响。结果通过MTT实验发现利多卡因处理第2 d后A组及B组神经细胞增殖水平显著低于对照组(P<0.05);第2 d后B组神经细胞增殖水平显著低于A组(P<0.05)。PI单染实验发现A组及B组的G0/G1期细胞比例显著高于对照组(P<0.05);A组及B组的S期细胞比例显著低于对照组(P<0.05);A组及B组的G2/M期细胞比例显著低于对照组(P<0.05)。Annexin V-FITC双染实验发现A组及B组细胞凋亡率均显著高于对照组(P<0.05);B组细胞凋亡率显著高于A组(P<0.05)。RT-PCR实验发现A组及B组的Mtor、CyclinD1基因水平均低于对照组(P<0.05);B组的Mtor、CyclinD1基因水平低于A组(P<0.05);A组及B组的Cleaved caspase3基因水平显著高于对照组(P<0.05);B组Cleaved caspase3基因水平显著高于A组(P<0.05)。Western blot实验发现A组及B组Mtor、CyclinD1蛋白水平均低于对照组(P<0.05);B组的Mtor、CyclinD1低于A组(P<0.05);B组Cleaved caspase3蛋白水平显著高于A组(P<0.05)。结论利多卡因可抑制发育期海马神经细胞生长,并导致神经细胞发生凋亡,利多卡因的神经毒性可能与抑制PI3K/Akt/Mtor信号通路有关。

Objective To study the effect and mechanism of lidocaine on the growth of hippocampal neurons in the developing period.Methods The experimental concentration of lidocaine was determined by concentration.dependent experiments.According to the different concentration of lidocaine,lidocaine was divided into 3 groups.The control group was treated with 0.9%saline without lidocaine.Group A was treated with 1μM lidocaine and group B with 10μM lidocaine.RT-PCR method was used to determine the gene level of mTOR,CyclinD1,Cleaved caspase3.MTT assay was used to detect the effect of lidocaine on the proliferation of developing hippocampal neurons;PI single staining was used to detect the effect of lidocaine on the cell cycle of developing hippocampal neurons;Annexin V-FITC double staining was used to detect the effect of lidocaine on the apoptosis of developing hippocampal neurons;Western blot was used to detect the effect of lidocaine on the expression of mTOR,CyclinD1 and Cleaved Caspase3 protein in hippocampal neurons during development.Results The MTT assay showed that the proliferation of nerve cells in group A and group B was significantly lower than that in the control group after the second day of lidocaine treatment(P<0.05).The proliferation of nerve cells in group B was significantly lower than that in group A after the second day(P<0.05).The proportion of G0/G1 phase cells in group A and group B was significantly higher than that in the control group(P<0.05).the proportion of S phase cells in group A and group B was significantly lower than that in the control group(P<0.05).The proportion of G2/M phase cells in group A and group B was significantly lower than that in the control group(P<0.05).The apoptosis rate of group A and group B was significantly higher than that of the control group(P<0.05);the apoptosis rate of group B was significantly higher than that of group A(P<0.05).RT-PCR showed that the levels of mTOR and CyclinD1 in group A and group B were lower than those in control group(P<0.05).The levels of mTOR and CyclinD1 in group B were lower than those in group A(P<0.05).The level of Cleaved caspase3 gene in group A and B group was significantly higher than that in control group,but higher in group B than in group A(P<0.05).Western blot analysis showed that the levels of mTOR and CyclinD1 in group A and group B were lower than those in control group(P<0.05),both of mTOR and CyclinD1 much higher ib group A compaired to group B(P<0.05).The level of cleaved caspase3 protein in group B was significantly higher than that in group A(P<0.05).Conclusion Lidocaine inhibits the growth of hippocampal neurons in developmental stages and causes neuronal apoptosis.The neurotoxicity of lidocaine could be related to the inhibition of PI3K/Akt/mTOR signaling pathway.