| Inflammation is a common and important pathological process, which can occur in various parts of the body’s tissues and organs. In the traditional view, inflammation refers to the defensive reaction of tissues with vessels to the damage factors. Trauma and most infectious diseases are inflammatory diseases, such as folliculitis, tonsillitis, pneumonia, nephritis, hepatitis, etc. The essential of inflammation is defense, primarily characterized by alteration, exudation and proliferation in the pathological aspect, and the main clinical manifestations is red, swollen, pain and so on. In recent years, many diseases that were thought to be irrelevant with inflammation including multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, type II diabetes, have been shown a close relation of inflammation to their pathogenesis. Therefore, regulating inflammation is regarded as an urgent research priority.NLRP3 inflammasome, one of the NLRs inflammasome family, is currently the most widely studied. The NLRP3 inflammasome is mainly expressed in the mononuclear macrophages. In recent years, inflammasomes have become a hot topic, because of its key role in various inflammatory diseases. Macrophages are important presenting cells in the immune system, playing an important role in both specific and nonspecific immunity and serving as an important object in the field of phagocytosis, cellular and molecular immunology. Macrophages are white blood cell organization, derived from by bone marrow stem cells. Macrophages are easy to be obtained, cultured and purified. In recent years, the role of macrophages in the systemic inflammatory response and NLRP3 inflammsome has attracted great attention.Adrenergic receptors (AR) are a kind of G protein coupled receptors (GPCRs), mediated by catecholamine. According to their different responses to norepinephrine, the adrenergic receptors can be sorted into a-adrenergic receptors and β-adrenergic receptors. P2 adrenergic receptors can modulate many physiological functions, such as activities of airway smooth muscle and vascular smooth muscle. β2 receptor is found to be express in macrophages, microglia, T cells, B cells and other immune cells. Stimulation of β2 receptors can affect the inflammatory response of these cells. In recent years, there are a large number of studies reporting that both short-acting and long-acting β2-AR agonists are capable of modulating inflammation of immune cells. Among these β2-AR agonists, salmeterol, exhibits the activity of anti-inflammation and has been used in clinical anti-inflammatory treatment of asthma and chronic obstructive pulmonary disease (COPD). However, there are no studies addressing the effects of β2-AR agonists in sepsis currently.In this study, we extracted and cultured primary bone marrow-derived macrophages (BMDM) from C57BL/6J mice and established inflammatory model in vitro, so as to investigate whether β2 receptor agonist salmeterol exerts an anti-inflammatory effect in the inflammatory response via regulating NLRP3 inflammasome. We also clarified its anti-inflammation roles in vivo by lipopolysaccharide (LPS) induced systemic inflammatory model in C57BL/6J mice. Our studies revealed a novel pharmacological effect of salmeterol, thus providing new ideas for the future treatment of systemic inflammation.Objective:To study the effect of β2 receptor agonist salmeterol on NLRP3 inflammation-mediated systemic inflammation and regulation mechanisms.Method:1. C57BL/6J mice were sacrificed and the primary bone marrow-derived macrophages were cultured, followed by LPS+ATP stimulate to establish the cellular inflammation model, while giving salmeterol (10-7 M～10-12 M) for protection at the same time, to clarify the effective concentration of anti-inflammatory effect of salmeterol.2. Protein immunoblotting (Western Blot) and enzyme-linked immunosorbent assay (ELISA) were used to detect the protein expression levels of the inflammatory component (NLRP3, caspase-1, IL-1β), to evaluate the salmeterol anti-inflammatory effect.3. On the above-mentioned in vitro culture of primary bone marrow-derived macrophages, we use LPS+ATP, poly (dA: dT), MDP, Flagellin, etc., tostimulate a variety of inflammasomes, while administering the effective concentration of salmeterol, then we use the Western Blot assay to detect the caspase-1, IL-1β expression levels, to determine whether the anti-inflammatory effect of salmeterol is mainly mediated by NLRP3 inflammasomes.4. We used cAMP detection kit for primary bone marrow-derived macrophages to detect the expression levels of cAMP tostudy the downstream signal pathways of anti-inflammatory effects of salmeterol.5. Western Blot was used in the detection of protein expression of β-arrestin 1 and β-arrestin 2.6. We interfered the expression of β-arrestin 1,2 by transferring siRNA into bone marrow-derived macrophages, to classify whether the anti-inflammatory effect of salmeterol is canceled.7. Co-immunoprecipitation (Co-IP) was used to observe the effect of salmeterol in the interaction of NLRP3 inflammasomes and β-arrestin 2.Results:1. In the primary BMDM cells, salmeterol (10-10 M～10-7 M) showed anti-inflammatory effects in a concentration-dependent manner. Furthermore, the anti-inflammatory effect of salmeterol is abolished by KH7, which is a cAMP inhibitor.2. Salmeterol reverse the inflammation caused by LPS+ATP, have no effect on the inflammation caused by poly(dA:dT) and Flagellin. So NLRP3 inflammasome played a major role in the anti-inflammatory effect of salmeterol.3. Salmeterol at 10-10M slightly elevated cAMP expression, higher concentration of salmeterol enhanced this effect.4. Salmeterol was able to reverse the down regulation of P-arrestin 2 in response to LPS+ATP. After knockdown of P-arrestin 2, the anti-inflammatory effect of salmeterol was suppressed.5. Salmeterol at 10-10M enhanced the interaction of β-arrestin 2 and NLRP3 protein.Conclusion:1. Salmeterol shows anti-inflammation effect via inhibiting NLRP3 inflammasome.2. High concentration (10-7 M) of salmeterol shows anti-inflammation effect through classical GPCR-cAMP pathway, and very low concentration (10-10 M) of salmeterol shows anti-inflammation effect through non-classical β-arrestin 2 pathway.The main innovations in this paper:1. We reveal the anti-inflammatory effect of β2-AR agonist salmeterol in systemic inflammation.2. This study demonstrates the different pathways of anti-inflammation effect in different concentrations of salmeterol. |
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