α-眼镜蛇神经毒素对小鼠的镇痛作用及脊髓背根神经节蛋白激酶A活性的影响

Analgesic effect ofα-cobratoxin on mice and its effect on protein kinase A activity of spinal dorsal root ganglion in mice

目的研究α-眼镜蛇神经毒素(α-cobratoxin,α-CbTX)对小鼠的镇痛作用及脊髓背根神经节(dorsal root ganglion,DRG)内蛋白激酶A(protein kinase A,PKA)活性的影响。方法健康雄性ICR小鼠(n=102)采用随机数字表法分为α-CbTX低、中、高剂量组(剂量分别为1 mg/kg、3 mg/kg、9 mg/kg,灌胃,每组n=21)、溶剂对照组(等体积0.9%氯化钠溶液,灌胃,n=21)和吗啡阳性对照组(3 mg/kg,腹腔注射,n=6)或阿司匹林阳性对照组(300 mg/kg,灌胃,n=12)。采用热板实验、醋酸扭体实验及福尔马林舔足实验评价α-CbTX的镇痛效应。采用福尔马林足底注射诱发疼痛30 min后,取L4~L6背根神经节,采用Western blot检测各组小鼠DRG内PKA C-α表达变化。采用SPSS 16.0进行统计分析,热板实验数据采用重复测量方差分析,其他实验数据用单因素方差分析,进一步两两比较采用LSD-t检验。结果在热板实验中,小鼠舔爪潜伏期的组别和时间的交互作用显著(F=8.902,P<0.05);小鼠给药后0.5 h,α-CbTX中剂量组[(11.83±1.47)s]、高剂量组[(14.33±12.1)s]舔爪潜伏期均长于溶剂对照组[(8.17±0.75)s](t=4.461,7.053,均P<0.05),α-CbTX中剂量组药效持续到给药后1.5 h(均P<0.05),α-CbTX高剂量组药效持续到给药后2 h(均P<0.05)。在醋酸扭体实验中,α-CbTX低、中、高各剂量组小鼠扭体次数[(34.50±3.62)次、(26.17±2.40)次、(13.83±3.76)次]均明显低于溶剂对照组[(42.50±4.59)次](t=3.938,8.040,14.112,均P<0.05)。在福尔马林实验Ⅱ相时间段,α-CbTX低、中、高各剂量组小鼠舔爪总时间[(71.17±6.46)s、(54.67±6.41)s、(40.50±3.89)s]均明显短于溶剂对照组[(98.67±11.50)s](t=6.950,11.120,14.700,均P<0.05)。在Western blot检测实验中,与溶剂对照组[(0.22±0.01)]比较,α-CbTX低、中、高各剂量组小鼠DRG中PKA C-α[(0.31±0.02)、(0.41±0.03)、(0.44±0.02)]表达均上调(t=3.140,6.471,7.492,均P<0.05)。结论α-CbTX具有明显的镇痛作用,其镇痛机制可能与激活蛋白激酶A的活性有关。

Objective To study the analgesic effect ofα-cobratoxin(α-CbTX)on mice and its effect on protein kinase A(PKA)activity of spinal dorsal root ganglion(DRG)in mice.Methods Healthy male ICR mice(n=102)were randomly divided into low-,medium-,and high-doseα-CbTX groups(1 mg/kg,3 mg/kg,9 mg/kg respectively,gavage,n=21),solvent control group(equivalent volume of 0.9%normal saline,gavage,n=21),morphine positive control group(3 mg/kg,intraperitoneal injection,n=6)or aspirin positive control group(300 mg/kg,gavage,n=12).The analgesic effect ofα-CbTX was evaluated by hot plate test,acetic acid twisting test and formalin foot licking test.Formalin plantar injection was used to induce pain and then the L4-L6 DRG was taken 30 minutes later.The expression of PKA C-αin L4-L6 DRG of mice were detected by Western blot.SPSS 16.0 software was used for statistical analysis.Repeated measurement ANOVA was used to evaluate the hot plate experimental data,and one-way ANOVA was used for other experimental data.LSD-t test was used for further pairwise comparison.Results In the hot plate test,the interaction between group and time of mice paw licking latency was significant(F=8.902,P<0.05).At 0.5 h after administration,the paw licking latencies ofα-CbTX medium-dose group((11.83±1.47)s)andα-CbTX high-dose group((14.33±12.1)s)were both longer than that of solvent control group((8.17±0.75)s)(t=4.461,7.053,both P<0.05).The efficacy ofα-CbTX medium dose group lasted until 1.5 h after administration(all P<0.05),and that ofα-CbTX high dose group lasted until 2 h after administration(all P<0.05).In the acetic acid writhing test,the writhing times in the low-,medium-and high-doseα-CbTX group((34.50±3.62)times,(26.17±2.40)times,(13.83±3.76)times))were significantly lower than that in solvent control group((42.50±4.59)times)(t=3.938,8.040,14.112,all P<0.05).In the period of the formalin test phaseⅡ,the total licking time ofα-CbTX low-,medium-and high-dose groups((71.17±6.46)s),(54.67±6.41)s,(40.50±3.89)s)were significantly shorter than that of the solvent control group((98.67±11.50)s)(t=6.950,11.120,14.700,all P<0.05).In the Western blot experiment,compared with solvent control group(0.22±0.01),the levels of PKA C-αin the DRG of mice in low-,medium-and high-doseα-CbTX groups((0.31±0.02),(0.41±0.03),(0.44±0.02))were up-regulated(t=3.140,6.471,7.492,all P<0.05).Conclusionα-CbTX has obvious analgesic effect,and its analgesic mechanism may be related to the activation of PKA.