Dynamic changes in Beclin-1, LC3B and p62 at various time points in mice with temporary middle cerebral artery occlusion and reperfusion (tMCAO).

ABSTRACT

Ischaemic stroke is attributable to cerebrovascular disease and is associated with high morbidity, disability, mortality and recurrence. Autophagy is a critical mediator and plays dual roles in ischaemic stroke. Autophagy can protect against ischaemic brain injury during the early stage of ischaemic stroke, while excessive autophagy can induce apoptosis and exacerbate brain injury. However, the time-dependent variations in autophagy in ischaemic stroke are unknown. C57BL/6 mice were used to establish a model of temporary middle cerebral artery occlusion and reperfusion (tMCAO). The neurological functional scores and infarct volumes were determined at 1 d, 3 d, 5 d, and 7 d after modelling. The levels of Beclin-1, LC3B, p62, GFAP, TNF-α, IL-6, IL-10, ROS, 4-HNE and 8-OHDG were measured by ELISA, RT-PCR, immunofluorescence analysis and western blotting. The morphology of autophagosomes of ischaemic penumbra was observed by transmission electron microscopy (TEM). Beclin-1, LC3B, ROS, 4-HNE, 8-OHDG, GFAP, TNF-α and IL-6 levels increased (P < 0.01), while p62 and IL-10 levels decreased (P < 0.01) after tMCAO compared to those in the sham group. Beclin-1, LC3B, ROS, 4-HNE, 8-OHDG, GFAP, TNF-α and IL-6 levels were reduced in tMCAO mice at 3 d, 5 d and 7 d (P<0.05), and p62 and IL-10 levels were enhanced (P < 0.05) compared to those at 1 d. In addition, Beclin-1 positively correlated with LC3B, GFAP, TNF-α, IL-6, ROS, 4-HNE and 8-OHDG (P < 0.05), and Beclin-1 negatively correlated with p62 and IL-10 (P < 0.05). The number of autophagosomes was consistent with the expression of autophagy marker proteins, both showing a steady decrease. In summary, autophagy was activated within 7 d of tMCAO induction, and it strengthened at 1 d and then weakened steadily from 3 to 7 d. In addition, this study verified that autophagy positively correlated with the inflammatory response and oxidative stress at 7 d after tMCAO.