清脑止痛胶囊对偏头痛模型大鼠热敏通道TRPV1/TRPA1通路的影响

Effect of Qingnao Zhitong Jiaonang on TRPV1/TRPA1 pathway in migraine model rats

目的 基于热敏通道瞬时受体电位通道香草醛亚型1(TRPV1)/瞬时受体电位销蛋白1(TRPA1)通路探究清脑止痛胶囊治疗偏头疼的作用机制。方法 取Wistar大鼠60只随机分为对照组、模型组、正天丸组(阳性对照,1.8 g·kg^(-1))和清脑止痛胶囊低、中、高剂量(0.35、0.70、1.40 g·kg^(-1))组。除对照组外,其余各组均用利血平0.7 mg·kg^(-1) sc注射法建立偏头痛大鼠模型。各组均于造模成功后ig给予相应剂量药物,每天1次,连续7 d。末次给药结束12 h后,观察各组大鼠挠头次数及行为学变化;采用vonFrey法测试大鼠眶周疼痛阈值;酶联免疫法检测脑组织中白细胞介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)、5-羟色胺(5-HT)水平变化;试剂盒法检测脑组织中一氧化氮(NO)、钙离子(Ca^(2+))水平;免疫荧光法检测TRPV1、TRPA1与神经元微管相关蛋白2 (MAP2)在脑组织中共表达水平;蛋白免疫印迹(Western blotting)法检测脑组织中TRPV1/TRPA1通路蛋白、降钙素基因相关肽(CGRP)、环氧化酶2(COX-2)表达水平。结果 与模型组比较,清脑止痛胶囊低、中、高剂量组及正天丸组大鼠挠头次数,脑干组织NO、Ca^(2+)、IL-1及TNF-α水平,TRPV1、TRPA1分别与MAP2阳性神经元共表达水平,TRPV1、TRPA1、CGRP、COX-2蛋白表达水平均显著降低(P<0.05),眶周疼痛阈值、5-HT表达显著升高(P<0.05),且上述指标变化均呈剂量相关性,高剂量组与正天丸组相比差异无统计学意义。结论 清脑止痛胶囊可能通过降低TRPV1/TRPA1通路介导的疼痛信号传导,改善偏头痛大鼠疼痛症状。

Objective To explore the mechanism of Qingnao Zhitong Jiaonang(QNZTJN) in the treatment of migraine based on transient receptor potential Vanilloild-1(TRPV1)/transient receptor potential ankyrin-1(TRPA1) pathway.Methods A total of 60 Wistar rats were randomly divided into control group,model group,positive control group(Zhengtian Pill,1.8 g·kg^(-1)),QNZTJN low,middle,and high dose(0.35,0.70,1.40 g·kg^(-1)) groups.Except the control group,the other groups were injected with reserpine 0.7 mg·kg^(-1) subcutaneously to establish migraine rat models.After successful modeling,the rats in each group were given corresponding doses of drugs by gavage for seven days,once a day.At 12 hours after the end of the last administration,the number of head scratching and behavioral changes of rats in each group were observed;the threshold of periorbital pain was measured by vonFrey method;the levels of interleukin-1(IL-1),tumor necrosis factor-α(TNF-α) and 5-hydroxytryptamine(5-HT) in brain tissue were detected by enzyme linked immunosorbent assay;the levels of nitric oxide(NO) and calcium ion(Ca^(2+)) in brain tissue were detected by kit method;the co expression levels of TRPV1,TRPA1 with neuronal microtubule associated protein 2(MAP2) were detected by immunofluorescence;the expression levels of TRPV1/TRPA1 pathway protein,calcitonin gene related peptide(CGRP)and cyclooxygenase 2(COX-2) were detected by Western blotting.Results Compared with model group,the number of head scratching,the levels of NO,Ca^(2+),IL-1 and TNF-α,the co-expression levels of TRPV1 and TRPA1 with MAP2 positive neurons,and the protein expression of TRPV1,TRPA1,CGRP and COX-2 decreased in the QNZTJN low,medium,high dose groups and positive control group(P < 0.05),the periorbital pain threshold and expression of 5-HT increased(P < 0.05),and the changes of above indexes in QNZTJN groups were dose-dependent.There was no significant difference between QNZTJN high-dose group and positive control group.Conclusion QNZTJN may improve the pain symptoms of migraine rats by reducing the pain signal transduction mediated by TRPV1/TRPA1 pathway.