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陈烽烽, 黎 磊, 陈向东, 等.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.四川大学学报(医学版),2019,50(1):20-26
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
  
中文关键词:  甲苯噻嗪 超极化激活环核苷酸门控离子通道 α2-肾上腺素受体激动剂 镇痛
英文关键词:Xylazine Hyperpolarization-activated cyclic nucleotide-gated ion channels α2-adrenergic receptor agonist Analgesia
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中文摘要:
      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40 mg/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100 μmol/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, Vh),计算Xyl对 Vh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30 mg/kg及40 mg/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 ~100 μmol/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 Vh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。
英文摘要:
      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 40 mg/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 100 μmol/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 HCN2 ion channels and the inhibition rate of xylazine on hyperpolarization-activated 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 30 mg/kg and 40 mg/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 HCN2 ion channels, and the hyperpolarization-activated currents inhibition rate of HCN1 by xylazine (12.5-100 μmol/L) was between (24.62±23.62)%-(62.40±15.48)%; V1/2 of HCN1 was between (-79.58±1.56) mV-(-98.95±3.57) mV. The Vh inhibition rate of HCN2 by xylazine (12.5-100 μmol/L) was between (29.19±17.82)%-(80.02±6.64)%; with V1/2 of HCN2 between (-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.
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