电针通过褪黑素受体2抑制脊髓背角星形胶质细胞分泌IL-17对神经病理性痛大鼠产生镇痛作用

Melatonin receptor 2 mediated spinal dorsal horn astrocytes IL-17 inhibition contributes to electroacupuncture analgesia in neuropathic pain rats

目的:观察褪黑素(Mel)受体2(MT2)是否参与电针对神经病理性痛大鼠的镇痛作用,以脊髓背角星形胶质细胞分泌白细胞介素17(IL-17)为研究切入点,探讨其可能机制。方法:先将18只大鼠分为假手术组、模型组、电针组,每组6只。采用坐骨神经慢性压迫法(CCI)制备大鼠神经病理性疼痛模型。电针组在造模后第7天电针"足三里""三阴交"。在造模前、造模后第7天和电针后60 min时检测大鼠患肢机械痛阈值(MWT)和热敏痛阈值(TPT);采用Western blot法检测脊髓背角MT2表达量,ELISA法检测Mel和IL-17含量,Western blot法和免疫组织化学法检测胶质纤维酸性蛋白(GFAP)表达量。再将30只大鼠分成假手术组、模型组、电针组、MT2拮抗剂(4-P-PDOT)组、二甲亚砜(DMSO)组,每组6只。4-P-PDOT组和DMSO组大鼠在电针前30 min分别鞘内注射10μL MT2拮抗剂4-P-PDOT(100μg)和10μL DMSO。检测大鼠患肢MWT和TPT;Western blot法和免疫组织化学法检测脊髓背角GFAP表达量,ELISA法检测IL-17含量。最后将30只大鼠分成假手术组、模型组、电针组、重组IL-17组、生理盐水组,每组6只。在电针前30 min,重组IL-17组和生理盐水组大鼠分别鞘内注射10μL重组IL-17蛋白(100 ng)和10μL 0.9%氯化钠溶液。检测各组大鼠患肢MWT和TPT。结果:造模后第7天,模型组及电针组大鼠MWT较造模前明显升高(P<0.05),TPT较造模前明显下降(P<0.05)。在电针后60 min时,与模型组比较,电针组MWT明显下降(P<0.05),TPT明显升高(P<0.05)。与假手术组比较,模型组大鼠脊髓背角GFAP表达量和IL-17含量明显增加,Mel含量和MT2表达量明显减少(P<0.05)。与模型组比较,电针组大鼠脊髓背角GFAP表达量和IL-17含量明显减少(P<0.05),Mel含量和MT2表达量明显增加(P<0.05)。与电针组和DMSO组比较,4-P-PDOT组大鼠MWT明显升高(P<0.05),TPT明显下降(P<0.05),GFAP表达量和IL-17含量也明显增加(P<0.05)。与电针组和生理盐水比较,重组IL-17组大鼠MWT明显升高(P<0.04),TPT明显下降(P<0.05)。结论:电针可增加CCI大鼠脊髓背角Mel含量和MT2表达量,抑制脊髓背角星形胶质细胞激活和IL-17释放。4-P-PDOT可逆转电针抑制CCI大鼠脊髓背角星形胶质细胞激活和IL-17释放,同时4-P-PDOT和重组IL-17可逆转电针对CCI大鼠的镇痛作用。推测电针可能是通过MT2抑制星形胶质细胞释放IL-17,从而对CCI大鼠产生镇痛作用。

Objective To investigate the effect of electroacupuncture(EA)on pain behaviors and expression of spinal dorsal horn melatonin receptor 2(MT2)and interleukin-17(IL-17)in neuropathic pain rats,so as to explore its mechanism underlying pain relief.Methods The present study includes 3 parts.In the first part,eighteen male SD rats were randomly divided into 3 groups:sham operation,model and EA groups,with 6 rats in each group.The neuropathic pain model was established by chronic constriction injury(CCI)of the right sciatic nerve.On the 7th day following modeling,EA was applied to the right"Zusanli"(ST36)and"Sanyinjiao"(SP6)(1 mA,2 Hz/100 Hz)for 30 min.The mechanical pain threshold(MWT)and thermal pain thre-shold(TPT)of the affected limb were detected before modeling,7 days following modeling and 60 min after EA.The expression of MT2 in spinal dorsal horn was detected by Western blot.The contents of melatonin(Mel)and IL-17 in the spinal dorsal horn were determined by ELISA.The expression of glial fibrillary acidic protein(GFAP)in the spinal dorsal horn was determined by Western blot and immunohistochemistry.In the second part,30 rats were divided into 5 groups:sham operation,model,EA,MT2 antagonist(4-P-PDOT),and dimethyl sulfoxide(DMSO)groups,with 6 rats in each group.Rats of the 4-P-PDOT and DMSO groups were intrathecal injection with 10μL MT2 antagonist 4-P-PDOT(100μg)and equivalent DMSO 30 min before EA.The MWT and TPT of affected limb were detected.The GFAP expression and IL-17 content in the spinal dorsal horn was detected by Western blot,immunohistochemistry and ELISA,respectively.In the third part,30 rats were randomly divided into 5 groups:sham operation,model,EA,recombinant IL-17,and normal saline groups,with 6 rats in each group.The recombinant IL-17 protein(100 ng,10μL)and the same amount of 0.9%sodium chloride solution were intrathecal injection into the rats of the recombinant IL-17 group and the normal saline group 30 min before the EA.The MWT and TPT of affected limb were measured.Results On the 7th day after modeling,the MWT of rats in the model group and the EA group were significantly higher,while TPT were lower than those before the modeling(P<0.05).At 60 min after EA,compared with the model group,the MWT and TPT of the EA group reversed significantly(P<0.05).The levels of GFAP and IL-17 were significantly increased,while the levels of Mel and MT2 were significantly decreased in the model group than in the sham operation group(P<0.05),and those were considerably reversed in the EA group than in the model group(P<0.05).Compared with the EA and DMSO groups,the MWT in the 4-P-PDOT group were significantly increased,while TPT were decreased(P<0.05),and the contents of GFAP and IL-17 were significantly increased(P<0.05).Compared to the EA and normal saline groups,MWT of the rats in the recombinant IL-17 group were significantly increased,while TPT decreased(P<0.05).Conclusion EA of ST36 and SP6 can alleviate neuropathic pain in CCI rats,which is closely related to its effect in inhibiting the release of IL-17 from astrocytes mediated by MT2.