鞘内阻断趋化因子配体12/趋化因子受体4趋化因子轴对选择性神经损伤模型大鼠前扣带皮层中胶质细胞原纤维酸性蛋白质表达的影响

Effect of intrathecal blocking of chemokine ligand 12/chemokine receptor 4 chemokine axis on glial fibrillary acidic protein expression in anterior cingulate cortex in rats with spared nerve injury

目的观察鞘内注射趋化因子受体(CXCR)4抑制剂AMD3100外周阻断趋化因子配体(CXCL)12/CXCR4趋化因子轴对选择性神经损伤(SNI)模型大鼠前扣带皮层(ACC)中胶质细胞原纤维酸性蛋白(GFAP)表达的影响。方法将96只健康雄性Sprague-Dawley大鼠,随机分为假手术未治疗组、假手术治疗组、SNI未治疗组、SNI治疗组,每组24只。SNI未治疗组和SNI治疗组采用结扎坐骨神经分支建立疼痛模型,假手术未治疗组和假手术治疗组暴露但不结扎坐骨神经分支;假手术治疗组和SNI治疗组术后鞘内连续注射AMD3100 10μL(含AMD3100 1μg,每天1次,连续2周),假手术未治疗组和SNI未治疗组在同时间点鞘内注射等量0.9%氯化钠溶液。于SNI前和首次注药后7、14、21、28、35d应用von-Frey细丝测量大鼠患肢机械性刺激缩足阈值(PWT)。每组分别于首次注药后14、21、28、35d各处死4只大鼠,采用Western印迹法检测ACC中GFAP蛋白质表达水平;每组分别于首次注药后14、28d各处死4只大鼠,采用免疫荧光法观察ACC中GFAP蛋白质表达情况。结果术前4组间PWT基础值的差异均无统计学意义(P值均>0.05),SNI未治疗组首次注药后7、14、21、28、35d的PWT值均显著低于假手术未治疗组和假手术治疗组同时间点(P值均<0.01),SNI治疗组首次注药后14、21、28、35d的PWT值均显著高于SNI未治疗组同时间点(P值均<0.01)。首次注药后28和35d,SNI未治疗组ACC中GFAP蛋白质相对表达量均显著高于假手术未治疗组和假手术治疗组同时间点(P值均<0.05),SNI治疗组ACC中GFAP蛋白质相对表达量均显著低于SNI未治疗组同时间点(P值均<0.05)。在首次注药后14d时,免疫荧光法观察4组大鼠ACC中GFAP蛋白质表达无明显异常;而在首次注药后28d时,SNI未治疗组大鼠ACC中GFAP蛋白质表达明显增多,主要表现为阳性细胞数增多,着色较深。结论鞘内阻断CXCL12/CXCR4趋化因子轴使SNI模型大鼠ACC中GFAP蛋白质表达下调,有效缓解神经病理性疼痛大鼠的疼痛症状。

Objective To observe the effect of periphery blocking of chemokine ligand 12/chemokine receptor 4 chemokine axis on the expression of glial fibrillary acidic protein （GFAP） in anterior cingulate cortex （ACC） in rats with spared nerve injury （SNI） through intrathecal injection of AMD3100. Methods Totally 96 healthy male Sprague-Dawle rats were randomly divided into 4 groups （n = 24）： sham untreated group, sham treated group, SNI untreated group and SNI treated group. Pain models were established by ligating the sciatic nerve in SNI untreated group and the SNI treated group. The sciatic nerve was exposed only in sham treated group and sham untreated group. AMD3100 （1 μg/10 pL）, a CXCR4 specific antagonist was intrathecally injected dailyfor two weeks in sham treated group and sham treated group. Meanwhile, normal saline （10 μL） was given to sham untreated group and SNI untreated group at the same time. The paw withdrawal threshold （PWT） to mechanical stimulation was measured with the von-Frey before SNI and 7, 14, 21, 28, and 35 days after the first injection. Four rats from each group were sacrificed on day 14, 21, 28, and 35 after the first injection. The expression of GFAP in ACC was detected by Western blotting and Results There were no significant differences in the baseline PWT among 4 groups （all P〉0. 05）. The PWT of the SNI untreated group were significantly lower than those of the sham untreated group and sham treated group on day 7, 14, 21, 28 and 35 after the first injection （all P〈0. 01）. The PWT of the SNI treated group were significantly higher than those of the SNI untreated group on day 14, 21, 28 and 35 after the first injection （all P〈0. 01）. On day 28 and 35 after the first injection, the relative expression of GFAP protein in ACC in the SNI untreated group were significantly higher than those in the sham untreated group and sham treated group on （all P〈0. 05）, and the relative expression of GFAP protein in SNI treated group were significantly lower than those in the SNI untreated group （all P〈0. 05）. The results of immunofluorescence showed that there were no significant changes in the expression ofGFAP in the four groups＇on the 14^th day after the first injection, and the expression of GFAP increased significantly on the 28th day after first injection in the sham untreated group, which was characterized by increased positive cells and deepened staining. Conclusion Intrathecal blocking of CXCL12/CXCR4 chemokine axis can down-regulate the GFAP expression in ACC in SNI rats and effectively alleviate pain symptoms in rats with neuropathic pain. （Shanghai Med J, 2018, 41 = 598-602）