Non-genomic Effect Of Glucocorticoid On Inflammatory Cells Via ROCK1

Glucocorticoid, secreted by the adrenal cortex, play an important role in the body’sdevelopment, differentiation, metabolism, anti-inflammatory, anti-allergic and immuneregulation under physiological and pathological conditions. In addition, GC exerts animportant role in different types of cells.Secretion of glucocorticoid increase under the regulation of hypothalamus-pituitary-adrenal axis （HPA） when body suffer a variety of harmful stimulates, such as stress,hypoxia, trauma and surgery etc. The mechanisms of GC is widely regarded as a genomiceffect for a long time. It means glucocorticoid get through cell membranes by assist oftransport protein or freely and bind with its receptor in the cytoplasm. Then in assist withHSP90molecular chaperone, glucocorticoid receptor transfer to the nucleus and bind withspecific response element which nearby target gene promoters, exerting its physiologicaland pharmacology role. But researchers found glucocorticoid could act though another waywhich took place within a very short time and could not be blocked by the inhibitors ofgene transcription and translation. It suggests that the rapid erffects are not related to theprocess of gene transcription and protein synthesis. It was called " non-genomic effect" or "quick effect ".Now therapeutic effects of glucocorticoid are generally considered to be mediated byfour different mechanisms,1) the classical genomic mechanism caused by the cytosolicglucocorticoid receptor （cGCR）;2) non-genomic effects which are also initiated by thecGCR;3)membrane-bound glucocorticoid receptor （mGCR）-mediated non-genomiceffects;4)non-specfic, non-genomic effects caused by interactions with cellular membranes.In acute inflammation, excessive activation of inflammatory cells are key factors inbody injury. Release of large quantities of content in neutrophil, enhancing phagocyticfunction in macrophage and increasing adhesion inflammation cell to vascular endothelial*The project supported by NSFC. No30470988" cells would cause serious injury to the body. High doses of glucocorticoid was used to rapidsuppression of occurrence and development of acute inflammation in clinic treatment, butits specific mechanism is unknown.In recent years, ROCK （Rho-associated coiled-coil protein kinase） as a key regulationfactor in inflammation caused more and more concern. ROCK belongs to serine/threonineprotein kinase family and exists two highly homologous of isoforms （ROKα/ROCK2�'�ROKβ/ROCK1）. It has been show that ROCK1play an important role in severalinflammatory diseases such as acute lung injury, cardiovascular disease and ischemic braindamage etc. Downregulated expression and activity of ROCK1, or using of ROCK inhibitor（Fasudil） could effectively alleviate inflammation. Moreover, experiments have shownROCK1mainly regulated adhesion of inflammatory cell.It still unknown whether glucocorticoid inhibit inflammation via ROCK1pathway andby which mechanism does it play, genomic or non-genomic.Our study used neutrophils and U937cells as a cell model which both play animportant role in the inflammatory response. We detected whether glucocorticoid inhibitsthe function of neutrophil and U937cells by non-genomic effects or not under stimulus ofPMA（phobrolmyristateacetate） and observed how ROCK1acted in this process.The main results and conclusion of the study are as below:1. Rat neutrophil was isolated by using a modification of discontinuous Ficoll-Hypaque gradient technique. This gradient centrifugation of rat peripheral blood method isable to separate a high purity and viability of neutrophils.2.10^-6M and10^-7M of dexamethasone have a distinct inhibit effect on neutrophil’srelease of superoxide anion（O₂-） and myeloperoxidase（MPO） under PMA’s stimulation in ashort time（5min,15min,30min）. And this effect is not reversible by applyingglucocorticoid receptor antagonists-mifepristone （RU-486） and protein synthesisinhibitors-cycloheximide （CHX）. Our results indicate that10^-6M and10^-7M ofdexamethason’s inhibitory effect on O₂-and MPO releasing by neutrophil is mediated bynon-genomic effect.3. ROCK specific inhibitor-Fasudil had no significant inhibitory effect onneutrophils’ releasing of O₂-and MPO in a short time.That suggested ROCK is not animportant regulating factor in this process. 4. Dexamethasone treatment can effectively suppress U937cells adhering ability tohuman umbilical vein endothelial cell （HUVEC） within30min. Mifepristone andcycloheximide cannot suppress this effect. It is indicated that this effect is also mediated bynon-genomic effect.5. Fasudil can also reduce U937cell adhesion ability significantly, suggesting thatROCK1plays an important role in this process. And this phenomenon is similar to theinhibitory effect of dexamethasone, from which we can speculate that dexamethasonesuppress U937monocyte adhesion by inhibiting ROCK1.6. ROCK1can be suppressed by dexamethasone and Fasudil in U937, and this effectcouldn’t be reversed by mifepristone and cycloheximide. Meanwhile, the expression ofROCK1protein does not change. It suggests that dexamethasone’s inhibitory ability toROCK1is also mediated by non-genomic effects.7. Dexamethasone had no significant effects on RhoA’s expression and activity inU937cells, suggesting that dexamethasone do not inhibit ROCK1function via RhoA.Conlusion:1. Higher concentrations of dexamethasone (10^-6M,10^-7M) could rapid suppressionby PMA stimulated neutrophils release the MPO and O₂-raher than lower concentrations ofdexamethasone and Fasudil. Inhibition of dexamethasone through non-genomic effect.2. ROCK1played a key role in the process of U937adhesion HUVEC.Dexamethasone could inhibit ROCK1activity rather than the protein expression to inhibitU937cell adhesion function.3. Dexamethasone inhibit ROCK1acivity though non-RhoA dependent way.