Conditioned culture medium of bone marrow mesenchymal stem cells promotes phenotypic transformation of microglia by regulating mitochondrial autophagy

Abstract

Objective To study the mechanism by which conditioned medium of bone marrow mesenchymal stem cells (BMSCs-CM) facilitates the transition of pro-inflammatory polarized microglia to an anti-inflammatory phenotype. Methods BV2 cells, a mouse microglia cell line, were transformed into a pro-inflammatory phenotype using lipopolysaccharide. The expression of phenotypic genes in BV2 cells was detected using real-time quantitative PCR (RT-qPCR). Enzyme-linked immunosorbent assay was used to measure inflammatory cytokine levels in BV2 cells co-cultured with BMSCs-CM. The expressions of mitophagy-associated proteins were determined using western blot. The mitochondrial membrane potential and ATP levels in BV2 cells were measured using JC-1 staining and an ATP assay kit, respectively. Additionally, we examined the proliferation, apoptosis, and migration of C8-D1A cells, a mouse astrocyte cell line, co-cultured with BV2 cells. Results After co- culture with BMSCs -CM, the mRNA expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase significantly decreased in pro-inflammatory BV2 cells, whereas the expression of CD206 and arginase-1 significantly increased. Moreover, TNF-α and interleukin-6 levels significantly decreased, whereas transforming growth factor-β and interleukin-10 levels significantly increased. Furthermore, co-culture with BMSCs-CM increased mitophagy-associated protein expression, ATP levels, mitochondrial and lysosomal co-localization in these cells and decreased reactive oxygen species levels. Importantly, BMSCs-CM reversed the decrease in the proliferation and migration of C8-D1A cells co-cultured with pro-inflammatory BV2 cells and inhibited the apoptosis of C8-D1A cells. Conclusion BMSCs-CM may promote the transition of polarized microglia from a pro-inflammatory to an anti-inflammatory phenotype by regulating mitophagy and influences the functional state of astrocytes.

Keywords

• Conditioned medium
• Bone marrow mesenchymal stem cells
• Microglia
• Polarization
• Mitochondrial autophagy