针刺通过α7nAChR/NF-κB p65信号通路减轻干眼眼表炎性反应

Acupuncture mitigates ocular surface inflammatory response viaα7nAChR/NF-κB p65 signaling in dry eye guinea pigs

目的:观察电针对干眼豚鼠眼表炎性反应及α7烟碱型乙酰胆碱受体(α7nAChR)/核因子κB(NF-κB)p65信号通路的影响,探讨电针治疗干眼的作用机制。方法:32只雄性英国三色短毛豚鼠随机分为空白组、模型组、电针组和假针刺组,每组8只。皮下注射氢溴酸东莨菪碱诱导干眼模型。电针组电针“攒竹”“太阳”及手针针刺双侧“睛明”“丝竹空”“瞳子髎”,15 min/次,1次/d,干预14 d;假针刺组采用钝针点刺穴位表面,取穴及时间同电针组,干预14 d。检测各组豚鼠造模前、造模后和干预后的泪膜破裂时间(BUT)、泪液分泌量(PRT)及角膜荧光素染色评分(Fl)等干眼相关眼表指标;用角膜共聚焦显微镜检测角膜上皮基质细胞周围活化免疫细胞密度;ELISA法检测角膜和泪腺中白细胞介素(IL)-4、IL-6、IL-10、肿瘤坏死因子α(TNF-α)的含量,免疫组织化学法及Western blot法检测角膜及泪腺中α7nAChR、NF-κB p65表达水平。结果:与空白组比较,模型组豚鼠BUT及PRT均明显减少(P<0.01),Fl、角膜上皮细胞周围活化的免疫细胞数密度明显升高(P<0.01),角膜及泪腺中IL-6、IL-10、TNF-α含量及泪腺中NF-κB p65阳性表达明显升高(P<0.01,P<0.05),泪腺中α7nAChR阳性表达明显降低(P<0.01),角膜中NF-κB p65核蛋白表达明显升高(P<0.01)。与模型组比较,干预后电针组BUT及PRT明显增加(P<0.01),Fl、角膜上皮中活化的免疫细胞密度降低(P<0.01,P<0.05),角膜及泪腺中IL-6、TNF-α含量降低(P<0.01),角膜及泪腺中IL-10含量及α7nAChR阳性表达及蛋白表达升高(P<0.01,P<0.05),NF-κB p65阳性表达及NF-κB p65核蛋白表达降低(P<0.01,P<0.05);假针刺组角膜中IL-10含量升高(P<0.01),角膜中TNF-α含量降低(P<0.05),泪腺中NF-κB p65阳性表达降低(P<0.01),泪腺中α7nAChR蛋白表达明显升高(P<0.01),角膜NF-κB p65核蛋白表达升高(P<0.05)。与假针刺组比较,电针组的BUT及PRT增加(P<0.05,P<0.01),Fl明显降低(P<0.01),角膜及泪腺中IL-4、IL-10升高(P<0.05,P<0.01),IL-6、TNF-α明显降低(P<0.01),角膜及泪腺中α7nAChR阳性表达明显升高(P<0.01),角膜中NF-κB p65阳性表达明显降低(P<0.01),角膜及泪腺中α7nAChR蛋白表达升高(P<0.05),角膜及泪腺中NF-κB p65核蛋白表达降低(P<0.01,P<0.05)。结论:电针可上调α7nAChR的表达,抑制NF-κB核转位,降低角膜活化的免疫细胞,减轻角膜及泪腺的炎性反应,改善干眼相关眼表指标。

Objective To observe the effect of electroacupuncture(EA)on the ocular surface inflammation andα7 nicotinic acetylcholine receptor(α7 nAChR)/nuclear factor kappa-B(NF-κB)p65 signal pathway in guinea pigs with dry eye,so as to explore its underlying mechanism.Methods A total of 32 male British tricolor short haired guinea pigs were randomized into blank control,model,EA and sham acupuncture groups,with 8 guinea pigs in each group.The dry eye model was established by subcutaneous injection of scopolamine hydrobromide solution(0.6 mg/0.2 mL each time,4 times a day for 10 days).Guinea pigs of the EA group was treated with EA at bilateral“Cuanzhu”(BL2)and“Taiyang”(HN5),and manual acupuncture at bilateral“Jingming”(BL1),“Sizhukong”(SJ23),“Tongziliao”(GB1)for 15 min,once daily for 14 days.For animals of the sham acupuncture group,a blunt needle was used to prick the skin surface of the acupoints,the acupoint selection and stimulation time were the same as those in the EA group.Before and after modeling and after the intervention,the breakup time(BUT)of lacrimal film,sodium fluorescein coloring(Fl)state of corneal epithelium and phenol red thread(PRT)moist length were recorded for assessing the severity of dry eye.The density of activated immune cells around the corneal epithelial stromal cells was determined by corneal confocal microscopy.The contents of interleukin-4(IL-4),IL-6,IL-10,tumor necrosis factorα(TNF-α)in the cornea and lacri-mal gland tissues were determined by ELISA,and the expression levels ofα7 nAChR and NF-κB p65 in the cornea and lacrimal gland were detected by immunohistochemistry and Western blot,separately.Results Compared with the blank control group,the corneal Fl,density of activated immune cells of corneal epithelium,contents of IL-6,IL-10 and TNF-αin both corneal and lacrimal gland tissues,NF-κB p65 cell positive rate and protein expression of lacrimal gland and corneal tissues were significantly increased(P<0.01,P<0.05),while the BUT,PRT and lacrimal glandα7 nAChR cell positive rate considerably decreased(P<0.01)in the model group.In comparison with the model group,the level of corneal Fl,density of the activated immune cells of corneal epithelium,contents of corneal and lacrimal IL-6 and TNF-α,and corneal and lacrimal NF-κB p65 cell positive rates and protein expressions were remarkably down-regulated in the EA group(P<0.01,P<0.05),rather than in the sham acupuncture group(P>0.05)except content of corneal IL-10,lacrimal NF-κB p65 cell positive rate and lacrimalα7 nAChR protein expression,whereas the levels of BUT,PRT,corneal and lacrimal IL-10 and corneal and lacrimalα7 nAChR cell positive rates and protein expressions significantly up-regulated in the EA group(P<0.01,P<0.05),rather than in the sham acupuncture group(P>0.05)except corneal TNF-αand corneal NF-κB p65 protein expression.Conclusion EA can improve corneal and lacrimal function in dry eye guinea pigs,which may be associated with its actions in increasing the expression ofα7 nAChR,inhibiting the nuclear translocation of NF-κB,and reducing the activated immune cells and inflammatory reaction.