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Abstract：
BACKGROUND: Inadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated.
METHODS: The proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot.
RESULTS: Metformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK).
CONCLUSIONS: Metformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.
METHODS: The proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot.
RESULTS: Metformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK).
CONCLUSIONS: Metformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.
摘要：
背景：神经再生不足和抑制性局部微环境是脊髓损伤（SCI）修复的主要障碍。内源性神经干细胞（NSC）的活化和分化命运调节是最有前途的修复方法之一。二甲双胍的抗氧化作用已被广泛研究,抗炎,抗衰老,和自噬调节特性在中枢神经系统疾病。然而,二甲双胍对内源性神经干细胞的影响尚待阐明。
方法：用CCK-8法评价神经干细胞的增殖和分化能力,EdU/Ki67染色和免疫荧光染色。采用WesternBlot和免疫荧光染色检测NSC中铁凋亡相关关键蛋白表达的变化。活性氧的水平，使用相应的测定试剂盒测量谷胱甘肽和组织铁。用透射电镜和JC-1荧光探针观察线粒体形态和膜电位的变化。通过BBB评分评估大鼠SCI后的运动功能恢复，LSS得分,CatWalk步态分析,和电生理测试。使用Western印迹检查AMPK途径的表达。
结果：二甲双胍在体外和体内均能促进神经干细胞的增殖和神经元分化。此外，在体外建立了使用erastin处理的神经干细胞的铁凋亡模型，二甲双胍治疗可以逆转关键的铁凋亡相关蛋白表达的变化，增加谷胱甘肽合成，减少活性氧的产生，改善线粒体膜电位和形态。此外，服用二甲双胍可改善SCI后大鼠的运动功能恢复和组织学结局。值得注意的是,加入化合物C后，二甲双胍的所有上述有益作用均完全消除，AMP激活蛋白激酶（AMPK）的特异性抑制剂。
结论：二甲双胍，由典型的AMPK依赖性调节驱动，促进内源性神经干细胞的增殖和神经元分化，同时抑制铁凋亡，从而促进SCI后运动功能的恢复。我们的研究进一步阐明了二甲双胍在SCI中的保护机制，为其作为SCI治疗剂的候选资格提供了新的机制见解。
方法：用CCK-8法评价神经干细胞的增殖和分化能力,EdU/Ki67染色和免疫荧光染色。采用WesternBlot和免疫荧光染色检测NSC中铁凋亡相关关键蛋白表达的变化。活性氧的水平，使用相应的测定试剂盒测量谷胱甘肽和组织铁。用透射电镜和JC-1荧光探针观察线粒体形态和膜电位的变化。通过BBB评分评估大鼠SCI后的运动功能恢复，LSS得分,CatWalk步态分析,和电生理测试。使用Western印迹检查AMPK途径的表达。
结果：二甲双胍在体外和体内均能促进神经干细胞的增殖和神经元分化。此外，在体外建立了使用erastin处理的神经干细胞的铁凋亡模型，二甲双胍治疗可以逆转关键的铁凋亡相关蛋白表达的变化，增加谷胱甘肽合成，减少活性氧的产生，改善线粒体膜电位和形态。此外，服用二甲双胍可改善SCI后大鼠的运动功能恢复和组织学结局。值得注意的是,加入化合物C后，二甲双胍的所有上述有益作用均完全消除，AMP激活蛋白激酶（AMPK）的特异性抑制剂。
结论：二甲双胍，由典型的AMPK依赖性调节驱动，促进内源性神经干细胞的增殖和神经元分化，同时抑制铁凋亡，从而促进SCI后运动功能的恢复。我们的研究进一步阐明了二甲双胍在SCI中的保护机制，为其作为SCI治疗剂的候选资格提供了新的机制见解。
