阿米洛利联合盐酸羟考酮注射液对癌痛模型小鼠脊髓CXCL13、脊髓背角c-fos表达的影响

Effects of amiloride combined with oxycodone hydrochloride injection on the expression of CXCL13 in spinal cord and C-fos in cornu dorsale medullae spinalis of mice models with cancer pain

目的探讨阿米洛利联合盐酸羟考酮注射液对癌痛模型小鼠脊髓趋化因子CXC配体13(CXCL13)及脊髓背角即刻早期基因c-fos表达的影响。方法选取60只BALB/c小鼠进行研究,将其随机分为五组,分别为正常组(A组)、皮肤癌痛组(B组)、生理盐水组(C组)、阿米洛利组(D组)及阿米洛利联合盐酸羟考酮注射液组(E组),每组12只。除A组外,其余组小鼠均采用小鼠一侧后跖面皮下注射Walker256癌细胞悬液的方法来建立皮肤癌痛模型。建模成功后,A组和B组小鼠不给于任何干预措施,C组给予经鞘内注射0.9%氯化钠注射液10μl,D组给予经鞘内注射阿米洛利溶液10μl,浓度为1.5μg/μl,E组在D组基础上同时给予静脉注射0.2 mg/kg盐酸羟考酮注射液。测定各组小鼠给药前后不同时间点的疼痛反应,测定结束后取脊髓组织,应用Western blot法及反转录-聚合酶链反应(RT-PCR)法测定脊髓CXCL13和脊髓背角c-fos表达情况。结果鞘内注射前30 min,B组、C组、D组、E组小鼠的机械痛阈值较接种前均明显降低(t分别=44.18、34.50、32.05、40.18,P均<0.05),且B组、C组、D组、E组小鼠鞘内注射后30 min、60 min、120 min和180 min机械痛阈值均明显低于A组(t分别=29.74、31.80、34.44、32.93;41.09、55.42、34.10、26.93;33.37、45.95、21.65、14.53;33.37、35.67、21.72、13.75,P均<0.05),E组鞘内注射后60 min、120 min和180 min的机械痛阈值明显高于D组(t分别=42.52、32.56、26.63,P均<0.05)。与A组比较,B组、C组、D组小鼠脊髓CXCL13蛋白表达、CXCL13 mRNA表达均上调(t分别=5.10、4.59、2.35;6.13、5.51、2.39,P均<0.05),而E组小鼠脊髓CXCL13蛋白表达、CXCL13 mRNA表达与A组比较,差异均无统计学意义(t分别=0.58、0.21,P均>0.05)。与A组比较,B组、C组、D组、E组小鼠脊髓背角c-fos蛋白表达、c-fos mRNA表达均上调(t分别=13.02、8.58、8.67、6.20;17.48、12.13、12.95、8.15,P均<0.05),但E组c-fos蛋白表达、c-fos mRNA表达明显低于B组、C组、D组(t分别=9.34、5.44、4.15;12.78、7.79、6.80,P均<0.05)。结论阿米洛利联合盐酸羟考酮注射液可用于皮肤癌小鼠癌痛过敏的缓解,该机制可能与两者联合可抑制脊髓和脊髓背角组织CXCL13、c-fos的表达有关。

Objective To investigate the effects of amiloride combined with oxycodone hydrochloride injection on the expression of CXC ligand 13(CXCL13)in spinal cordand immediate early genes(IEGs)c-fos in spinal cord of mice models with cancer pain.Methods Totally 60 BALB/c mice were randomly divided into 5 groups:normal group(group A),skin cancer pain group(group B),normal saline group(group C),amiloride group(group D),and amiloride combined with oxycodone hydrochloride injection group(group E).Except group A,the mice in other groups were subcutaneously injected with Walker 256 cancer cell suspension on the posterior plantar surface of mice to establish skin cancer pain model.The mice in group A and group B did not give any intervention.The mice in group C were injected with normal saline intrathecally,the mice in group D were injected with amiloride solution intrathecally,and the mice in group E were injected with amiloride solution and 0.2 mg/kg oxycodone hydrochloride injection.The pain response were measured at different time points before and after administration.The spinal cord tissues were taken after administration.The expressions of CXCL13 and c-fos in spinal cord dorsal horn were detected by Western blot and RT-PCR.Results The mechanical pain threshold of mice in group B,group C,group D and group E at 30 minutes before intrathecal injection were significantly lower than those before intrathecal injection(t=44.18,34.50,32.05,40.18,P<0.05).The mechanical pain threshold of mice in group B,group C,group D and group E at points of 30 min,60 min,120 min,180 min after intrathecal injection were significantly lower than those in group A(t=29.74,31.80,34.44,32.93;41.09,55.42,34.10,26.93;33.37,45.95,21.65,14.53;33.37,35.67,21.72,13.75,P<0.05).The mechanical pain threshold of group E at points of 60 min,120 min,180 min after intrathecal injection were significantly higher than those of group D(t=42.52,32.56,26.63,P<0.05).Compared with group A,the expression of CXCL13 protein and mRNA was up-regulated in group B,group C and group D(t=5.10,4.59,2.35;6.13,6.51,2.39,P<0.05).There was no significant difference in CXCL13 protein and mRNA expression between group E and group A(t=0.58,0.21,P>0.05).Compared with group A,the expression of c-fos protein and mRNA in spinal dorsal horn of mice in group B,group C,group D and group E were up-regulated(t=13.02,8.58,8.67,6.20;17.48,12.13,12.95,8.15,P<0.05).The c-fos protein and mRNA in group E were significantly lower than those in group B,group C,and group D(t=9.34,5.44,4.15;12.78,7.79,6.80,P<0.05).Conclusion Thecombination of amiloride and oxycodone hydrochloride injection can alleviate cancer hyperalgesia in skin cancer mice,which may be related to the inhibition of the expression of CXCL13 and c-fos in spinal cord and spinal dorsal horn.