脊髓IL-17调控大鼠神经病理性痛的机制与外周神经相关蛋白的关系

Relationship between mechanism of spinal interleukin-17 regulating neuropathic pain and peripheral nerve-related proteins in rats

目的评价脊髓IL-17调控大鼠神经病理性痛的机制与外周神经相关蛋白的关系.方法取鞘内置管成功的雄性SD大鼠36只,10~12周龄,体重240~260 g,采用随机数字表法分为4组(n=9):假手术组(Sham组)、神经病理性痛(NP组)、空载体(BV)组和IL-17 siRNA重组慢病毒组(siRNA组).鞘内置管成功后5 d,采用坐骨神经慢性束缚性损伤(CCI)法制备神经病理性痛模型.CCI后7 d时Sham组和NP组分别鞘内注射生理盐水20μl,BV组和siRNA组分别鞘内注射空载体和IL-17 siRNA-GFP重组慢病毒1×107TU各10μl,再用生理盐水10μl冲管,每天1次,连续4 d.分别于CCI前1 d和CCI后1、7、10、11、12、13、14 d时测定热缩足潜伏期(TWL)和机械缩足反应阈(MWT).于CCI后14 d时取手术侧近端坐骨神经组织,提取总蛋白,进行同位素标记相对和绝对定量和液质联用-串联质谱分析,筛选差异表达蛋白.结果与Sham组比较,其余3组CCI后各时点MWT降低,TWL缩短(P<0.05).与NP组和BV组比较,siRNA组CCI后10~14 d时MWT升高,TWL延长(P<0.05).NP组和BV组上述指标比较差异无统计学意义(P>0.05).筛选出了58个差异表达蛋白,其中21个蛋白表达显著上调,37个蛋白表达显著下调.结论筛选出58个外周神经相关蛋白与脊髓IL-17调控大鼠神经病理性痛的机制有关.

Objective To evaluate the relationship between the mechanism of spinal interleukin-17 (IL-17) regulating neuropathic pain (NP) and peripheral nerve-related proteins in rats. Methods Thirty-six healthy adult male Sprague-Dawley rats, in which IT catheters were successfully implanted, aged 10-12 weeks, weighing 240-260 g, were divided into 4 groups ( n=9 each) using a random number table method: sham operation group (Sham group), NP group, blank vector group (BV group), and IL-17 siRNA recombinant lentivirus group ( siRNA group). NP was induced by chronic constriction injury ( CCI) to the sciatic nerve at 5 days after IT catheters were successfully implanted. At day 7 after CCI, normal sa-line 20 μl was intrathecally injected in Sham and NP groups, and in BV and siRNA groups, blank vector IL-17 siRNA-GFP and recombinant lentivirus 1 × 107 TU 10 μl were intrathecally injected, respectively, the catheter was flushed with normal saline 10 μl once a day for 4 consecutive days. The mechanical paw withdrawal threshold ( MWT) and thermal paw withdrawal latency ( TWL) were measured on day 1 before CCI and days 1, 7, 10, 11, 12, 13 and 14 after CCI. Proximal sciatic nerve tissues on the operated side were obtained at 14 days after CCI, the total protein was extracted, and the isobaric tags for relative and absolute quantification and liquid chromatography-tandem mass spectrometry were performed to stratify the differentially expressed proteins. Results Compared with group Sham, the MWT was significantly de-creased and TWL was shortened at each time point after CCI in the other three groups (P<0. 05). Com-pared with NP and BV group, the MWT was significantly increased and TWL was prolonged at days 10-14 after CCI in group siRNA ( P<0. 05). There was no significant difference in the parameters mentioned above between group NP and group BV (P>0. 05). Fifty-eight differentially expressed proteins were identified, and among the 58 proteins, the expression of 21 proteins was significantly up-regulated and the expression of 37 proteins was down-regulated. Conclusion Fifty-eight peripheral nerve-related proteins identified are related to the mechanism of spinal IL-17 regulating neuropathic pain in rats.