The Mechanism And Inhibitory Effect Of Physiological Concentration Of Glucocorticoids On Myocardial Inflammation Induced By Lipopolysaccharides

BackgroundPharmacological dose of exogenous glucocorticoids (GCs) are clinically the most effective anti-inflammatory agents for the treatment of severe inflammatory diseases such as asthma and rheumatoid arthritis, however, it is limited in clinical application due to its side effects.It was generally believed that physiological concentration of glucocorticoid mainly regulated metabolism in the past. Recently studies found endogenous GCs also exert an anti-inflammatory effect. GCs act by binding to cytosolic glucocorticoids receptor (GR), which upon binging become activated and rapidly translocate to the nucleus to suppress the multiple inflammatory genes that are activated in chronic inflammatory diseases. Most inflammatory proteins are transcriptionally controlled by nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). It has been suggested that the transactivation of the pro-inflammatory transcription factor mainly depends on post-translational mechanisms and histone acetyltransferase (HAT) and histone deacetylase (HDAC) play a central role in modulating the activities of these transcription factors. Pro-inflammatory transcription factor bind to and activate co-activator molecules, which then acetylate core histones to switch on gene transcription. GCs suppress inflammation mainly by reversing histone acetylation of activated inflammatory genes and recruitment of HDAC to the activated transcription complex. Recent studies have reported that HDAC function are reduced in patients with chronic obstructive pulmonary disease (COPD) that respond poorly to GCs treatment. Oxidative stress produced by smoking can reduce HDAC activity and HDAC2 expression of lung tissue from patients with COPD and cancel exogenous GCs anti-inflammatory sensitivity. These findings indicated the molecular mechanism of exogenous GCs and GCs resistance in the controlling of lung inflammatory responses. Based on exogenous GCs'studies above-mentioned, we put forward a hypothesis whether endogenous, physiological concentration of GCs are also able to regulate inflammatory response, especially during the progression of myocardial inflammatory disorders . Our previous studies found average level of serum corticosterone in normal rats was approximately 127ng/ml. Through regulating the activity and expression of HDAC, endogenous GCs were able to inhibit myocardial inflammatory response induced by LPS in rats. However, the cellular and molecular anti-inflammatory mechanisms of endogenous GCs involved in the myocardial inflammatory disorders are poorly elucidated. Similarly, whether enhanced oxidative stress can down-regulate endogenous GCs function via inhibiting cardiac HDAC activity and expression and whether antioxidant, as a cardioprotective agent, may restore anti-inflammatory sensitivity of endogenous GCs through improving HDAC function, are not yet illustrated. To answer aforementioned questions, we firstly used immunohistochemistry approach to locate the exact cells involved in myocardial inflammation induced by LPS in rats. Our results showed that cardiac fibroblasts were a major cellular source for myocardial pro-inflammatory cytokines production in rats. To test cellular and molecular mechanisms of anti-inflammatory resistance of endogenous GCs, we applied xathine oxdiase/xathine (XO/X) system induced reactive oxygen species (ROS) and investigated the effect of physiological concentration of hydrocortistone and antioxidant allopurinol on inflammatory response stimulated by LPS in cultured adult rat cardiac fibroblasts.Part oneAnti-inflammatory effect and resistant mechanism of endogneous glucocorticoids on adult rat cardiac fibroblasts induced by lipopolysaccharide.ObjectiveTo investigate the role of endogenous GCs on myocardial inflammation of rats induced by LPS and locate the major target cells; to explore the mechanism of GCs resistance induced oxidative stress in vitro.MethodIn vivo, Male Sprague-Dawley rats were randomly divided into five groups: control, LPS-treated, lateral adrenalectomy (ADX), ADX+LPS-treated and ADX+ LPS-treated before GCs pre-treatment. Hearts were harvested for histological and immunohistochemical evaluation.In vitro, adult rat cardiac fibroblasts were isolated and cultured. After the cultured ARCFs were were treated