Formononetin Enhances Autophagy Flux in The Penumbra of Cerebral Ischemia and Improves Nerve Damage

刺芒柄花素增强脑缺血半暗带自噬流改善神经损伤

Objective Formononetin(FOR),a traditional Chinese medicine,has been widely used for nerve protection and nerve function rehabilitation after cerebral stroke.However,the role of FOR in autophagic lysosome function in cerebral ischemiareperfusion damage has not been investigated.This study aimed to explore whether the therapeutic benefits of FOR were influenced by the regulation of autophagy flux.Methods Male Sprague-Dawley rats were separated into sham,model,and MCAO+FOR(30 mg/kg)groups after undergoing middle cerebral artery occlusion(MCAO)and ischemia-reperfusion(I/R).Then,the brain tissues in the ischemic penumbra were obtained to detect the proteins in autophagic/lysosomal pathway with antibodies of Beclin-1,LC3,SQSTM1/P62,Ubiquitin,LAMP-2,Cathepsin B(CTSB)and Cathepsin D(CTSD)by Western blot and immunofluorescence,respectively.Meanwhile,the therapeutic effectiveness was evaluated by measuring infarct volume,neurological impairments,and neuronal necrosis.Results The findings of this study demonstrate that FOR treatment exhibits a dual effect by enhancing the autophagic activities of Beclin-1 and LC3 in neurons,while simultaneously improving the autophagic clearance function,as evidenced by reinforced lysosomal activities of LAMP-2,CTSB,and CTSD,as well as reduced autophagic accumulation of Ubiquitin and P62 in the MCAO+FOR group compared to the MCAO group.Additionally,7 d of FOR treatment dramatically reduced neurological deficits,infarct volume,and neuronal death caused by cerebral ischemia.Conclusion These findings suggest that the neuroprotective mechanism of FOR therapy in accelerating recovery from ischemic stroke may involve the increase of autophagy flux in the penumbra.

目的刺芒柄花素(formononetin,FOR)常被用于脑卒中后的神经保护和神经功能康复,其在脑缺血再灌注(ischemia-reperfusion,I/R)损伤中的自噬溶酶体功能尚未见报道。本研究旨在探讨刺芒柄花素的治疗效果是否受到自噬流调节方式的影响。方法雄性Sprague-Dawley(SD)大鼠在接受大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)和I/R后被分为假组、模型组和MCAO+FOR(30 mg/kg)组。然后获得缺血半暗带的脑组织,分别用Beclin-1、LC3、SQSTM1/P62、Ubiquitin、LAMP-2、Cathepsin B、Cathepsin D抗体通过蛋白质印迹法(Western blot,WB)和双重免疫荧光检测自噬/溶酶体通路中的关键蛋白质。同时评估脑梗死体积、神经功能损伤和神经元坏死情况,以评价FOR的干预效应。结果研究表明,与MCAO组相比,FOR干预除了能促进神经元中Beclin-1和LC3的自噬活性外,还能够改善自噬清除功能,如LAMP-2、Cathepsin B、Cathepsin D的溶酶体活性增强,以及Ubiquitin和P62的自噬积累减少。另外,FOR干预7 d后显著减少了脑缺血导致的神经功能缺损、梗死体积以及神经元死亡。结论FOR的干预治疗可能是通过增强自噬流促进脑缺血半暗带恢复的神经保护机制。