鞘内注射LXM-10对神经病理性疼痛大鼠Fos蛋白和P2X_3受体表达的影响

Effects of Intrathecal Administration LXM-10 on the Expression of FOS Protein and P2X_3 Receptor in Neuropathic Pain Rat Models

目的研究鞘内注射新型镇痛药LXM—10(2,4—dimethyl—9—β—phenylethyl—3—oxo—6,9—diazaspiro[5,5]undecane chloride,2,4—E乙烷基—9—β—苯乙酸—3—oxo—6,9—二氮杂螺环—[5,5]十二烷基盐酸盐,LXM—10)对神经病理性疼痛(neuropathic pain,NPP)大鼠脊髓背角和背根神经节Fos蛋白和P2X_3受体表达的影响。方法选取SD大鼠108只,随机分为假手术组(S组)、对照组(C组)和药物组(L组),对照组和药物组制备坐骨神经慢性压迫模型(chronic constriction injury of the sciatic nerve,CCI—SN)。假手术组、药物组和对照组根据给药时间不同分为3个亚组(n=12只),药物组和对照组分别在CCI—SN造模成功后1、5、12d开始向蛛网膜下腔注射生理盐水10μl,连续3d,分别设为C3组、C7组、C14组,或向蛛网膜下腔注射LXM-10 6μg/kg,连续3d,分别设为L3组、L7组、L14组;假手术组仅在相应时间点向鞘内注射生理盐水,设为S3组、S7组、S14组。连续注射3d后于末次给药后2h处死动物,取腰膨大脊髓节段和腰4～6背根神经节分别进行免疫组化实验检测Fos蛋白和P2X_3受体的表达情况。结果对照组各时间点Fos蛋白随着时间延长表达增强,Fos蛋白主要表达于脊髓背角。药物组在各时间点脊髓背角Fos蛋白免疫反应阳性(Fos-like immunoreactivity,F-LI)细胞均较相应时间点对照组明显减少((L3组 vs C3组,P=0.003;L7组 vs C7组,P=0.023;L14组 vs C14组,P=0.005)。背根神经节中Fos蛋白变化趋势同脊髓背角(L3组 vs C3组,P=0.002;L7组 vs C7组,P=0.003;L14组vsC14组,P=0.002)。P2X_3在CCI—SN造模成功后3～14d,表达逐渐升高,主要在背根神经节表达。在脊髓背角药物组各时间点P2X_3阳性细胞表达均较对照组对应时间点降低(L3组 vs C3组,P=0.043;L7组vs C7组,P=0.008;L14组 vs C14组,P=0.005)。在背根神经节鞘内给药后,各时间点P2X_3阳性细胞表达明显减少(L5组 vs C3组,P=0.054;L7组 vs C7组,P=0.001;L14组 vs C14组,P=0.003)。结论对于NPP大鼠,CCI—SN后脊髓背角和背根神经节中Fos蛋白和P2X_3受体表达均明显增强。LXM—10鞘内给药可明显降低二者表达。

Objective To investigate the changes of Fos protein and P2X~ receptor＇s expression in rats with neuropathic pain in spinal cord dorsal horn and dorsal root ganglion （DRG） after intrathecal injection of new analgesic drugs LXM- 10. Methods One hundred and eight adult SD rats were randomly divided into three groups （n=36 each）： the sham operation group （S group）, the control group （C group） and the LXM-10 group （L group）. The control group and XM-10 group were made into the chronic constriction injury of the sciatic nerve（CCI-SN） model. The operation group, control group and XM-10 group had been divided into three sub-groups （n-12 each） according to the injection time points. On the day 1, 5, 12 after CCI-SN, control group and XM-10 group were subarachnoid injected with corresponding drugs： normal saline 10pl for C3 group, C7 group, C14 group, or LXM-10 6.0pg/kg for L3 group, L7 group and L14 groupcontinuous 3 days, respectively. The sham operation group were intrathecally injected with normal saline 10pl for S3 group, S7 group, S14 group. After intrathecally administrating relevant drugs （normal saline or LXM-10） for 3 days, the rats were sacrificed in two hours after the last administration, and their spinal cord and L4-6 DRG were collected immediately. The expression of Fos protein and P2X3 receptor were measured with immunohistochemistry tests. Results The expression of Fos protein in the control group at each time point increased with time, and it was mainly expressed in the spinal cord dorsal horn. Compared with the control group at each time point,the Fos-like immunoreactivity（F-LI） cells in LXM-10 group decreased significantly. （The L3 group compared with the C3 group, P=0.003; the L7 group compared with the C7 group, P=0.023; the L14 group compared with the C14 group, P〈0.005.） The changes of Fos protein expression in DRG was similar to relevant spinal cord. （The L3 group compared with the C3 group, P=0.002; the L7 group compared with the C7 group, P=0.003; the L14 group compared with the C14 group, P=0.002.） The expression of P2X3 was up-regulated in all CCI-SN groups. The P2X3 positive expression could be easily observed in L4-6 DRG. Compared with the control group, the expression of P2X3 positive cells in the LXM-10 group at each time point decreased in the spinal cord dorsal horn. （L3 group compared with C3 group, P=0.043; L7 group compared with C7 group, P=0.008; L14 group compared with C14 group, P=0.005.） After intrathecal administration in DRG, the expression of P2X3-positive cells significantly reduced at each time point. （L3 group compared with C3 group,P=0.034; L7 group compared with C7 group, P=0.001; L14 group compared with C 14 group, P=0.003.） Conclusion For the neuropathic pain rats, after CCI of rats＇ sciatic nerve, both Fos protein and P2X3 receptor＇s expression were up-regulated in the dorsal horn of spinal cord and DRG. Intrathecally administrated LXM-10 could significantly inhibit the expression of Fos and P2X3 proteins. Intrathecal administration LXM-10 can significantly reduce the expression of the two.