葡萄糖酸钙对间斑寇蛛毒液致痛大鼠脊髓致痛因子表达的影响

Inhibitory effect of calcium gluconate on induced pain factor in Latrodectus tredecimguttatus venom induced pain in rats

目的探讨葡萄糖酸钙对间斑寇蛛毒液致痛大鼠脊髓中致痛因子依赖于钙离子的钾离子通道蛋白（IK1）、降钙素基因相关肽（CGRP）、环氧合酶2（COX-2）、嘌呤能受体（P2X3）相对表达量的影响,研究葡萄糖酸钙在疼痛中的作用。方法将雄性SD大鼠80只随机分为福尔马林、10、40、100、300μL/kg毒液致痛组（40只）和钙剂治疗组（40只）,其中治疗组大鼠预先尾部静脉注射10%葡萄糖酸钙2 m L,然后两组大鼠均以5%福尔马林溶液150μL和4种剂量（10、40、100、300μL/kg）的间斑寇蛛毒液进行足底皮下注射,24 h后处死后,取L4~5脊髓标本,采用免疫组化、Western blot和荧光实时定量PCR技术分别从组织学、蛋白以及分子水平分析两组P2X3、CGRP、COX-2和IK1相对表达量。结果 （1）免疫组化结果显示300μL/kg毒液剂量时,治疗组P2X3和CGRP的表达显著低于致痛组,差异具有统计学意义（P〈0.05）,COX-2和IK1的表达差异无统计学意义（P〉0.05）。（2）Western blotting结果显示,与致痛组各剂量相比,10μL/kg剂量治疗组大鼠脊髓中P2X3和IK1表达量明显升高,CGRP和COX-2表达量降低,差异有统计学意义（P〈0.05）;40μL/kg剂量治疗组COX-2表达量降低,CGRP、P2X3和IK1的表达差异无统计学意义（P〉0.05）;100μL/kg剂量治疗组P2X3、CGRP和IK1表达量明显降低,COX-2的表达量升高,差异具有统计学意义（P〈0.05）;300μL/kg剂量治疗组大鼠脊髓中P2X3、CGRP、COX-2、IK1表达量明显降低,差异具有统计学意义（P〈0.05）。（3）荧光实时定量PCR结果显示：10μL/kg治疗组大鼠脊髓中P2X3和CGRP mRNA的表达高于致痛组,COX-2和IK1 mRNA的表达低于致痛组,差异有统计学意义（P〈0.05）;40μL/kg治疗组P2X3和IK1 mRNA的表达高于致痛组,CGRP mRNA的表达低于致痛组,差异有统计学意义（P〈0.05）,COX-2 mRNA的表达差异无统计学意义（P〉0.05）;100μL/kg治疗组P2X3、CGRP、和COX-2mRNA的表达均高于致痛组,IK1 mRNA的表达低于致痛组,差异有统计学意义（P〈0.05）;300μL/kg治疗组P2X3、CGRP、COX-2和IK1 mRNA的表达均高于致痛组,差异具有统计学意义（P〈0.05）。各致痛组间P2X3、CGRP、COX-2和IK1 mRNA表达差异无统计学意义（P〉0.05）。（4）3种方法结果显示福尔马林致痛组与治疗组致痛因子的表达差异无统计学意义（P〉0.05）。结论新疆间斑寇蛛毒的致痛机制可能与大鼠脊髓中致痛因子CGRP、P2X3、COX-2和IK1蛋白的高表达有关,葡萄糖酸钙能显著抑制部分高浓度毒素组致痛因子CGRP、P2X3、COX-2和IK1蛋白的表达量,缓解疼痛的传递。

Objective This study were designed to explore the inhibitory effect of calcium gluconate on the relative expression amount of induced pain factors including potassium ion channel protein calcium ion de-pendent （IK1）, calcitonin gene related peptide （CGRP）, cyclooxygenase 2 （COX-2）, purinergic receptor （PZX3） ia the rat spinal cord induced by Latrodectus tredecimguttatus venom, and to study the treatment effect of calcium gluconate on pain. Methods 80 male SD rats were randomly divided into pain group, （40） and calcium treatment group （40）, the treatment group rats were given vein injection of 2 mL of 10% calcium gluconate pre tail; the other two groups of rats were given injection of 150μL of 5% formalin so- lution and Latrodectus tredecimguttatus toxin of 4 kinds of concentrations （10, 40, 100, 300/μL/kg）. The rats were killed after 24 h and the L4--5 spinal cord was collected. The relative expression of CGRP, COX- 2, P2X3, IK1 was analyzed by using immunohistochemical method, Western blotting method, and real- time fluorescence quantitative PCR technology. Results Compared with the induced pain group, the ex- pression of induced pain factor in the treatment group decreased significantly （P 〈0.05） by immunohisto- chemical method and Western blotting method in a dose of 300 μL/kg, The rest of the concentration of treatment group compared with the induced pain were significantly different （P 〈0.05）, but no regularity. The results of real time fluorescent quantitative PCR are contrary to the previous two. Conclusion The possible mechanisms of pain induced by Xiniiang Latrodectus tredecimguttatus venom may be related to the level of relative expression of pain factors in rat spinal cord including CGRP P2X3, COX-2, IK1, and calcium gluconate could significantly inhibit the relative expression of CGRP, P2X3, COX-2, IK1 induced by high concentration toxin and slower the pain transfer.