The Role And Mechanism Of MiR-132 Increases The Cholinergic Anti-inflammatory Pathway In Alveolar Macrophages

Objective:To observe whether miR-132 can enhance the inhibitory effect of acetylcholine(ACh) on the lipopolysaccharides(LPS) induced inflammation in alveolar macrophages(AM), and explore the regulatory mechanism of miR-132 on AM inflammatory responses.Methods:AM were cultured in vitro, and were treated with LPS, LPS+ACh, LPS+ACh+Physostigmine(Phy); AM were transfected with miR-132 mimic or inhibitor, and were treated with LPS or LPS+ACh. Enzyme linked immunosorbent assay(ELISA)was used to detection the production tumor necrosis factortor-?(TNF-?),interleukin-1?(IL-1?) and interleukin-6(IL-6) in the supernatant of AM. Real time-qualified polymerase chain reaction(RT-qPCR) was used to detect the expression of miR-132 and acetylcholinesterase(AChE) in the cells. True Choline esterase assay kit was used to detect the activity of AChE in supernatant. Western Blot was used to detect the expression of AChE, nuclear factor-?B(NF-?B) p65,signal transducer and activator of transcription 3(STAT3), phosphorylated-STAT3(p-STAT3). Immunofluorescence was used to detect the nuclear translocation of NF-?B p65.Results:1. The levels of TNF-?, IL-1? and IL-6 were gradually increased at 3h, 6h, 12 h,24h after LPS treatment, compared with control groups. The expression of miR-132 gradually increased at 6h after LPS treatment. The levels of AChE mRNA, protein and activity were higher than those in control group at 12 h after LPS treatment. The level of AChE mRNA has no obvious changes at 24 h after LPS treatment while the levels of AChE protein and activity were reduce.2. 0.01 ?M, 0.1 ?M, 1 ?M ACh had less influence on the secretion of TNF-?, IL-1? and IL-6 induced by LPS, 10 ?M and 100 ?M ACh significantly reduced the inflammatory cytokines in the supernatant. The anti-inflammatory effects of ACh was significantly enhanced after Phy treatment.3. MiR-132 mimic or miR-132 inhibitor transfection does not affect AM inflammatory reaction induced by LPS. When AM were pretreated with ACh,overexpression of miR-132 can enhance the anti-inflammatory effects of ACh on LPS induced inflammation in AM, blockage of miR-132 can weaken the anti-inflammatory effects of ACh on LPS induced inflammation in AM.4. Transfection of miR-132 mimic or inhibitor had no effect on the level of AChE mRNA. MiR-132 mimic can inhibit the protein and activity levels of AChE,miR-132 inhibitor can increase the protein and activity levels of AChE. MiR-132 can enhance the inhibition effects of ACh on LPS induced NF-?B nuclear translocation in AM; MiR-132 can enhance the inhibition effects of ACh on LPS induced STAT3 activation in AM.Conclusion:MiR-132 can enhance the anti-inflammatory effect of ACh on LPS induced inflammation. The mechanism may be miR-132 targeted AChE in AM, enhanced the anti-inflammatory effect of ACh, which is related with suppression of NF-?B and STAT3 activation, thus inhibited the excessive activation of AM.