TRPA1通过PLC/PKC信号通路对偏头痛大鼠的行为学和疼痛敏感性的影响

Effects of TRPA1 on behavior and pain sensitivity in migraine rats via PLC/PKC signaling pathway

目的 探讨瞬时受体电位锚蛋白1(TRPA1)通过磷酯酶C(PLC)/蛋白激酶C(PKC)信号通路对偏头痛大鼠的行为学和疼痛敏感性的影响。方法 将SD大鼠随机分为对照组、模型组、TRPA1敲低组(腺病毒包装的TRPA1 siRNA载体)、TRPA1空载组(空慢病毒载体)、TRPA1敲低+PMA(PKC激活剂)组,每组12只,分组以腺病毒包装的TRPA1 siRNA载体、空慢病毒载体及PMA干预处理24 h后,模型组与给药干预组皮下注射10 mg/kg硝酸甘油以制备偏头痛大鼠模型,对照大鼠组皮下注射等剂量0.9%氯化钠溶液,然后以qRT-PCR实验检测除TRPA1敲低+PMA组外其余5组大鼠脑组织及血液中TRPA1 mRNA的表达;观察5组大鼠行为,记录其耳红消失时间及一段时间内挠头次数、爬笼次数;检测5组大鼠机械性疼痛及温度痛阈;酶标仪检测5组大鼠血清促炎因子前列腺素E2(PGE2)、白介素(IL)-6及抗炎因子IL-10水平;免疫印迹实验检测5组大鼠脑组织PLC/PKC通路相关蛋白表达。结果 与对照组比较,模型组、TRPA1空载组大鼠缩头阈值、热刺激潜伏期、血清IL-10水平显著降低(P<0.05),脑组织及外周血中TRPA1表达、耳红消失时间、挠头次数、爬笼次数、动态痛觉超敏评分、血清PGE2、IL-6水平、脑组织PLC/PKC通路蛋白p-PLC/PLC、p-PKC/PKC显著升高(P<0.05)。与模型组比较,TRPA1敲低组大鼠缩头阈值、热刺激潜伏期、血清IL-10水平升高(P<0.05),脑组织及外周血中TRPA1表达、耳红消失时间、挠头次数、爬笼次数、动态痛觉超敏评分、血清PGE2、IL-6水平、脑组织PLC/PKC通路蛋白p-PLC/PLC、p-PKC/PKC降低(P<0.05);TRPA1空载组大鼠各指标无显著变化(P>0.05)。与TRPA1敲低组比较,TRPA1敲低+PMA组大鼠缩头阈值、热刺激潜伏期、血清IL-10水平降低(P<0.05),耳红消失时间、挠头次数、爬笼次数、动态痛觉超敏评分、血清PGE2、IL-6水平、脑组织PLC/PKC通路蛋白p-PKC/PKC升高(P<0.05)。结论 TRPA1可通过调控PLC/PKC信号通路介导大鼠偏头痛的发生,下调TRPA1表达可通过抑制PLC/PKC信号通路激活减轻炎症,以降低大鼠疼痛敏感性,最终改善头疼症状。

Objective To explore effects of transient receptor potential ankyrin 1(TRPA1)on the behavior and pain sensitivity of migraine rats via phosphatase C(PLC)/protein kinase C(PKC)signaling pathway.Methods SD rats were randomly divided into the control group,model group,TRPA1 knockdown group(TRPA1 siRNA vector packaged by adenovirus),TRPA1 no-load group(no-load lentiviral vector),TRPA1 knockdown+PMA(PKC activator)group with 12 rats per group,Rats in the model group and administration intervention group were subcutaneously injected with 10mg/kg nitroglycerin after treatment lasting 24h with adenovirus packaged TRPA1 siRNA vector,no-load lentiviral vector and PMA to prepare a migraine rat model,the rats in the control group were subcutaneously injected with the same dose of 0.9%normal saline,and then expressions of TRPA1 mRNA in brain tissues and bloods of rats except the TRPA1 knockdown+PMA group were detected by qRT PCR experiments.The behaviors of rats in groups were observed,and the disappearance time of ear redness and number of head scratching and frequency of cage climbing at intervals were recorded.The mechanical pain and thermal pain threshold were detected.The level of serum pro-inflammatory factor prostaglandin E2(PGE-2),interleukin(IL-6)and anti-inflammatory factor IL-10 were detected by microplate reader.The expression of PLC/PKC pathway-related proteins in brain tissues was detected by western blotting.Results Compared to the control group,the head retraction threshold,thermal stimulus latency and serum IL-10 level in the model group and TRPA1 no-load group were significantly decreased(P<0.05),TRPA1 expression in the brain tissue and peripheral blood,disappearance time of ear redness,number of head scratching,frequency of cage climbing,dynamic allodynia score,serum PGE-2,IL-6 level,brain tissue PLC/PKC pathway protein p-PLC/PLC and p PKC/PKC were significantly increased(P<0.05).Compared to the model group,the head retraction threshold,thermal stimulus latency and serum IL-10 level of TRPA1 knockdown group were significantly increased(P<0.05),TRPA1 expression in brain tissue and peripheral blood,disappearance time of ear redness,number of head scratching,frequency of cage climbing,dynamic allodynia score,serum PGE-2,IL-6 level,brain tissue PLC/PKC pathway proteins p-PLC/PLC,and p-PKC/PKC were significantly decreased(P<0.05);and no significant changes in indexes in the TRPA1 no-load group were noted(P>0.05).Compared to the TRPA1 knockdown group,the head retraction threshold,thermal stimulus latency and serum IL-10 level in the TRPA1 knockdown+PMA group were decreased(P<0.05),the disappearance time of ear redness,number of head scratching,frequency of cage climbing,dynamic allodynia score,serum PGE-2,IL-6 level,and brain tissue PLC/PKC pathway protein p-PKC/PKC were increased(P<0.05).Conclusion TRPA1 is capable of mediating the attack of migraine rats by regulating PLC/PKC signaling pathway,and down-regulated expression of TRPA1 can reduce inflammation by inhibiting the activation of PLC/PKC signaling pathway,thereby reducing pain sensitivity in rats and finally improving their headache symptoms.