基于NGF/p38 MAPK/TRPV1信号通路探讨热敏灸治疗膝骨关节炎的外周敏化效应机制

Mechanism of peripheral sensitization on heat-sensitive moxibustion for knee osteoarthritis based on NGF/p38 MAPK/TRPV1 signaling pathway

目的:观察热敏灸对膝骨关节炎(KOA)兔背根神经节NGF/p38 MAPK/TRPV1信号通路的影响,探讨其治疗KOA的作用机制。方法:36只普通级雄性新西兰兔,采用随机数表法随机分为空白组(6只)、假手术组(6只)、造模组(24只)。采用木瓜蛋白酶溶液注射膝关节腔法造模15 d,假手术组关节腔内注射等量的0.9%氯化钠溶液。造模成功后,选取6只为模型组,其余艾灸犊鼻穴,每日1次,每次40 min,共7 d,根据兔艾灸过程中温度变化分为热敏灸组和非热敏灸组,各6只;空白组、假手术组、模型组不予干预措施。干预结束后,观察各组兔行为学变化;采用ELISA法检测血清神经生长因子(NGF)含量;Western blot法检测各组大鼠背根神经节p-p38 MAPK、TRPV1蛋白的表达。结果:造模后,与空白组比较,假手术组、模型组、非热敏灸组、热敏灸组兔Lequesne MG评分均显著升高(P<0.01)。干预后,与模型组比较,非热敏灸组及热敏灸组Lequesne MG评分显著下降(P<0.05,P<0.01)。与空白组比较,模型组血清NGF含量显著上升(P<0.01);与模型组比较,非热敏灸组、热敏灸组血清NGF含量显著下降(P<0.01);与非热敏灸组比较,热敏灸组血清NGF含量显著下降(P<0.05)。与空白组比较,模型组p-p38 MAPK、TRPV1蛋白含量显著升高(P<0.01);与模型组比较,非热敏灸组及热敏灸组p-p38 MAPK、TRPV1蛋白表达显著下降(P<0.01);与非热敏灸组比较,热敏灸组p-p38 MAPK、TRPV1蛋白表达显著下降(P<0.01)。结论:热敏灸可能通过减少局部NGF的产生,从而抑制NGF/p38 MAPK/TRPV1通路的激活,降低炎性局部的痛敏反应而发挥其改善KOA的作用。

Objective: To observe the effect of heat-sensitive moxibustion on NGF/p38 MAPK/TRPV1 signaling pathway in rabbit dorsal root ganglion of knee osteoarthritis(KOA), and to explore the potential mechanism of heat-sensitive moxibustion for KOA. Methods: A total of 36 New Zealand male rabbits were randomly divided into a blank group(6 rabbits),a sham operation group(6 rabbits) and a model establishment group(24 rabbits) by random number table method. Papain solution was injected into the joint cavity for 15d, and normal saline solution was injected into the joint cavity of the sham operation group. After successful modeling, rabbits in the model establishment group were randomly divided into model group(6 rabbits) and moxibustion group(18 rabbits). In the moxibustion group, suspending moxibustion was applied at Dubi(ST 35), once a day, 40 min each time, for 7 days totally. According to the temperature changes, rabbits in the moxibustion group were divided into a heat-sensitive moxibustion group and a non-heat-sensitive moxibustion group, 6 rabbits in each group.No intervention was given in the blank group, sham operation group and model group. After the intervention, the behavioral changes of rabbits were observed. Serum NGF was detected by ELISA. Western blot method was used to detect the protein expression of p38 MAPK and TRPV1 in dorsal root ganglion. Results: After modeling, the Lequesne MG scores of rabbits in sham group, model group, non-heat-sensitive moxibustion group and heat-sensitive moxibustion group were all higher than those in blank group(P<0.01). After intervention, compared with the model group, the Lequesne MG score of the non-heatsensitive moxibustion group and the heat-sensitive moxibustion group decreased significantly(P<0.05, P<0.01). Compared with the blank group, the serum NGF content in the model group increased significantly(P<0.01). Compared with the model group,the serum NGF content in non-heat-sensitive moxibustion group and heat-sensitive moxibustion group decreased significantly,and the difference was statistically significant(P<0.01). Compared with the non-heat-sensitive moxibustion group, the serum NGF content in the heat-sensitive moxibustion group decreased significantly, and the difference was statistically significant(P<0.05). Compared with the blank group, the protein contents of p-p38 MAPK and TRPV1 in the model group were increased obviously, and the difference was statistically significant(P<0.01). Compared with the model group, the protein expression of p-p38 MAPK and TRPV1 decreased obviously in the non-heat-sensitive moxibustion group and the heat-sensitive moxibustion group, and the difference was statistically significant(P<0.01). Compared with the non-heat-sensitive moxibustion group,the protein expression of p-p38 MAPK and TRPV1 in the heat-sensitive moxibustion group decreased significantly, and the difference was statistically significant(P<0.01). Conclusion: Heat-sensitive moxibustion may inhibit the activation of NGF/p38 MAPK/TRPV1 signaling pathway by reducing the production of local NGF, reduce the inflammatory local pain-sensitive response, and thus play its role in improving KOA.