电针对神经病理性疼痛大鼠脊髓P2X3受体表达的影响

Effect of electroacupuncture on P2X3 receptor expression in spinal cord of rats with neuropathic pain

目的探讨电针对神经病理性疼痛大鼠脊髓P2X，受体表达的影响。方法清洁级雄性SD大鼠60只，体重180g-220g，采用随机数字表法分为6组，每组10只：假手术组（Sham组）仅分离坐骨神经，不结扎；神经病理性疼痛组（CCI组）采用坐骨神经慢性压迫性损伤法制备神经病理性疼痛模型；2Hz穴位电针组（LEA组）于坐骨神经结扎（chronic constrictive injury，CCI）后第4天开始电针治疗，刺激大鼠术侧足三里-阳陵泉穴，频率2Hz连续波，强度≤1．5mA，每天1次，30min／次，连续6d；15Hz穴位电针组（HEA组）电针频率为15Hz，余同LEA组；2Hz非穴位电针组（NA-LEA组）电针刺激大鼠术侧足三里．阳陵泉旁5mm非穴位点，余同LEA组；15Hz非穴位电针组（NA-HEA组）电针刺激大鼠术侧足三里．阳陵泉旁5mm非穴位点，余同HEA组。各组术前1d（T0）和术后3、5、7、9d（T1-4）测定大鼠后肢机械缩足反射阂值（mechanical withdrawal threshold，MWT）和热缩足反射潜伏期（thermal withdrawal latency，TWL）；于术后第10天处死大鼠，取右侧L4-6脊髓节段，Western-blot和反转录酶壤合酶链锁反应法分别检测大鼠脊髓P2X，受体蛋白和mRNA表达。结果与Sham组比较，其余各组MWT和TWL降低（P〈0．05），脊髓P2X3受体蛋白和mRNA表达升高（P〈0．05）；与CCI组比较，LEA组和HEA组L2-4时MWT和TWL升高（P〈0．05），脊髓P2X3受体蛋白和mRNA表达降低（P〈0．05）；与LEA组比较，T4时HEA组MWT和TWL升高，脊髓P2X，受体蛋白和mRNA表达增多（P〈0．05）。结论电针足三里-阳陵穴可明显增加神经病理性疼痛大鼠的机械痛和热痛阈值，其机制可能与降低脊髓背角P2X3受体表达有关。

Objective To investigate the effect of electroacupuncture（EA） on P2X3 receptor expression in the spinal cord of rats with neuropathic pain （NP）. Methods Sixty healthy male Sprague Dawley （SD） rats weighing 180 g-220 g were randomly divided into 6 groups （n=10 each）： sham operation group （Sham group）, CCI group, CCI＋2Hz-Acupoint-EA （LEA group）, CCI＋ 15Hz-Acupoint-EA （HEA group）, CCI＋2Hz-Nonacupoint-EA （NA-LEA group）; CCI＋15Hz-Nonacupoint-EA （NA -HEA group）. Chronic constriction injury （CCI） was induced by exposing the right sciatic nerve and placing 4 ligatures on the sciatic nerve at 1 mm intervals with 4-0 silk thread. In sham operation group the right sciatic nerve was exposed but not ligated. Electrical stimulation at aeupoints was performed at Zusanli （ST36） and Yanlingquan （GB34） once a day from 4th day after CCI to the successive 6 days （30 min/d）. Nonacupoints were performed 5 mm nearby the acupoints of Yanglingquan and Zusanli. EA variables were set as alternating strings of continues frequency （2 Hz or 15 Hz）, and the intensity was adjusted to induce moderate muscle contract of the hindlimb （≤ 1.5 mA）. The thermal withdrawal latency（TWL） was measured using hot plate test and mechanical withdrawal threshold （MWT） with yon Frey filaments was measured for injured ipsilateral paw the day before and at the 3rd, 5th, 7th days after operation （T0-4）. The right lumbar segment （L4-6） of spinal cord was removed on the 10th day after induction of CCI for determination of the protein and mRNA expression of P2X3 in the spinal cordl using western-blotting and reverse transcription-polymerase chain reaction （RT-PCR）, respectively. Results In comparison with sham group, after CCI, MWT and TWL decreased significantly and P2X3 protein and mRNA levels were significantly increased in all other groups （P〈0.05）. Compared with group CCI, MWT and TWL increased at T24 and the protein and mRNA level of P2X3 expression decreased significantly in LEA and FlEA group （P〈0.05）. MWT and TWL was significantly higher and P2X3 protein and mRNA expression were lower in group LEA than those in group HEA （P〈0.05）. Conclusions Down-regulation of P2X3 receptor expression in spinal cord is involved in the mechanism of analgesic effects of electroacupuncture on neuropathic pain.