间斑寇蛛毒致痛大鼠脊髓神经元细胞中钙离子激活的内向整流钾通道蛋白表达

Protein expression of calcium-activated inwardly rectifying potassium channel in spinal cord neuron in rat pain model induced by Latrodectus tredecimguttatus venom

目的分析不同浓度间斑寇蛛毒致痛大鼠脊髓灰质神经元细胞中钙离子激活的内向整流钾通道（IK1）的蛋白表达变化。方法选取36只雄性sD大鼠，采用分层随机抽样法分为对照组（4只）和毒素组（32只），毒素组分别于右后足趾部皮下注射10、40、100、300μl／kg间斑寇蛛粗毒液，每种浓度各8只。观察毒素组大鼠注射毒液后的缩足反应。对照组不做处理。24h后将所有大鼠断颈处死，取样第4—5腰椎脊髓样本，采用苏木精一伊红（HE）染色法观察脊髓神经元细胞结构，采用链霉菌抗生物素蛋白一过氧化物酶连结（SP）法观察神经元细胞中IKl蛋白的表达情况，采用实时荧光定量聚合酶链反应（PCR）检测IK1 mRNA的表达水平。分析不同浓度下IK1 mRNA及蛋白表达的变化。采用Pearson相关性检验分析大鼠缩足次数与毒液浓度的相关性。结果注射间斑寇蛛毒15min后毒素组大鼠1h内的缩足次数分别为（44±16）、（64±9）、（69±19）、（91±35）次，Pearson相关性分析显示不同浓度下的1h缩足次数与毒液浓度呈正相关（F2=0．548，P〈0．05）。对照组及10、40、100、300μl／kg毒素组大鼠脊髓神经元细胞中平均IK1蛋白阳性细胞数分别为（2．0±1．0）、（5．0±2．0）、（6．0±1．0）、（6．0±2．0）、（7．0±1．0）个；各毒素组平均阳性细胞数均明显高于对照组，40、100、300μl／kg毒素组明显高于10μl／kg毒素组（均P〈0．05），40、100、300μl／kg毒素组之间差异无统计学意义（P〉0．05）。对照组及10、40、100、300bd／kg毒素组大鼠脊髓中IK1 mRNA的相对表达量分别为（0．56±0．16）、（0．62±0．27）、（1．27±0．10）、（1．53±0．29）、（1．61±0．86）；各毒素组IK1 mRNA表达水平均明显高于对照组，40、100、300bd／kg毒素组明显高于10μl／kg毒素组（均P〈0．05），40、100、300μl／kg毒素组之间差异无统计学意义（P〉0．05）。结论问斑寇蛛毒具有急性致痛作用，与毒素呈浓度依赖性；其疼痛传递机制依赖于钙离子激活的IK1 mRNA及蛋白在脊髓神经元细胞中的表达。

Objective To observe the protein expression of calcium-activated inwardly rectifying potassi- um channel （IK1） in spinal cord neuron in rat pain model induced by Latrodectus tredecimguttatus venom. Methods Totally 36 male SD rats were randomly divided into control group（4 rats） and toxin group（32 rats） ; rats in toxin group were given subcutaneous injection of 10, 40, 100, 300 μL/kg Latrodectus tredecimguttatus venom at toes of right rear foot, respectively 8 rats for each concentration. Times of paw-withdrawal reactions were observed 15 min after injection. After 24 h, all rats were executed and spinal cord of 4-5 lumbar vertebra were sampled. Hematoxylin-eosin（HE） staining method was used to observe the structure of spinal cord neuron; strep- tomyces avidin-peroxidase link（SP） method was used to observe the protein expression of IK1 ; real-time fluores- cent quantitative polymerase chain reaetion（PCR） was used to detect mRNA expression of IK1. Pearson correlation test was used to analyze the correlation between paw-withdrawal reactions and the concentration of Latrodectus tredecimguttatus venom. Results Times of paw-withdrawal reactions 15 rain after the injection of Latrodectus tre- decimguttatus venom in 10, 40, 100, 300 μl/kg toxin groups were （43.7 ± 15.5 ）, （64. 3 ±9. 1 ） , （69.3 ±19.0）, （91.3± 34. 6）times respectively; Pearson correlation test showed positive correlation between paw-with- drawal reactions and the concentration of venom（ r2 = 0. 548, P 〈 0. 05 ）. Spinal cord neuron numbers of IK1 pro- tein positive expression in control group and 10, 40, 100, 300 μl/kg toxin groups were （ 2.0 ±1.0 ）, （ 5.0 ± 2. 0） , （6.0 ± 1.0） , （6. 0 ±2, 0）, （7.0 ± 1.0） respectively; neuron numbers of IK1 protein positive expression in 4 toxin groups were all significantly higher than control group（P 〈 0. 05 ） ; neuron numbers in 40, 100,300 μl/kg toxin groups were significantly higher than those in 10 μl/kg group（ P 〈 0. 05） ; differences of IK1 protein positive expression neuron number among 40, 100, 300 μl/kg toxin groups were not significant （ P 〉 0. 05 ）. Relative quantities of IK1 mRNA expression in spinal coM in control group and 10, 40, 100,300 μl/kg toxin groups were （0.56 ±0.16）,（0.62 ±0.27）,（1.27 ±0.10）,（1.53 ±0.29）,（1.61 ±0.86） respectively; IK1 mRNA expressions in 4 toxin groups were all significantly higher than those in control group（ P 〈 0. 05 ） ; IKI mRNA ex- pressions in 40, 100, 300 μl/kg toxin groups were significantly higher than those in 10μl/kg group（ P 〈 0. 05 ） ; differences of IK1 mRNA expression relative quantity between 40, 100, 300 μl/kg groups were not significant （P〉 0. 05 ）. Conclusions Latrodectus tredecimguttatus venom can cause acute pain effect, which is concentration dependent. The mechanism of pain transmission is related to calcium-activated IK1 mRNA and protein expressions in spinal cord neuron.