二甲双胍通过TET2-Foxo3a途径对脊髓损伤的抗凋亡机制研究

Metformin inhibits apoptosis by regulating TET2-Foxo3a pathway after spinal cord injury

目的通过组织学、RT-PCR及功能评估等实验方法,探讨二甲双胍对脊髓损伤大鼠抗凋亡的调控机制。方法建立大鼠脊髓损伤模型,通过Basso-Beattie -Bresnahan locomotor rating scale(BBB)评分和斜板试验评估大鼠后肢运动功能的恢复。通过TTC染色比较脊髓组织坏死面积的改变。分离提取大鼠脊髓组织,通过Dot-blot分析、免疫组化检测DNA的羟甲基化水平。通过qPCR、Western Blot检测TET2mRNA及蛋白表达水平,并通过抑制TET2的表达来检测脊髓损伤范围的改变。通过免疫印迹及免疫共沉淀检测TET2与Foxo3a的相互作用。通过RT-PCR分析检测Foxo3a下游相关基因表达水平的变化。结果与SCI+NS组相比,SCI+MET组使用二甲双胍治疗后脊髓组织的坏死面积明显减少,BBB评分及斜板试验评分更高(P<0.05)。同时我们发现与对照组相比,SCI大鼠24h后TET2mRNA和蛋白水平升高,DNA的5-hmC水平升高。而SCI后使用二甲双胍治疗,TET2mRNA和蛋白水平、5-hmC水平进一步升高。与SCI+MET组相比,使用了SC-1后DNA的5-hmC水平降低,坏死的面积较前增加。SCI后Bim,P27kip,Bax的mRNA水平显着增加。二甲双胍治疗后,Bim,Bax的mRNA水平较SCI+NS组降低(P<0.05)。SCI损伤后Bim,P27kip,Bax的相对5-hmC水平显着增加。二甲双胍治疗后Bim和Bax的相对5-hmC水平降低(P<0.05)。结论二甲双胍能促进TET2和Foxo3a的相互作用,提升整体DNA的5-hmC水平,抑制相关凋亡基因的表达,从而改善脊髓损伤后的组织损伤及神经功能恢复。

Objective Through TTC staining,immunohistochemical analysis,RT-PCR and hind limb motor function evaluation and other experimental methods,to explore the regulatory mechanism of metformin on anti-apoptosis in rats with spinal cord injury(SCI).Methods Establish a rat spinal cord injury model.Through Basso-Beattie-Bresnahan locomotor rating scale(BBB)and cant test to evaluate the recovery of hindlimb motor function in rats.The changes of necrotic area of spinal cord tissue were compared by TTC staining.Extraction of rat spinal cord tissue,by Dot blot analysis and immunohistochemical detection of the hydroxyl of DNA methylation level.By qPCR,Western Blot detection TET2mRNA and protein expression level,and the changes in the scope of spinal cord injury were detected by inhibiting the expression of TET2.The interaction between TET2 and Foxo3a was detected by immunoblotting and immunoprecipitation.Through RT-PCR assay Foxo3a downstream related changes in the level of gene expression.Results Compared with the SCI+NS group,the necrotic area of the spinal cord tissue was reduced after metformin treatment,and the BBB score and the incline test score were higher(P<0.05).At the same time,we found that the levels of TET2mRNA and protein increased significantly after SCI at 24 h,and the 5-hmC level of DNA increased.The levels of TET2mRNA and protein and 5-hmC increased further after the use of metformin.After using SC-1,compared with the SCI+MET group,the level of 5-hmC decreased and the area of infarction increased.After SCI,the mRNA levels of downstream genes Bim,P27kip,Bax increased significantly.After metformin treatment,the mRNA levels of Bim and Bax were lower than those in the SCI+NS group(P<0.05).After SCI,the 5-hmC levels of downstream genes Bim,P27kip,Bax increased significantly.After metformin treatment,the 5-hmC levels of Bim and Bax were lower than those in the SCI+NS group(P<0.05).Conclusion Metformin can promote the interaction between TET2 and Foxo3a,increase the 5-hmC level of the overall DNA,and inhibit the activation of related apoptosis genes,thereby improving tissue damage and nerve function recovery after spinal cord injury.