Abstract
The mortality rate for patients experiencing sepsis is decreasing; however, an effective therapeutic strategy requires further investigation. Increasing evidence has supported the idea that dysregulated microRNAs (miR) participate in the development of sepsis. Meanwhile, macrophages are crucial players in various inflammatory responses and diseases. The objective of the current study was to investigate the associated molecular mechanisms of action of miR-15a-5p on inflammatory responses in lipopolysaccharide (LPS)-stimulated mouse macrophages and the macrophage cell line RAW264.7. RAW264.7 macrophages were stimulated with LPS for 4 h, and ELISAs were subsequently used to measure the expression levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, in RAW264.7 macrophages. The expression levels of miR-15a-5p in RAW264.7 macrophages were detected after the stimulation of LPS using reverse transcription quantitative-PCR. The results indicated that the IL-1β, IL-6, TNF-α and miR-15a-5p levels were significantly increased compared with the control group. The Target gene prediction database (TargetScan) and dual-luciferase reporter assays were subsequently employed, and TNF-α induced protein 3-interacting protein 2 (TNIP2) was confirmed as a direct target for miR-15a-5p. Additionally, it was found that the TNIP2 expression levels were decreased in RAW264.7 macrophages following LPS treatment compared with controls. The present study also examined the effects of miR-15a-5p inhibitor on inflammatory cytokine expression levels and the activation of the NF-κ signaling pathway. These results demonstrated that miR-15a-5p inhibitor reduced the secretion of inflammatory cytokines and inhibited NF-κ pathway activation by targeting TNIP2. This may be associated with the progression of sepsis. Meanwhile, a LPS-induced mouse model of sepsis was established to examine the regulation of TNIP2 and miR-15a-5p during inflammation. In the animal model, miR-15a-5p inhibitor significantly suppressed the secretion of inflammatory factors. The levels of creatin, blood urea nitrogen, aspartate aminotransferase and alanine aminotransferase in the serum of LPS-treated mice were also found to be decreased in the miR-15a-5p inhibitor treatment group, while the protective effects of miR-15a-5p inhibitor on sepsis were eliminated by TNIP2-small interfering RNA combination therapy. In conclusion, the present findings indicated that miR-15a-5p may be involved in the inflammatory process during sepsis by activating the NF-κ pathway and targeting TNIP2. This suggests that miR-15a-5p inhibitor may be a novel anti-inflammatory agent and therapeutic strategy for sepsis.