SIRT2在大鼠脊髓缺血再灌注损伤中的作用

Effect of SIRT2 in rats spinal cord ischemia-reperfusion injury

目的探讨SIRT2对大鼠脊髓缺血再灌注损伤(spinal cord ischemia-reperfusion injury,SCII)的神经保护作用。方法实验分2部分,共54只SD雄性大鼠,第一部分实验动物共30只。应用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)及Western blot检测脊髓组织SIRT2随再灌注时间改变mRNA水平及蛋白水平表达变化。其中24只实验动物分为4组:空白对照组,缺血1 h再灌注后6 h、12 h和24 h组,每组6只。应用免疫荧光染色检测空白对照组及再灌注24 h组SIRT2表达及定位情况,实验大鼠共6只,每组3只。第二部分,SIRT2特异性抑制剂AGK-2的应用。将24只大鼠随机分为4组,进行4种不同处理,分别为假手术组(S组),DMSO组(P组),AGK-2 1 mmol/L组(T1组)、AGK-2 5 mmol/L组(T2组)。分别评价缺血后大鼠运动功能和脊髓氧化应激水平。结果再灌注6 h,12 h,24 h后,RT-PCR及Western blot结果显示随再灌注时间延长SIRT2基因及蛋白水平表达明显升高(P<0.05);行为学评分显示T2组神经损害更为严重(P<0.05),MDA水平T2组较S组明显升高,P组与T1组无明显差异。SOD活力与MDA水平相对应。结论 SIRT2随脊髓再灌注时间延长表达明显增加,表明SIRT2可能通过降低脊髓组织中的氧化应激水平减轻SCII。

Objective To research the neuroprotective effect of SIRT2 in rats with spinal cord ischemia-reperfusion injury （SCII） and the possible mechanism of this protective effect. Methods A total of 54 SD rats were divided into 2 groups. The first part of the 30 rats was randomly divided into the following subgroups： control, ischemia-reperfusion （I/R） after 6 h, 12 h and 24 h （6 rats per time point）. The levels of mRNA and protein expression of SIRT2 in spinal cord tissue were tested by reverse transcription-polymerase chain reaction （RT-PCR）and Western blot analysis. The SIRT2 expression and localiza- tion in the control group and in the I/R-24 h group was tested by irnmunofluoreseence staining （3 rats per group）. The second part, to investigate the effect of treatment with the specific SIRT2 inhibitor AGK-2, consisted of 24 rats, which were divided into 4 groups with different treatments ： sham group （ S group）, DMSO group （ P group）, AGK-2 at 1 mmol/L group （ T1 group）, and AGK-2 at 5 mmol/L group （＇12 group）. Neurological function and spinal cord oxidative stress levels after isehe- mia injury in the rats was evaluated separately. Results RT-PCR and Western blot analysis showed that after reperfusion 6 h, 12 h and 24 h post-ischemia, the mRNA and protein levels of SIRT2 in the spinal cord were significantly up-regulated （P 〈 0.05） as compared to the sham group. The behavioral scores display that the nerve damage in theT2 group was signifi- cantly more severe than in the P group （ P 〈 0.05 ）. MDA levels in the T2 group were significantly higher than in the S group （P 〈 0.05 ）, whereas P group and T1 group showed no significant differences （ P 〉 0.05 ）. SOD levels is corresponding to MDA levels. Conclusion SIRT2 expression increases significantly as the time between ischemia and reperfusion prolonging. SIRT2 plays an europrotective role in SCII, possibly through the reduction of oxidative stress in the spinal cord to relieve SCII.