蛛网膜下腔注射SRT1720对慢性坐骨神经结扎大鼠脊髓SIRT1表达和NF-κB去乙酰化调节的影响

Effects of intrathecal SRT1720 injection on expressions of SIRT1 and deacetylation of NF- κB in spinal cord in rats with chronic constriction injury

目的：探讨蛛网膜下腔注射SRT1720对慢性坐骨神经结扎（CCI）大鼠脊髓沉默信息调节因子2相关酶1（SIRT1）表达和活性的影响，以及对NF-κB p65去乙酰化的调节作用。方法将120只雄性SD大鼠采用随机数表法分成5组，每组24只，即假手术组（S）组、坐骨神经结扎组（CCI组）、二甲亚砜（DMSO）组、坐骨神经结扎＋低剂量SRT1720治疗组（CCI＋LD组）和坐骨神经结扎＋高剂量SRT1720治疗组（CCI＋HD组）。其中CCI、DMSO、CCI＋LD和CCI＋HD组大鼠提前8d蛛网膜下腔预置给药导管后建立CCI模型。DMSO组从手术日起，每天通过蛛网膜下腔预置细管给予10μl DMSO，而CCI＋LD组和CCI＋HD组每天分别给予SRT172010μg和20μg（溶于10μl DMSO）。在CCI前1d、CCI后1、3和7d各组分别处死大鼠6只，取L4~5段手术侧脊髓组织，采用荧光法测定沉默信息调节因子2相关酶1（SIRT1）活性，Western blot法测定SIRT1、NF-κB、乙酰化- NF-κB p65（Lys310）水平，并计算各组NF-κB的乙酰化水平（乙酰化- NF-κB p65/NF-κB）。结果与S组比较，CCI和DMSO组在CCI后各时点SIRT1水平和酶活性均显著下降（均P＜0.01），相反，NF-κB、乙酰化- NF-κB p65水平和NF-κB乙酰化水平均显著升高（均P＜0.01）；而与 CCI组比较，CCI＋LD和CCI＋HD组在CCI后3d、7d SIRT1水平和酶活性均较高（均 P＜0.01），NF-κB、乙酰化- NF-κB p65（Lys310）水平则较低。其中SRT1720高剂量组在CCI后7d，SIRT1水平和酶活性显著高于低剂量组（P＜0.01），而NF-κB的乙酰化水平则更低（P＜0.01）。结论 SRT1720蛛网膜下腔给药可改善CCI导致的脊髓SIRT1水平和酶活性的降低，抑制NF-κB p65的过高表达和乙酰化。

Objective To investigate the effect of intrathecal SRT1720 injection on expression and activity of SIRT1, and deacetylation of NF- κBp65 in rats with chronic constriction injury （CCI）. Methods One hundred and twenty male SD rats were randomly divided into five groups with 24 animals in each group：Sham group （S group）, CCI group, dimethylsulfoxide （DMSO） group, CCI＋LD group and CCI＋HD group. Lumbosacral intrathecal （IT） catheter was placed 8d before CCI operation in DMSO, CCI＋LD and CCI＋HD groups. CCI was induced in al animals except those in S group. 10μl DMSO was intrathecal y injected q.d via the catheter in DMSO group; 10μg and 20μg SRT1720 dissolved in 10μl DMSO was given to rats in CCI＋LD and CCI＋HD groups. Six rats were sacrificed randomly before and at 1, 3, 7 d after CCI or sham surgery in each group. The L4~5 spinal cord segments were removed for detection of deacetylase activity of SIRT1 by fluorometric kit. The proteins levels of （SIRT1） and NF- κB/Ac- NF- κB p65 were determined by Western blot analysis. Results The deacetylase activity and protein levels of SIRT1 were significantly decreased after operation in CCI and DMSO group at postoperative d1, 3 and 7, and the proteins level of NF- κB/Ac- NF- κB p65 were significantly increased（P〈0.01）, compared with S group（P〈0.01）. Compared with CCI group, the deacetylase activity and protein level of SIRT1 increased, and NF- κB/Ac- NF- κB p65 protein level decreased both in CCI＋LD group and CCI＋HD group at postoperative d3 and d7 （P〈0.01）. Furthermore, the activity and protein levels of SIRT1 were higher in CCI＋HD group and the proteins levels of Ac- NF- κB p65 were lower （P〈0.01） than those in CCI＋LD group at postoperative d7 （P〈0.01）. Conclusion SRT1720 intrathecal injection can improve the down- regulation of spinal SIRT1 deacetylase activity and protein levels in CCI rats, furthermore, it also can inhibit over- expression and acetylation of NF- κB p65.