Rapid Effect Of A Novel Type Of Glucocorticoids On Mast Cells Degranulation And Its Mechanism

Glucocorticoids (GCs) are steroid hormones secreted by adrenal cortex zona fasciculate cells. They regulate the growth-developmental, metabolic, and immune functions in body and play a pivotal role in stress. And they are also widely used in anti-allergic, anti-inflammatory and immunosuppressive conditions.The mechanism of GCs'action has been well studied in life science with military significance and practical value. Over the passed decades, it was widely assumed that GCs work solely through regulating gene expression and synthesis of protein, which needs several hours or days to take its biological effect. In addition to the classical "genomic" mechanism, there is increasing recognition of alternative modalities of GCs' action that is independent from modulating gene expression and for this reason defined as "nongenomic mechanism". These are characterized by a rapid response (seconds to minutes) and insensitivity to inhibitors of gene transcription and protein synthesis.Nongenomic effects of GCs are well studied in neuroendocrinology, though GCs have been employed mainly in anti-allergic, anti-inflammatory and immunosuppressive conditions. In the treatment of allergic diseases, GCs are ones of the most potent agents available. Antigen-induced degranulation of mast cells plays a crucial role in the attack of allergic reaction. Cross-linking of the high affinity IgE receptor by antigen induces mast cells exocytosis, finally resulting in a cascade of morphological and biochemical reactions, and attack of allergic reaction. We have certified that GCs could exert rapid effects on them and insensitivity to inhibitors of gene transcription and protein synthesis. The present experiment selected mast cellls as experimental cells to explore the effects and possible molecular mechanisms of GCs on antigen-induced degranulation of mast cells in the allergic reaction.Over the passed decades, it was widely assumed that GCs work solely through regulating gene expression and synthesis of protein, which needs several hours or days to take its biological effect. In addition to the classical "genomic" mechanism, there is increasing recognition of alternative modalities of GCs'action that is independent from modulating gene expression and for this reason defined as "nongenomic mechanism". These are characterized by a rapid response (seconds to minutes) and insensitivity to inhibitors of gene transcription and protein synthesis.Long-term application of GCs easily leads to serious complications, including hyperglycemia, hypokalemia, hypocalcemia, hypertension, and adrenal dysfunction, serious secondary infection, bone necrosis, and so on. The GCs'nongenomic mechanism can guide us to synthesize the hydrophilic GCs' derivative to avoid the side effects of GCs.Based on the above ideas, the subject took the following studies:1. To synthesize and identify the GCs' derivative:hydrocortisone as raw materials and Phenylalanineas a carrier, we synthesized hydrocortisone-21-Phenylalanine ester hydrochloride (HP). Then we used nuclear magnetic resonance and mass spectrometry to identify its structure.2. We explored the effects of GCs'derivative HG to antigen-induced degranulation of mast cells:In vitro, we established the model of antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells, we examined GCs'(HG) nongenomic effect on antigen-induced histamine release of mast cells at different concentration and time.3. And we studied the membrane fluidity in mast cells to get more information about the molecular mechanism involved in.4.. The activation of phospho-Syk and phospho-ERK was detected by western blot analysis; We measured the histamine release when the Syk inhibitor called Piceatannol was added.5. Intracellular levels of cAMP in RBL-2H3 were measured by Radioimmunoassay.6. GRE-luciferase reporter assay was widely used to assess the activation of classical glucocorticoid receptor. To check whether HG act via genomic mechanisms, we determined the activation of GRE by GRE-luciferase reporter assay.The main results were as follows:1. We synthesized HP;1H-NMR and mass spectrometry results showed that the synthetic GCs'derivative structure was correct.2. In vitro, GCs(HG) could dose-dependently inhibit antigen-induced histamine release of mast cells with time-course character, and neither GCs nuclear receptor antagonist nor protein synthesis inhibitor could block the rapid action, which confirmed the existence of nongenomic mechanism.3. GCs could dose-dependently increase the microviscosity and reduce the membrane fluidity of mast cells; and GCs nuclear receptor antagonist could not block the rapid action.4. GCs and the derivative could inhibit the phosphorylation of Syk and ERK, and it could not be blocked by RU486. The antagonist of Syk and GCs both could inhit the histamine release from mast cells, and this could indicate Syk may be the key molecular in mast cell's degranulation.5. There was no significant difference between HG pretreatment and control group. It suggested that cAMP was not the key signal in the GC's rapid nongenomic effect on degranulation of mast cells.6. In the time-response study with 10-7 M HH, a significant increase in GRE-Luc activity was observed from 15 min. However, the activation could not be observed in 10-7 M HG group. These results indicated that HG could not active classic genomic effects from RBL-2H3 cells in 30 min.GCs and HG could rapidly inhibited Syk phosphorylation of antigen-induced mast cells and neither GCs nuclear receptor antagonist nor protein synthesis inhibitor could block the effect. GCs and Piceatannol could both inhibit the histamine release, which confirmed that Syk is the key molecular in the degranulation of mast cells.GCs and HG could rapidly inhibited Erk phosphorylation of antigen-induced mast cells and GCs nuclear receptor antagonist could not block the effect.The study proposed the new molecular mechanism of GCs' rapid effect on mast cells:GCs could rapidly influence the signal pathway in the activation of mast cells including the phosphorylation of Syk and ERK, and then decrease the membrane fluidity and release of inflammation mediator, finally resulting in the inhibition of type I allergic reaction independent of classic genomic mechanism. And the new synthetic hydrophilic glucocorticoids derivatives had the same effect, we speculate glucocorticoids play the role through nongenomic mechanisms. In addition, further study might raise the possibility of new therapeutic strategies for immune diseases, which will expand the research of GCs' nongenomic effect from the basic theoretical research to the fields of application.