Zhuang Wenting, Huang Zhiwen, Yu Liling, Yu Meilin, He Hongyun, Deng Yihao
School of Basic Medical Sciences, Kunming University of Science and Technology, Kunming 650500, China.
School of Basic Medical Sciences, Kunming University of Science and Technology, Kunming 650500, China.
Biochem Pharmacol. 2025 Feb;232:116715. doi: 10.1016/j.bcp.2024.116715. Epub 2024 Dec 11.
Our previous study demonstrated that Berberine (BBR) significantly enhances autophagic flux, alleviating ischemic neuronal injury by restoring autolysosomal function, but how BBR augmented autolysosomal functions remained elusive. N-ethyl-maleimide sensitive factor (NSF) is considered as a major ATPase to reactivate soluble NSF attachment protein receptors (SNAREs), which directly mediate autophagosome-lysosome fusion. However, NSF was dramatically inactivated by ischemia to hamper membrane-membrane fusion, leading to autophagic/lysosomal dysfunction in neurons. This study was to investigate whether BBR-ameliorated autophagic flux was exerted by reinforcing NSF activity, which subsequently boosted autophagosome-lysosome fusion in ischemic neurons. Rat model of ischemic stroke and neuronal ischemia model of HT22 cells were prepared by middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD), respectively. BBR was intraperitoneally administrated with 100 mg/Kg/d for 3 days before MCAO and was treated with 90 μM in HT22 neurons for 12 h, respectively. The results illustrated that NSF activity was markedly reinforced to facilitate autophagosome-lysosome fusion in penumbral cells and OGD HT22 neurons by BBR treatment. Consequently, the ischemia-created autophagic/lysosomal dysfunction was greatly restored to alleviate ischemic injury. Thereafter, NSF activity in OGD HT22 neurons was altered by transfection with NSF-overexpressing lentiviruses and siRNA-mediated knockdown, respectively. The data showed that BBR-enhanced autophagic flux and it-induced neuroprotection were greatly counteracted by NSF knockdown. By contrast, NSF overexpression synergistically boosted autophagosome-lysosome fusion and further attenuated neuronal death upon BBR treatment. Therefore, our study indicates that BBR-conferred neuroprotection against ischemic stroke is induced through facilitating autophagosome-lysosome fusion, by which enhancing autophagic flux in ischemic neurons.
我们之前的研究表明，黄连素（BBR）可显著增强自噬通量，通过恢复自溶酶体功能减轻缺血性神经元损伤，但BBR增强自溶酶体功能的机制仍不清楚。N-乙基马来酰亚胺敏感因子（NSF）被认为是一种主要的ATP酶，可重新激活可溶性NSF附着蛋白受体（SNAREs），后者直接介导自噬体-溶酶体融合。然而，NSF在缺血时会显著失活，从而阻碍膜-膜融合，导致神经元自噬/溶酶体功能障碍。本研究旨在探讨BBR改善自噬通量是否是通过增强NSF活性来实现的，而NSF活性增强随后会促进缺血神经元的自噬体-溶酶体融合。分别通过大脑中动脉闭塞（MCAO）和氧-葡萄糖剥夺（OGD）制备了缺血性中风大鼠模型和HT22细胞的神经元缺血模型。在MCAO前3天，BBR以100mg/Kg/d的剂量腹腔注射，在HT22神经元中分别用90μM处理12小时。结果表明，BBR处理可显著增强NSF活性，促进半暗带细胞和OGD处理的HT22神经元中的自噬体-溶酶体融合。因此，缺血导致的自噬/溶酶体功能障碍得到极大恢复，从而减轻缺血性损伤。此后，分别通过转染NSF过表达慢病毒和siRNA介导的敲低来改变OGD处理的HT22神经元中的NSF活性。数据显示，NSF敲低可极大地抵消BBR增强的自噬通量及其诱导的神经保护作用。相比之下，NSF过表达可协同促进自噬体-溶酶体融合，并在BBR处理后进一步减轻神经元死亡。因此，我们的研究表明，BBR对缺血性中风的神经保护作用是通过促进自噬体-溶酶体融合来诱导的，从而增强缺血神经元中的自噬通量。