with (1-104 ng/ml) LPS or 102 ng/ml LPS at different time points, TNF-α, IL-1βand IL-6 levels were measured by ELISA. After 24h of pre-treatment with XO/X system, ROS levels were measured by a ROS-specific probe, 2',7'-dichlorofluorescein diacetate (H2DCF-DA). ARCFs were pre-treated with XO/X system before incubated with 127ng/ml hydrocortisone, and then were treated with 102 ng/ml LPS, cytokines assay were measured by ELISA, HDAC activity by Fluoroimetric HDAC Activity Assay Kit, HDAC2 expression were detected by Western-blot.Result1. Cardiac fibroblasts were target cells and major production of pro-imflammatory cytokines in the heart induced by LPS in vivo.2. LPS stimulated TNF-α, IL-1βand IL-6 production in a time-effective and concentration-dependent manner; physiological concentration of GCs inhibited LPS-stimulated cytokines production of ARCFs. Xathine oxidase/xanthine system induced ROS production and reduced HDAC activity and HDAC2 expression in cultured ARCFs. Oxidative-stress weaken the inhibitive effect of physiological concentration of GCs on LPS-induced inflammatory response of ARCFs and antioxidant may restore GCs'anti-imflammatory functon.Brief summary1. Endogenous GCs maybe exert a cardioprotective effect through inhibiting myocardial inflammatory response. Cardiac fibroblasts were the target cells.2. Physiological concentration of GCs inhibited inflammatory response of cardiac fibroblasts induced by LPS.3.Oxidative stress caused the resistance of GCs anti-inflammatory effects by down-regulating ARCFs'HDAC activity and HDAC2 expression. Antioxidants may partly restore GCs anti-inflammatory function.Part twoPhysiological concentrations of glucocorticoids inhibit proliferation of adult rat cardiac fibroblasts induced by pro-inflammatory cytokines: roles of ERK1/2 and NF-κBObjectiveTo investigate the effect of physiological concentration of GCs on ARCFs proliferation induced by diverse inflammatory mediators and growth factors, and examined the implicated proliferation signaling pathways.Method1. After incubation with increasing concentration of TNF-α(10-105 pg/ml), IL-1β(10-104 pg/ml), PDTC (an antioxidant and inhibitor of NF-κB, 1μM, 30min pre-treatment) and U0126 ( an inhibitor of MEK1 activation, 100nM, 30min pre-treatment) for 48h, cell proliferation was examined by measuring relative obsorbance of CCK-8.2. The GR antagonist RU486 (100nM) were added to cells 30min before GCs addition (and therefore 60 min before mitogen stimulation), cell proliferation was examined by measuring relative obsorbance of CCK-8.3. ARCFs were pre-incubated with 127ng/ml hydrocortisone and 100nM U0126 for 30min before 24h of TNF-α(104pg/ml) stimulation, we measured the expression of cyclinD1 by Western-blot.4. ARCFs were treated with 104 pg/ml TNFαand IL-1βat different time points, respectively, the expression of p-ERK1/2, p-IκB, NF-κB were detected by Western-blot.Result1.Pro-inflammatory cytokines induced proliferation of ARCFs in a concentration-dependent manner.2.GCs inhibited the proliferation of ARCFs stimulated by pro-inflammatory cytokines via a GR-dependent action. U0126 and PDTC inhibited ARCFs proliferation stimulated by cytokines.3. GCs inhibited increases in the levels of the cell cycle protein CyclinD1 stimulated by TNF-α.4. Pro-inflammatory cytokines activated proliferation pathway protein NF-κB and ERK1/2 in a time-effective manner.5. Inhibitory effect of physiological concentration of GCs on ARCFs proliferation signal pathway induced by pro-inflammatory cytokines. Brief summaryActivated ERK1/2 and NF-κB contributed to the proliferation of cultured ARCFs induced by pro-inflammatory cytokines, and GCs blocked above-mentioned pathway protein through GR signaling pathway to inhibit ARCFs proliferation.Conclusion1. Cardiac fibroblasts were the target cells of myocardial inflammatory response inhibited by endogenous GCs.2. Endogenous GCs maybe participate in the regulation of myocardial inflammatory response through regulating ARCFs'HDAC function. Oxidative stress caused the GCs resistance of anti-inflammatory effects by down-regulating ARCFs'HDAC activity and HDAC2 expression.2. Physiological concentration of GCs has a anti-proliferative effect on ARCFs via GC-GR pathway, this inhibition may be explained by inhibiting ERK1/2 activation, by reducing cyclin D1 protein levels to arrest G1-phase, by inhibiting NF-κB nulear translocation and increasing degradation of IκB.