Glucocorticoid Inhibits Neutrophilic Inflammation And Improves Acute Stress Tolerance By Rapid Nongenomic Mechanisms

Glucocorticoids (GCs) regulate a variety of growth, metabolic, developmental, andimmune functions and play a pivotal role in preserving basal and stress-related homeostasis.They also represent one of the most widely prescribed anti-inflammatory andimmunomodulatory drugs during the past70years. Though the molecular mechanisms ofGCs have been extensively studied, it is still remains largely unclear. It is believedtraditionally that GCs exert most of their effects genomically. Latest findings suggest thatnongenomic activity might become relatively more important in mediating the therapeuticeffects of high-dose pulsed glucocorticoid administration.Glucocorticoid pulse therapy involves intravenous administration of high dose ofprednisolone (0.5-2g) or its equivalents. As the glucocorticoid plasma and tissueconcentrations achieved under treatment with glucocorticoid megadoses are far beyondthose required for cytosolic receptor saturation, additional nongenomic effects have beenpostulated to account for their therapeutic action. Glucocorticoid pulse therapy is effectivefor many acute diseases, such as rheumatoid arthritis, acute spinal cord trauma, acute lunginjury and chronic obstructive pulmonary disease (COPD). These diseases are usuallyinvolved in the invasion of human polymorphonuclear neutrophils (PMN). PMN comprise50-70%of circulating leukocytes. In the inflammatory response, few cells play asprominent a role as the neutrophil. Human neutrophil contains three main lysosomalgranules, azurophil granules, specific granules and gelatinase granules, and secretoryvesicles. Specific proteolytic and digestive enzymes capable of destroying extracellularmatrix and bacterial debris are stored inside these granules, which therefore are involved inimmune and inflammatory processes as well as in a variety of diseases and tissue injuries.Our previous study found that GCs could inhibit PMN degranulation rapidly throughnongenomic mechanisms and PDE4may be one of the molecular targets of GCs. In the1stpart of this study, we investigated the in vivo effects of GCs on PMN degranulation in amice systemic inflammation model, verified again the nongenomic effects of GCs on PMNdegranulation by employing amino acid linked new GCs analogues, detected the acutecytotoxicity of6α-Methylprednisolone on PMN, and explored the further mechanismsinvolved in by detecting [Ca2+]i, membrane lipid rafts, membrane fluidity. We veryfied thenongenomic effects by employing RU486and cycloheximide (CHX).Though GCs are widely used in the treatment of inflammatory and autoimmune states, this inherent chemical should not exist only for clinical use. There may existimportant physilogical significance in stress response concerning nongenomic mechanismof GCs.Nongenomic effects are charactared as short latency, and insensitive to inhibitorsof DNA transcription or protein synthesis (e.g. actinomycin D or cycloheximide) comparedwith genomic effects. It is believed traditionally that GCs exert most of their effectsgenomically. Upon ligand binding, glucocorticoids receptor (GCR) translocates into thenucleus of cells, where it modulates the transcriptional activity ofglucocorticoid-responsive genes in either of two ways: by binding to specific sequences inthe promoter region of target genes, the glucocorticoid-response elements (GREs), orthrough protein-protein interactions with other transcription factors, such as nuclearfactor-κB (NF-κB), activator protein-1(AP-1), and several signal transducers andactivators of transcription (STATs). It costs at least30minutes for genomic effects of GCstake action. So it is considered that the sympathetic-adrenal medullary (SAM) system maytake action at the very beginning of acute stress reactions. Secretion of the catecholaminesis regulated by mental influence on the hypothalamus in the central nervous system, whichactivates the sympathetic adrenal medullary (SAM) system. The cardiovascular andneuroendocrine functions activated by catecholamines mobilize energy to the muscles,heart, and brain and, at the same time, reduce blood flow to the skin, internal organs andgastrointestinal system. This is an efficient means for coping with