| Backgrounds and Objectives: Inflammation is a fundamental physiological process that is crucial for survival, but at the same time is one of the major causes of human mobility and mortality. Recent studies indicate the vagus nerve can modulate the immune response and control inflammation through the cholinergic anti-inflammatory pathway. Acetylcholine, the principal neurotransmitter of the vagus nerve, can inhibit the production of pro-inflammatory cytokines from macrophages throughα7-nicotinic acetylcholine receptor. Nicotine, a more selective cholinergic agonist, is more efficient than acetylcholine at inhibiting the production of pro-inflammatory cytokines. A growing number of studies indicate theα7nAChRs are crucial to the regulation of systemic inflammation. However, the subcellular mechanism has remained unknown. Stimulation of macrophages with endotoxin induces the transcription of a number of pro-inflammatory cytokines and neither acetylcholine nor nicotine decreases the mRNA level of these cytokines, suggesting that nicotine controls the contents of these cytokines at post-transcriptional level. And as we all know, miRNAs are short, noncoding RNA species, which can repress target mRNA at post-transcriptional level. So our hypothesis is miRNAs might play a major role in the cholinergic anti-inflammatory pathway. The objective of our study is to investigate if miRNAs involved in the cholinergic anti-inflammatory pathway and study their possible anti-inflammatory mechanisms.Methods: The study was based on the model of LPS-induced inflammation. In animals and macrophage models, we used the means of whole animal and molecular biological experiment to investigate the role of miRNAs (miR-124a had been found) in cholinergic anti-inflammatory pathway. Then, we observed the effect of miR-124a on the expression of IL-6 and TNF-ααin vitro. Further more, we verified the anti-inflammatory effect of miR-124a through the over-expression of miR-124a or the knockdown of miR-124a in vivo. At last, we preliminarily studied the mechanism of the anti-inflammatory effect of miR-124a.Results:1. In the nicotinic anti-inflammation model of RAW264.7 macrophages, we found the characteristic of nicotine in anti-inflammatory effect, nicotine could down-regulate both the content of IL-6 mRNA and protein, but only reduce TNF-αprotein level. The expression level of miR-124a was obviously up-regulated afterα7nAChR was activatied. Then, we investigated the time-effect relationship of nicotine and observed the up-regulation of miR-124a is earlier than the down-regulation of IL-6 and TNF-α. Over-expression of miR-124a reduced the level of IL-6 and TNF-αprotein in a dose-depended way. Over-expression of miR-124a down-regulated the level of IL-6 mRNA but not the level of TNF-αmRNA. The anti-inflammatory effect of nicotine was attenuated after miR-124a was knocked down.2. In animal experiment, we vestigated the dosage of LPS to induce shock in Blab/c mice and the anti-shock dosage of nicotine. Then, we observed the expression of miR-124a was obviously up-regulated by nicotine in heart, spleen, brain, and serum. In vivo, miR-124a mimics extended survival time of endotoxemia mice and the anti-shock effect of nicotine was attenuated when miR-124a was knocked down.3. We constructed IL-6, TNF-αand STAT3 3'-UTR luciferase reporter, and observed miR-124a mimics markedly decreased STAT3 3'-UTR luciferase level, but has no effect on IL-6 or TNF-α3'-UTR luciferase reporter. In RAW264.7 macrophages, miR-124a mimics not only decreased the level of STAT3 protein, but also inhibited the up-regulation of p-STAT3 induced by LPS through the down-regulation of STAT3 protein expression. The same as miR-124a, nicotine could inhibit the up-regulation of p-STAT3 induced by LPS. And nicotine didn't affect the level of STAT3 mRNA. After STAT3 was knocked down in RAW264.7 macrophages, IL-6 induced by LPS was decreased both in protein level and in mRNA level, but TNF-αwas not affected neither in mRNA level nor in protein level. Further more, we observed IL-6 induced by LPS was down-regulated both in protein level and in mRNA level in STAT3 knockout MEF cells. The down-regulation effect of miR-124a mimics on IL-6 was attenuated after STAT3 was knocked down in RAW264.7 macrophages.4. TNF-αprotein stability was decreased not only by nicotine but also by miR-124a mimics.Conclusion: miR-124a was up-regulated by nicotine through activation ofα7nAChR, and miR-124a inhibited the production of IL-6 mRNA and protein by the down-regulation of STAT3 and p-STAT3. Repression of TNF-αby miR-124a was through another mechanism, which needed to be studied more.
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