大鼠神经病理性痛时外周神经BIP与Nav1.8的关系

Relationship between BIP and Nav1.8 in peripheral nerve in a rat model of neuropathic pain

目的评价大鼠神经病理性痛时外周神经免疫球蛋白重链结合蛋白(BIP)与电压门控性钠离子通道亚型1.8(Nav1.8)的关系。方法SPF级健康雄性SD大鼠44只,体重210~260 g。采用坐骨神经慢性缩窄损伤(CCI)法制备神经病理性痛模型。实验Ⅰ采用随机数字表法将20只大鼠分为2组(n=10):假手术组(Sham组)和CCI组。实验Ⅱ采用随机数字表法将24只大鼠分为3组(n=8):假手术组(Sham组)、CCI+生理盐水组(CCI+NS组)和CCI+BIP抑制剂HA15组(CCI+H组)。于术后第4天开始,CCI+NS组腹腔注射0.9%生理盐水1 ml,CCI+H组腹腔注射HA150.7 mg/kg,连续3 d(1次/d)。实验Ⅰ和Ⅱ均于术前1 d、术后1、3、d及7 d(T0-T4)时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL),术后第7天行为学测试完成后采用Western blot法检测背根神经节和坐骨神经BIP与Nav1.8的表达。实验Ⅰ于术后第7天行为学测试结束后采用免疫荧光检测背根神经节和坐骨神经BIP和Nav1.8的表达及共定位情况,采用免疫共沉淀实验评价BIP和Nav1.8相互作用情况。结果实验Ⅰ与Sham组比较,CCI组T1-T4时MWT降低,TWL缩短,背根神经节Nav1.8表达下调,BIP表达上调,坐骨神经Nav1.8和BIP表达上调(P<0.05),CCI组背根神经节和坐骨神经BIP与Nav1.8存在明显共定位,背根神经节BIP可以免疫共沉淀出Nav1.8,Nav1.8也能免疫共沉淀出BIP。实验Ⅱ与Sham组比较,CCI+NS组T1-T4时MWT降低,TWL缩短,背根神经节Nav1.8表达下调,BIP表达上调,坐骨神经Nav1.8和BIP表达上调(P<0.05);与CCI+NS组比较,CCI+H组T3,4时MWT升高,TWL延长,背根神经节和坐骨神经Nav1.8和BIP表达下调(P<0.05)。结论BIP可介导外周神经Nav1.8的再分布,参与大鼠神经病理性痛的病理生理机制。

Objective To evaluate the relationship between immunoglobulin heavy chain-binding protein(BIP)and Nav1.8 in peripheral nerve in a rat model of neuropathic pain.Methods Forty-four SPF healthy male Sprague-Dawley rats,weighing 210-260 g,were used in this study.Neuropathic pain was induced by chronic constriction injury(CCI)in anesthetized rats.The experiment was performed in two parts.ExperimentⅠTwenty rats were divided into 2 groups(n=10 each)using a random number table method:sham operation group(group Sham)and group CCI.ExperimentⅡTwenty-four rats were divided into 3 groups(n=8 each)using a random number table method:sham operation group(group Sham),CCI plus normal saline group(group CCI+NS)and CCI plus BIP inhibitor HA15 group(group CCI+H).Starting from 4th day after surgery,0.9%normal saline 1 ml was intraperitoneally injected in group CCI+NS,and HA150.7 mg/kg was intraperitoneally injected in group H,once a day for 3 consecutive days.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 1 day before surgery and 3,5 and 7 days after surgery(T0-T4),and the expression of BIP and Nav1.8 in dorsal root ganglion(DRG)and sciatic nerve was detected by Western blot on 7th day after completion of behavioral testing in two groups.The expression and colocalization of BIP and Nav1.8 in DRG and sciatic nerve were determined by immunofluorescence on 7th day after completion of behavioral testing in groupⅠ,and the interaction between BIP and Nav1.8 was evaluated by co-immuno-precipitation.Results ExperimentⅠCompared with group Sham,the MWT was significantly decreased,and TWL was shortened at T1-T4,the expression of Nav1.8 in DRG was down-regulated,the expression of BIP was up-regulated,and the expression of Nav1.8 and BIP in sciatic nerve was up-regulated in group CCI(P<0.05),and BIP and Nav1.8 on the sciatic nerve were co-localized,BIP could co-precipitate Nav1.8 from DRG,and Nav1.8 could also coprecipitate BIP in group CCI.ExperimentⅡCompared with group Sham,the MWT was significantly decreased,and TWL was shortened at T1-T4,the expression of Nav1.8 in DRG was down-regulated,the expression of BIP was up-regulated,and the expression of Nav1.8 and BIP in sciatic nerve was up-regulated in group CCI+NS(P<0.05).Compared with group CCI+NS,the MWT was significantly increased,and TWL was prolonged at T3,4,and the expression of Nav1.8 in DRG was down-regulated in group CCI+H(P<0.05).Conclusion BIP can mediate the redistribution of Nav1.8 in peripheral nerve and is involved in the pathophysiological mechanism of neuropathic pain in rats.