过表达SIRT1对脊神经结扎大鼠病理性疼痛的影响及机制

Influence of SIRT1 over-expression on pathological pain in spinal nerve ligation rats

目的探讨过表达沉默信息调节因子1(SIRT1)对脊神经结扎(SNL)大鼠病理性疼痛的影响及机制。方法将大鼠随机分为假手术组、Cont-shRNA组、SIRT1-shRNA组,每组18只。采用L5脊神经结扎法制备SNL模型,假手术组仅暴露脊神经不进行结扎处理,经鞘内注射25μL生理盐水,每天注射1次,连续7d;Cont-shRNA、SIRT1-shRNA组SNL模型制备成功后鞘内分别注射25μL的Cont-shRNA、SIRT1-shRNA,均每天注射1次,连续7d。于第1、3、7天时,每组取6只,记录大鼠机械刺激缩足反应阈值(PWT)、热刺激缩足反应潜伏期所用时间(PTWL),采用免疫印记法检测脊髓组织中SIRT1、乙酰化组蛋白H3(Ac-H3)蛋白表达,免疫荧光染色法观察星形胶质细胞活化情况;ELISA法检测脊髓组织中TNF-α、IL-1β、IL-6表达;电镜下观察脊髓神经元超微结构,免疫印记法检测脊髓组织中凋亡相关蛋白表达。结果与假手术组相比,第1、3、7天Cont-shRNA组SIRT1蛋白表达降低(P均<0.05),Ac-H3蛋白表达升高(P均<0.05),PWT、PTWL降低(P均<0.05),胶质纤维酸性蛋白(GFAP)阳性细胞数量升高(P均<0.05);TNF-α、IL-1β、IL-6水平升高(P均<0.05);GFAP蛋白表达升高(P均<0.05),Bcl-2蛋白表达降低(P均<0.05),Bax、cleaved-Caspase-3蛋白表达升高(P均<0.05)。与Cont-shRNA组相比,SIRT1-shRNA组SIRT1蛋白表达升高(P均<0.05),Ac-H3蛋白表达降低(P均<0.05),PWT升高(P均<0.05),GFAP阳性细胞数量降低(P均<0.05);TNF-α、IL-1β、IL-6表达降低(P均<0.05);GFAP、Bax、cleaved-Caspase-3蛋白表达降低(P均<0.05),Bcl-2蛋白表达升高(P均<0.05)。结论过表达SIRT1可减轻SNL大鼠病理性疼痛,其机制可能与降低Ac-H3蛋白表达、抑制星形胶质细胞活化、降低神经元凋亡有关。

Objective To investigate the effect of silent information regulator 1 (SIRT1) over-expression on pathological pain in spinal nerve ligation (SNL) rats and its mechanism. Methods Rats were randomly divided into sham operation group, Cont shRNA group, and SIRT1 shRNA group with 18 rats in each group. SNL models were prepared by L 5 spinal nerve ligation, only spinal nerve was exposed without ligation in shamoperation group, and they were was intrathecally injected with 25 μL normal saline once a day for 7 consecutive days;after the preparation of the SNL models in the Cont shRNA and SIRT1 shRNA group, 25 μL of Cont shRNA and SIRT1 shRNA were intrathecally injected, once a day for 7 consecutive days. On the 1st, 3rd and 7th days, 6 rats were taken from each group, the expression of SIRT1 and acetylated histone H3 (Ac-H3) in the spinal cord tissues was detected by Western blotting, and mechanical pain threshold (PWT) and thermal pain threshold (PTWL) were measured to evaluate the behavioral changes in rats;the activation of astrocytes was observed by immunofluorescence staining;the expression of TNF-α, IL-1β and IL-6 in the spinal cord tissues was detected by ELISA;the ultrastructure of spinal cord neurons was observed under electron microscopy and the expression of apoptosis-related proteins was detected by immunoblotting. Results Compared with sham operation group, the expression of SIRT1 protein decreased ( P < 0.05), the expression of Ac H3 protein increased ( P < 0.05), the expression of PWT and PTWL decreased ( P < 0.05), the number of glial fibrillary acidic protein (GFAP) positive cells increased ( P < 0.05), the levels of TNFα, IL-1β and IL-6 increased ( P < 0.05), the expression of GFAP protein ncreased ( P < 0.05), Bcl 2 protein expression decreased ( P < 0.05), while Bax and cleaved Caspase 3 protein expression increased ( P < 0.05) in the Cont shRNA group on day 1, 3 and 7;compared with the Cont shRNA group, SIRT1 protein expression increased in the SIRT1 shRNA group ( P < 0.05), Ac H3 protein expression decreased ( P <0.05), and PWT increased ( P < 0.05 ), the number of GFAP-positive cells decreased ( P < 0.05);the expression of TNF-α, IL-1β and IL-6 decreased (all P < 0.05);the expression of GFAP, Bax and cleaved-Caspase 3 protein decreased ( P < 0.05), Bcl 2 protein expression increased ( P < 0.05). Conclusion Over-expression of SIRT1 can alleviate pathological pain in SNL rats, and the mechanism may be related to decreasing the expression of Ac-H3 protein, inhibiting the activation of astrocytes and decreasing the apoptosis of neurons.