Objective: To observe the effect of electroacupuncture (EA) on ocular surface sensory neuralgia and the expression of P2X3 receptor (P2X3R) and protein kinase C(PKC)in cornea and trigeminal ganglion (TG) in dry eye disease (DED) guinea pigs, so as to explore its mechanism underlying improvement of ocular surface sensory neuralgia in DED.
Methods: Male British tricolor short haired guinea pigs were randomly divided into control, model, medication (pranoprofen), EA and sham acupuncture groups, with 8 guinea pigs in each group. The dry eye model was induced by subcutaneous injection of scopolamine hydrobromide solution (0.6 mg/0.2 mL,once daily) for 10 d. Guinea pigs in the medication group were treated by applying pranoprofen eye drops to eyes, 1 drop for one eye each time, three times a day. Guinea pigs of the EA group received EA stimulation (4 Hz/20 Hz,1 mA) of bilateral "Cuanzhu" (BL2) and "Taiyang" (HN5) and acupuncture at "Jingming" (BL1) "Sizhukong" (TE23), "Tongziliao" (GB1) for 15 min, once a day. Guinea pigs in the sham acupuncture group received blunt stimu-lation at the surface of the same acupoint with the tip of the acupuncture needle, once a day. All the treatments were conducted for 14 d. The corneal epithelium fluorescein staining score (0-3 points) was given according to the number of fluorescence-positive dots and flake-like coloration, the corneal mechanical perception thread (CMPT) detected using a corneal perception meter, and the palpebral fissure height measured. The number of sensory neurons in the cornea and TG was determined by using cholera toxin subunit B conjugated with Alexa Fluor 488 fluorescence labelling, and the expression levels of P2X3R and PKC in the cornea and TG detected by using immunohistochemistry and Western blot, separately.
Results: Compared with the control group, the corneal fluorescein staining score, immunoactivity and expression of P2X3R proteins in both cornea and TG, PKC proteins in TG were significantly increased (P<0.01), whereas the CMPT and the height of palpebral fissure and the number of TG neurons significantly decreased in the model group (P<0.05,P<0.01). In comparison with the model group, the fluorescein staining score in the medication and EA groups, the immunoactivity and expression of P2X3R in cornea and TG in the EA group, and that of TG PKC in the EA group and the sham acupuncture groups were significantly decreased (P<0.05, P<0.01), while the height of palpebral fissure and CMPT after EA and the number of labelling TG sensory neurons were remarkably increased in the EA group (P<0.01) rather than in the medication and sham acupuncture groups (P>0.05).
Conclusion: EA can alleviate the damage of corneal epithelium and sensory neurons in dry eye model guinea pigs, which may be related to its functions in down-regu-lating the expression of P2X3R and PKC in the cornea and TG.
目的：观察电针对干眼豚鼠眼表感觉神经痛及角膜和三叉神经节(TG)中嘌呤信号P2X3受体和蛋白激酶C(PKC)表达的影响, 探究电针缓解干眼眼表感觉神经痛的机制。方法：将40只雄性英国三色短毛豚鼠随机分为空白组、模型组、普拉洛芬组、电针组和假针刺组, 每组8只。皮下注射氢溴酸东莨菪碱溶液连续10 d诱导干眼模型, 并且在干预期间保持造模操作直至干预结束。普拉洛芬组予普拉洛芬滴眼, 3次/d, 每次1滴；电针组针刺双侧“睛明”“攒竹”“丝竹空”“瞳子髎”“太阳”, 其中“攒竹”“太阳”给予电针干预, 15 min/次, 每天1次；假针刺组采用钝针点刺穴位表面, 取穴与电针组相同。3组均干预14 d。检测各组动物造模前、造模后和干预后眼表感觉神经痛相关指标；用霍乱毒素亚单位B结合荧光素488标记角膜和TG中感觉神经元；免疫组织化学法和Western blot法检测角膜和TG中P2X3受体和PKC的表达。结果：造模后与空白组比较, 模型组的角膜荧光素染色评分显著升高(P<0.01), 睑裂高度明显降低(P<0.01), 角膜机械知觉阈值差异无统计学意义(P>0.05)。干预后与空白组比较, 模型组豚鼠角膜荧光素染色评分升高(P<0.01), 角膜机械知觉阈值和睑裂高度明显降低(P<0.05, P<0.01)；与模型组比较, 电针组和普拉洛芬组的角膜荧光素染色评分降低(P<0.01, P<0.05), 电针组的睑裂高度和角膜机械知觉阈值显著升高(P<0.01)。与空白组比较, 模型组TG中被标记的阳性神经元数量明显降低(P<0.01)；与模型组比较, 电针组TG中被标记的阳性神经元数量明显升高(P<0.01)。与空白组比较, 模型组角膜和TG中P2X3受体, TG中PKC表达水平显著升高(P<0.01)；与模型组比较, 电针组角膜和TG中P2X3受体, TG中PKC表达明显降低(P<0.01)。结论：电针可减轻干眼模型豚鼠角膜和感觉神经元损伤, 提高角膜机械知觉阈值, 该作用可能与电针抑制角膜和TG中P2X3受体和PKC的表达有关。.
Keywords: Dry eye; Electroacupuncture; P2X3 receptor; Protein kinase C; Sensory neuralgia.