Fig. 11

Autophagy activation reversed the protective effect of VD3 on alveolar epithelial cells against hypoxic injury. The cells were divided into 5 groups: normoxia group, hypoxia group, hypoxia + VD3 group, hypoxia + VD3 + mitochondrial autophagy inhibitor (5 μM Mdivi-1) group and hypoxia + VD3 + mitochondrial autophagy agonist (50 μM CCCP) group. A mRNA levels of LC3B-II, p62, and Beclin-1 were determined through RT-qPCR. B and C The expression of LC3 I, LC3 II, p62, and Beclin-1 in lung were assessed using Western blot analysis. Load control: β-actin. D and E PINK1, Parkin, and Mfn1 protein expression were measured by Western blot analysis. COXIV, mitochondria loading control. F LDH activity was measured at 490 nm using the LDH cytotoxicity kit. G Expression levels of cellular supernatant TNF-α, IL-6 and IL-1β were determined by ELISA. H and I The expression of C3, C3a, and C5 in type II alveolar epithelial cells was analyzed by Western blot. Load control: β-actin. Means and standard deviations (SD) were used to represent the data. Statistical analyses (two group comparisons) were performed using Student t-test. A one-way ANOVA with Tukey post hoc test of means was used for multiple group comparisons. ^** P < 0.01 vs Normoxia, ^# P < 0.05 vs Hypoxia, ^## P < 0.01 vs Hypoxia, ^&P < 0.05 vs Hypoxia + VD3, ^&&P < 0.01 vs Hypoxia + VD3