大鼠神经病理性痛时脊髓kindlin-1/Wnt3a信号通路与炎症反应的关系

Relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain

目的评价大鼠神经病理性痛时脊髓kindlin-1/Wnt3a信号通路与炎症反应的关系。方法清洁级健康雄性SD大鼠24只,10~12周龄,体重250~280 g,采用随机数字表法分为4组(n=6):假手术组(SH组)、神经病理性痛组(NP组)、kindlin-1 shRNA组(K组)和Wnt3a抑制组(W组)。采用慢性坐骨神经压迫法建立小鼠神经病理性痛模型。于术前21 d时K组鞘内注射kindlin-1 shRNA腺病毒载体10μl,SH组、NP组和W组鞘内注射病毒空载体10μl。于术后1~3 d时W组鞘内注射Wnt抑制剂IWP-210μl,SH组、NP组和K组鞘内注射人工脑脊液10μl。分别于术前1 d、术后4和7 d时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。术后7 d痛阈测定结束后,处死大鼠取L4-6脊髓组织,采用ELISA法测定TNF-α和IL-1β含量,采用Western blot法检测kindlin-1和Wnt3a表达。结果与SH组比较,NP组、K和W组术后4和7 d时MWT降低,TWL缩短,脊髓TNF-α和IL-1β含量升高,NP组和W组脊髓kindlin-1和Wnt3a表达上调,K组脊髓Wnt3a表达上调(P<0.05);与NP组比较,K组和W组术后4和7 d时MWT升高,TWL延长,K组脊髓TNF-α和IL-1β含量降低,kindlin-1和Wnt3a表达下调,W组脊髓TNF-α和IL-1β含量降低,Wnt3a表达下调(P<0.05),kindlin-1表达差异无统计学意义(P>0.05)。结论脊髓kindlin-1通过上调Wnt3a的表达,进而调控炎症反应,参与大鼠神经病理性痛的维持。

Objective To evaluate the relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain(NP).Methods Twenty-four clean-grade healthy male Sprague-Dawley rats,aged 10-12 weeks,weighing 250-280 g,were divided into 4 groups(n=6 each)using a random number table method:sham operation group(SH group),NP group,kindlin-1 shRNA group(K group)and Wnt3a inhibition group(W group).NP was induced by chronic constrictive injury in anesthetized animals.At 21 days before operation,kindlin-1 shRNA adenovirus vector 10μl was intrathecally injected in group K,and empty viral vector 10μl was intrathecally injected in SH,NP and W groups.Wnt inhibitor IWP-210μl was intrathecally injected in group W,and artificial cerebrospinal fluid 10μl was intrathecally injected in SH,NP and K groups at 1-3 days after operation.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 1 day before operation and 4 and 7 days after operation,respectively.At the end of pain threshold measurement at 7 days after operation,the animals were sacrificed and the lumbar segments(L4-6)of the spinal cord were obtained for determination of the contents of tumor necrosis factor(TNF)-αand interleukin(IL)-1β(IL-1β)(using enzyme-linked immunosorbent assay)and the expression of kindlin-1 and Wnt3a(by Western blot).Results Compared with group SH,MWT was significantly decreased,TWL was shortened at 4 and 7 days after operation,and the contents of TNF-αand IL-1βin spinal cord were increased in NP,K,and W groups,the expression of kindlin-1 and Wnt3a was up-regulated in NP and W groups,and expression of Wnt3a was up-regulated in group K(P<0.05).Compared with group NP,MWT was significantly increased and TWL was prolonged at 4 and 7 days after operation in K and W groups,the contents of TNF-αand IL-1βin spinal cord were decreased,and the expression of kindlin-1 and Wnt3a was down-regulated in group K,the contents of TNF-αand IL-1βin spinal cord were decreased,and the expression of Wnt3a was down-regulated in group W(P<0.05),and no significant change was found in kindlin-1 expression(P>0.05).Conclusion Spinal kindlin-1 regulates the inflammatory response by up-regulating the expression of Wnt3a,and it is involved in the maintenance of NP in rats.