Berberine enhances autophagic flux to alleviate ischemic neuronal injury by facilitating N-ethylmaleimide-sensitive factor-mediated fusion of autophagosomes with lysosomes.

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.