甲苯噻嗪通过抑制超极化激活环核苷酸门控离子通道电流产生镇痛作用机制研究

Xylazine Produced Analgesic Effect via Inhibits Hyperpolarization-activated Cyclic Nucleotide-gated Ion Channels Currents

目的探讨甲苯噻嗪(xylazine,Xyl)通过抑制超极化激活环核苷酸门控阳离子通道(hyperpolarization-activated cyclic nucleotide-gated,HCN)电流产生镇痛作用的机制。方法对HCN亚型通道1(HCN1)基因敲除小鼠(HCN1-/-)及HCN1野生型小鼠(HCN1+/+)分别腹腔注射生理盐水及Xyl(10、20、30和40mg/kg),通过进行机械痛觉测试及甩尾试验方法检测Xyl的镇痛效果,计算出最大镇痛效应百分比(percent maximum possible effect,%MPE);HEK 293细胞转染HCN1质粒和HCN亚型通道2(HCN2)质粒,分为对照组及不同浓度Xyl(12.5、25、50和100μmol/L)实验组,利用全细胞膜片钳记录表达HCN1和HCN2离子通道的HEK 293细胞超极化激活电流(hyperpolarization-activated currents,Ih),计算Xyl对Ih的抑制率。结果HCN1+/+小鼠和HCN1-/-小鼠注射Xyl后对机械痛觉刺激和热辐射刺激反应的%MPE均为随着Xyl质量浓度增加而加大,当Xyl质量浓度为30mg/kg及40mg/kg时,HCN1-/-小鼠机械痛觉刺激测试的%MPE为(62.06±14.72)%和(69.92±16.09)%;甩尾试验%MPE为(52.50±1.97)%和(64.74±6.34)%。HCN1+/+小鼠机械痛觉刺激测试的%MPE为(75.47±8.06)%和(86.35±11.31)%;甩尾试验%MPE为(57.83±4.82)%和(74.98±9.35)%,等量Xyl对HCN1+/+小鼠与HCN1-/-小鼠的镇痛作用(机械痛觉刺激和甩尾试验)差异有统计学意义(P<0.05)。全细胞膜片钳试验结果显示,Xyl对HCN1及HCN2离子通道电流均产生了抑制作用,Xyl(12.5～100μmol/L)对HCN1的Ih的抑制率为(24.62±23.62)%～(62.40±15.48)%;HCN1的半数激活电压(V1/2)为:(-79.58±1.56)mV～(-98.95±3.57)mV。Xyl对HCN2的Ih的抑制率为(29.19±17.82)%～(80.02±6.64)%;HCN2的V1/2为:(-102.17±1.36)mV～(-117.48±2.38)mV。结论 Xyl对HCN1+/+小鼠的镇痛效果比HCN1-/-小鼠更好。Xyl可能是通过抑制HCN离子通道电流,从而产生镇痛作用。

Objective To investigate the analgesic mechanism of xylazine by inhibiting the activation of hyperpolarized cyclic nucleotide-gated(HCN)ion channels.Methods HCN subchannel 1(HCN1)knockout mice(HCN1^-/-)and HCN1 wild type mice(HCN1^+/+)were intraperitoneally injected with physiological saline and xylazine(10,20,30,and 40mg/kg).Mechanical pain test and tail flick test were used to test the analgesic effect of xylazine by using the percentage of the maximal possible effect(%MPE);The control group and test groups of different concentrations of xylazine(12.5,25,50,and 100μmol/L)were set up using HEK 293 cells transfected HCN1 plasmid and HCN subchannel 2(HCN2)plasmid,respectively.The activated current of hyperpolarized HEK 293 cells expressing HCN1 and HCN2ion channels and the inhibition rate of xylazine on hyperpolarizationactivated currents were recorded using a whole cell patch clamp.Results The results demonstrated that%MPE of the mechanical pain stimuli test and the thermal radiation stimuli test increased with the higher concentration of xylazine injected for both HCN1^+/+mice and HCN1^-/-mice.When injecting xylazine by 30mg/kg and 40mg/kg,the%MPE of mechanical pain stimuli test for HCN1^-/-mice were %MPE=(62.06±14.72)% and %MPE=(69.92±16.09)%,respectively;and the percentages of tail flick tests were(52.50±1.97)% and % MPE=(64.74±6.34)%,respectively.But for HCN1^+/+mice,the percentages of mechanical pain stimuli test were %MPE=(75.47±8.06)% and%MPE=(86.35±11.31)%;respectively,and the percentage of tail flick tests were%MPE=(57.83±4.82)% and(74.98±9.35)%.The analgesic effect results of the mechanical pain test and tail flick test of HCN1^+/+mice were significantly different from HCN1^-/-mice(P<0.05).Whole-cell patch clamp test results showed that xylazine had inhibitory effects on the currents of HCN1 and HCN2ion channels,and the hyperpolarization-activated currents inhibition rate of HCN1 by xylazine(12.5-100μmol/L)was between(24.62±23.62)%-(62.40±15.48)%;V1/2of HCN1 was between(-79.58±1.56)mV-(-98.95±3.57)mV.The Ih inhibition rate of HCN2 by xylazine(12.5-100μmol/L)was between(29.19±17.82)%-(80.02±6.64)%;with V1/2of HCN2between(-102.17±1.36)mV-(-117.48±2.38)mV.Conclusion Xylazine showed better analgesic effect on HCN1^+/+mice than HCN1^-/-mice.Xylazine can produce analgesic effect by inhibiting HCN ion channel currents.