PGC-1α/SIRT3信号通路在右美托咪定预处理大鼠脊髓缺血再灌注损伤中的作用及机制研究

The Role and Mechanism of PGC-1α/SIRT_(3) Signaling Pathway in Dexmedetomidine Pretreatment of Spinal Cord Ischemia-reperfusion Injury in Rats

目的:基于过氧化物酶体增殖物受体γ共激活因子1α(PGC-1α)/线粒体去乙酰化酶3(SIRT3)信号通路探讨右美托咪定预处理大鼠脊髓缺血再灌注损伤中的作用及机制。方法:将96只SPF级SD大鼠随机平均分为假手术组(sham组)、脊髓缺血再灌注组(模型组)、脊髓缺血再灌注+右美托咪定组(实验组)以及脊髓缺血再灌注+右美托咪定+PGC-1α阻断剂(DPA组),共四组,每组24只。采用开胸夹闭主动脉弓15min构建脊髓缺血再灌注损伤大鼠模型,sham组只开胸但不做夹闭处理。实验组与DPA组于建模前25min鞘内注射右美托咪定,DPA组同时注射20μmol/L SR-18292,sham组和模型组则注射等量生理盐水。分别于再灌注6、8、12和24h时每组随机抽取6只大鼠,评估大鼠后肢运动功能,采用脊髓运动功能(BBB)评分,HE染色观察大鼠脊髓病理学变化,细胞凋亡原位检测(TUNEL)脊髓组织细胞凋亡,并计算细胞凋亡指数(AI),蛋白免疫印迹(WB)法和qRT-PCR检测大鼠脊髓组织SIRT3、PGC-1α蛋白质和mRNA的表达。结果:与sham组相比,其余三组再灌注各时点BBB评分均显著降低(P<0.05);与模型组比较,实验组和DPA组再灌注各时点BBB评分显著升高(P<0.05);与实验组比较,DPA组再灌注各时点BBB评分显著降低(P<0.05)。与sham组比较,其余三组AI显著升高,神经元数量显著降低(P<0.05);与模型组比较,实验组和DPA组AI显著降低,神经元数量增加(P<0.05);与实验组比较,DPA组AI显著升高,神经元数量显著降低(P<0.05)。与sham组比较,模型组和DAP组PGC-1α和SIRT3 mRNA及蛋白质的表达量显著降低(P<0.05),实验组PGC-1α和SIRT3 mRNA表达量显著升高,PGC-1α蛋白质的表达量显著降低(P<0.05),SIRT3蛋白质的表达量差异不显著(P>0.05);与模型组比较,实验组和DPA组PGC-1α和SIRT3 mRNA及蛋白质的表达量显著增加(P<0.05);与实验组比较,DPA组PGC-1α和SIRT3 mRNA及蛋白质的表达量显著降低(P<0.05)。结论:右美托咪定预处理大鼠脊髓缺血再灌注损伤能够有效激活PGC-1α/SIRT3信号通路,并抑制大鼠脊髓神经细胞凋亡,促进神经运动功能恢复,改善大鼠脊髓组织的受损情况。

Objective:To investigate the role and mechanism of dexmedetomidine pretreatment in spinal cord ischemia-reperfusion injury in rats based on peroxisome proliferator-activated receptorγcoactivator 1α(PGC-1α)/mitochondrial deacetylase 3(SIRT3)signaling pathway.Methods:96 SPF SD rats were randomly divided into sham operation group(sham group),spinal cord ischemia-reperfusion group(model group),spinal cord ischemia-reperfusion+dexmedetomidine group(experimental group)and spinal cord ischemia-reperfusion+dexmedetomidine+PGC-1αblocker(DPA group),a total of four groups,24 rats in each group.A rat model of spinal cord ischemia-reperfusion injury was established by clamping the aortic arch for 15 minutes.The sham group only underwent thoracotomy without clamping.The experimental group and the DPA group were intrathecally injected with dexmedetomidine 25 minutes before modeling.The DPA group was also injected with 20μmol/L SR-18292.The sham group and the model group were injected with the same amount of normal saline.6 rats were randomly selected from each group at 6,8,12 and 24 hours after reperfusion.The motor function of the hind limbs of the rats was evaluated.The spinal cord motor function(BBB)score was used.The pathological changes of the spinal cord were observed by HE staining.The apoptosis of the spinal cord tissue was detected by TUNEL,and the apoptosis index(AI)was calculated.The expression of SIRT3 and PGC-1αprotein and mRNA in the spinal cord tissue of rats was detected by Western blotting(WB)and qRT-PCR.Results:Compared with the sham group,the BBB scores of the other three groups were significantly decreased at each time point of reperfusion(P<0.05).Compared with the model group,the BBB scores of the experimental group and the DPA group were significantly increased at each time point of reperfusion(P<0.05).Compared with the experimental group,BBB score of DPA group decreased significantly at each time point of reperfusion(P<0.05).Compared with the sham group,AI was significantly increased and the number of neurons was significantly decreased in the other three groups(P<0.05).Compared with the model group,the AI of the experimental group and the DPA group decreased significantly,and the number of neurons increased(P<0.05).Compared with the experimental group,AI was significantly increased and the number of neurons was significantly decreased in the DPA group(P<0.05).Compared with the sham group,the expression levels of PGC-1αand SIRT3 mRNA and protein in the model group and DAP group were significantly decreased(P<0.05).The expression levels of PGC-1αand SIRT3 mRNA in the experimental group were significantly increased,the expression of PGC-1αprotein was significantly decreased(P<0.05),and the expression of SIRT3 protein was not significantly different(P>0.05).The expression of PGC-1αand SIRT3 mRNA and protein was significantly increased in the experimental and DPA groups compared with the model group(P<0.05).Compared with the experimental group,the expressions of PGC-1αand SIRT3 mRNA and protein in DPA group were significantly decreased(P<0.05).Conclusion:Dexmedetomidine pretreatment of spinal cord ischemia-reperfusion injury in rats can effectively activate the PGC-1α/SIRT3 signaling pathway,and inhibit the apoptosis of spinal cord neurons in rats,promote the recovery of nerve motor function,and improve the damage of spinal cord tissue in rats.