miR-132 inhibits lipopolysaccharide-induced inflammation in alveolar macrophages by the cholinergic anti-inflammatory pathway.

OBJECTIVE: Although microRNA-132 (miR-132) has been shown to be involved in the inflammatory regulation, its role in sepsis-induced lung injury is unknown. We hypothesized that miR-132 attenuated lipopolysaccharide (LPS)-induced inflammation of alveolar macrophages by targeting acetylcholinesterase (AChE) and enhancing the acetylcholine (ACh)-mediated cholinergic anti-inflammatory response. METHODS: The LPS-treated rat alveolar macrophage cell line NR8383 was used as the inflammatory model. To assess the effect of miR-132, alveolar macrophages were transfected with miR-132 mimic or inhibitor. RESULTS: We found that miR-132 was upregulated in LPS-stimulated alveolar macrophages. Induction of AChE mRNA showed an inverse pattern with respect to AChE protein and activity, suggesting posttranscriptional regulation of AChE. Utilizing miR-132 mimic transfection, we found that overexpression of miR-132 enhanced the ACh-mediated cholinergic anti-inflammatory reaction by targeting AChE mRNA in LPS-treated alveolar macrophages. Blockage of miR-132 using miR-132 inhibitor reversed the Ach action upon LPS-induced release of inflammatory mediators and reduction in AchE protein/activity. Moreover, in the presence of ACh, upregulation of miR-132 suppressed LPS-induced nuclear translocation of NF-κB and production of STAT3 and phosphorylated STAT3, while downregulation of miR-132 enhanced the nuclear translocation of NF-κB. CONCLUSION: We propose that miR-132 functions as a negative regulator of the inflammatory response in alveolar macrophages by potentiating the cholinergic anti-inflammatory pathway, and represents a potential therapeutic leverage point in modulating inflammatory responses.

[The effect of transfected microRNA-146a on expression of interleukin-1 receptor-associated kinase 1 and tumor necrosis factor receptor-associated factor 6 in alveolar macrophages].

OBJECTIVE: To observe the effect of transfected microRNA-146a (miR-146a) on expression of interleukin-1 receptor-associated kinase 1 (IRAK-1) and tumor necrosis factor receptor-associated factor 6 (TRAF-6) in alveolar macrophages, and to explore the regulatory mechanism of miR-146a in the inflammatory response of alveolar macrophages. METHODS: Alveolar macrophages NR8383 were cultured and divided into two groups: transfected miR-146a mimic group was transfected 50 nmol/L Pre-miR miR-146a precursors and the negative control group was transfected Cy3-labeled Pre-miR negative control. Cells were collected at 24 hours after transfection. The miR-146a and the mRNA expression of IRAK-1 and TRAF-6 were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR), and the protein expression of IRAK-1 and TRAF-6 was assayed by Western Blot. RESULTS: Compared with negative control group, the expression of miR-146a was upregulated by (24.55±6.14) fold compared with miR-146a mimic group (t=-9.353, P=0.001). The mRNA expressions of IRAK-1 and TRAF-6 in miR-146a mimic group were upregulated by (1.16±0.10) fold (t=2.701, P=0.054) and (1.19±0.16) fold (t=2.032, P=0.112) , respectively, compared with that of negative control group, but the protein levels of IRAK-1 and TRAF-6 were decreased by 73.0% (t=-9.353, P=0.001) and 64.1% (t=-6.839, P=0.002), respectively . CONCLUSIONS: miR-146a mimic was successfully transfected into the alveolar macrophage NR8383. The overexpression of miR-146a in alveolar macrophages can down-regulate the expression of IRAK-1 and TRAF-6 in protein translation levels, and its mechanism may be related with inhibition of protein translation.