stress. Nevertheless,studies on the relative importance to life of cortex and medulla of the1adrenal glands haverevealed that the adrenal medulla, in contrast to the cortex, is not essential for life. Almostany type of stress, whether physical or neurogenic, causes an immediate and markedincrease in ACTH secretion by the anterior pituitary gland, followed within minutes bygreatly increased adrenocortical secretion of cortisol. Even though we know that cortisolsecretion often increases greatly in stressful situations, we are not sure why this is ofsignificant benefit to the animal. Thus we infer that the nongenomic effects of GCs mayplay vital roles at the early phage of acute stress. However, there still lacks powerfullaboratory basis. So, we set out the2ndpart of this study to investigate the effects of stressdose of corticosterone (CORT) on Acute Weight-Loaded Forced Swimming (AWLFS)exhaustive stress in adrenalectomized mice. We investigated changes on the exhaustiontime, pathological injury, ultrastructure and biomaker (NO, blood glucose, lactic acid andATP in skeletal muscle) in vivo and in vitro. We veryfied the nongenomic effects byemploying RU486. Main results:ⅠNongenomic mechanisms involved in inhibitory effects of glucocorticoids onneutrophil degranulation1. Mice pretreated with high-dose Methylprednisolone succinate (0.04mg/g bodywt) prevented fMLP induced MPO and LF release from circulation neutrophils in5minutes.2. Amino acid linked new GCs analogues showed inhibitory effects on PMNdegranulation..3.6α-Methylprednisolone (MP) megadoses have no cytotoxicity on PMN in15minutes.4. MP accelerated the draw back of fMLP elevated [Ca2+]i rapidly. And neitherRU486nor CHX could block this effect.5. Both MP and fMLP shows inhitory effects on PMN membrane fluidity. Thecombine use of MP and fMLP increased PMN membrane fluidity.6. MP inhibited fMLP induced lipid rafts aggregation, and could not be blocked byRU486/CHX. While interrupting lipid rafts by MβCD had no effect oninhibitory effects of MP on PMN degranulationⅡGlucocorticoid Improves Acute Weight-Loaded Forced Swimming exhaustive StressTolerance by rapid nongenomic mechanisms1. CORT prolonged the exhaustion time in AWLFS mice in less than20minutes,and this effect could not be blocked by RU486.2. There are less break and lysis in skeletal muscle fibers of CORT group than inthose of control group. And electron microscope shows mitochondrial pyknosis andhigh density, dilatation and vacuolization of sarcoplasmic reticulum vesicles, loss of Z discand M line in control group compared with in GCs group.3. There is lower blood nitric oxide, higher blood glucose and skeletal muscle tissueATP in GCs group than in control group in AWLFS mice. L-NAME, inhibitor ofNOS, prolonged exhaustion time in combined use with GCs. GCs also inhibitedACh induced NO release from C2C12myotube cells independentglucocorticoids recepter (GCR). And GCs showed inhibitory effects on glucoseuptake in C2C12myotube cells.Conclusions:1. Nongenomic mechanisms exist in glucocorticoid pulse therapy; nongenomic inhibition of neutrophil degranulation may take action in therapy of GCs pulsetherapy in many acute diseases, such as rheumatoid arthritis, acute spinal cordtrauma, acute lung injury and COPD. Glucocorticoids accelerated the draw back offMLP elevated [Ca2+]i may be parts of mechanism. Lipid rafts, membrane fluidityand cytotoxicity should not answer for nongenomic effects of GCs on neutrophildegranulation.2. Glucocorticoids nongenomic mechanisms exist in stress reaction. Rapid release oflarge amounts GCs during acute stress has physilogical significance; CORTimproves AWLFS exhaustive tolerance via rapid inhibition of NO systemactivation and hence increase of ATP in skeletal muscle cells (SKMCs) and relieveof NO's inhibition on muscle force, and also may via increasing blood glucosethrough inhibition of glcose uptaken by SKMCs.This study indicates that pharmacological dose of GCs shows nongenomicanti-inflammatory mechanisms. Nongenomic regulatory mechanisms exist in physiologicaleffects of GCs, rapid inhibition of NO system activation may be the key point of thisnongenomic regulatory mechanism. This study provides new laboratory evidences and newdirections for developing novel GCs analogues to improve stress tolerance and effecs/riskratio.