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
Acute lung injury (ALI) is a severe condition often accompanied by extensive inflammation, increased autophagy, reduced phagocytosis, and excessive production of inflammatory mediators by pulmonary macrophages. These factors contribute to pulmonary edema and worsen the condition. Autophagy, a cellular process that degrades and recycles damaged organelles and proteins, plays a dual role in cell survival and death. While moderate autophagy protects cells from stress conditions like hypoxia and starvation, excessive autophagy can lead to cell death. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have emerged as a potential therapeutic option due to their ability to repair injured tissues and regulate autophagy. This study investigates the role of BM-MSCs in modulating autophagy in RAW264.7 macrophages under oxygen-glucose deprivation/restoration (OGD/R) conditions and explores the underlying signaling pathways.
The study established a co-culture system of RAW264.7 macrophages with BM-MSCs under OGD/R conditions to mimic ischemia/reperfusion injury (IRI). Autophagy was assessed using recombinant adenovirus (Ad-mCherry-GFP-LC3B) to visualize autophagic activity. Western blotting was employed to measure autophagy-related proteins, including light chain 3 (LC3)-I, LC3-II, and p62. Additionally, microarray expression analysis was conducted to identify differentially expressed genes, with a focus on heme oxygenase-1 (HO-1), a downstream molecule of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.
Under OGD/R conditions, RAW264.7 cells exhibited increased autophagy, as evidenced by a higher LC3-II/LC3-I ratio (1.27 ± 0.20 vs. 0.44 ± 0.08) and decreased p62 expression (0.77 ± 0.04 vs. 0.95 ± 0.10). Co-culture with BM-MSCs significantly reduced the LC3-II/LC3-I ratio (0.68 ± 0.14 vs. 1.27 ± 0.20) and upregulated p62 expression (1.10 ± 0.20 vs. 0.77 ± 0.04). These findings suggest that BM-MSCs can downregulate autophagy in macrophages under OGD/R conditions.
The study also explored the role of the PI3K/Akt signaling pathway in autophagy regulation. Under OGD/R conditions, the expression of PI3K (0.40 ± 0.06 vs. 0.63 ± 0.10) and the p-Akt/Akt ratio (0.39 ± 0.02 vs. 0.58 ± 0.03) were significantly reduced. Co-culture with BM-MSCs upregulated PI3K expression (0.54 ± 0.05 vs. 0.40 ± 0.06) and the p-Akt/Akt ratio (0.52 ± 0.05 vs. 0.39 ± 0.02). These results indicate that BM-MSCs activate the PI3K/Akt pathway to modulate autophagy in macrophages.
To further confirm the involvement of the PI3K/Akt pathway, RAW264.7 cells were treated with LY294002, a PI3K inhibitor. Pre-treatment with LY294002 blocked the BM-MSC-mediated downregulation of autophagy, as evidenced by an increased LC3-II/LC3-I ratio (1.29 ± 0.15 vs. 0.89 ± 0.08). This confirms that BM-MSCs regulate autophagy through the PI3K/Akt pathway.
Microarray expression analysis identified HO-1 as a differentially expressed gene downstream of the PI3K/Akt pathway. HO-1 mRNA and protein expression were upregulated in RAW264.7 cells co-cultured with BM-MSCs under OGD/R conditions (mRNA: 0.89 ± 0.17 vs. 0.40 ± 0.07; protein: 0.70 ± 0.09 vs. 0.48 ± 0.02). This suggests that HO-1 plays a role in the BM-MSC-mediated regulation of autophagy.
The study also identified other autophagy-related genes, such as Mapk3 (extracellular regulated protein kinases 1/2, ERK1/2) and Bnip3/Bnip3l (Nip3-like protein X, Nix), which may implicate additional signaling pathways like the mitogen-activated protein kinase/ERK and hypoxia-inducible factor-1a/Bnip3/Beclin-1 pathways. These findings highlight the complexity of autophagy regulation and suggest that multiple pathways may be involved.
In conclusion, this study demonstrates that BM-MSCs modulate autophagy in RAW264.7 macrophages under OGD/R conditions via the PI3K/Akt/HO-1 signaling pathway. The findings provide insights into the mechanisms underlying BM-MSC-mediated autophagy regulation and suggest potential therapeutic targets for ALI. Further research is needed to explore the communication between BM-MSCs and macrophages, the role of HO-1 in autophagy regulation, and the involvement of other signaling pathways.
doi.org/10.1097/CM9.0000000000001133
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