Identification and validation of metformin protects against PM-induced macrophages cytotoxicity by targeting toll like receptor pathway.

Fine particle matter (PM) has been extensively reported to contribute to the pathogenesis of pulmonary diseases. Recently, metformin has been reported to attenuate PM associated respiratory and cardiovascular injury, but the underling mechanism has not been discovered. Here, we performed comprehensively bioinformatics analysis and fully validation experiment to investigate the protection role of metformin and underling mechanism with RNAseq profile in GEO database. A combination of various bioinformatics tools including edgeR, principal component analysis (PCA), K-Means clustering, Gene Set Enrichment Analysis (GSEA), GO and KEGG enrichment were performed to identify the TLRs/MyD88/NF-κB axis functional as the key signaling transduction during PM associated toxicity. PM activated TLRs/MyD88/NF-κB pathway and resulted in significantly generation of IL-6, TNF-α, mitochondrial damage, decreasing of cell viability and increased LDH activity in RAW264.7 cells. Metformin significantly attenuated the production of IL-6, mitochondrial damage, cell viability and LDH activity by limiting TLRs/MyD88/NF-κB pathway. The siRNA against AMPKα2 or negative control were transfected to RAW264.7 cells to identify whether metformin protects PM-induced cytotoxicity in an AMPKα2-dependent manner. Pretreatment with metformin significantly attenuated PM induced decreasing of cell viability and increased LDH activity, as well as inhibited the TLRs/MyD88/NF-κB pathway in both siControl or siAMPKα2 cells. Taken together, our results indicate that metformin protects against PM-induced mitochondrial damage and cell cytotoxicity by inhibiting TLRs/MyD88/NF-κB signaling pathway in an AMPKα2 independent manner.