Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions

Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.