Effects Of Glucocorticoids On Respiratory Burst Of Macrophages And Its Nongenomic Mechanism

Glucocorticoids (GCs) are steroid hormones secreted by adrenal cortex zona fasciculate cells. Their physiological functions include sugar, protein and fat metabolism regulation, the function of the circulatory system, the function of the immune system, and so on. When the body encountered strong stress, GCs secrete growing rapidly, and enhance the body's ability to resist the stress to maintain organs and tissues for the normal function of activities. Thus, GCs are considered as the key position of stress reaction. In addition to physiological function, GCs have been employed mainly in anti-allergic, anti-inflammatory and immunosuppressive conditions. GCs'medicinal dose is well in excess of their physiological dose, and the effects are not fully consistent, which makes an appeal of research on GCs'mechanism.In the 1980s, the "Nerve-Endocrine-Immune Network" concept make GCs'functions on the immune system become a hot spot of research. On the one hand, GCs in pharmacological dose significantly inhibit immune function. On the other hand, GCs in physiological dose or"stress dose"may play a broad and complex regulation on immune functions. At present, the research of GCs on the mechanism of the immune system is in the high-dose, long time, but less in the low-dose, short-term. GCs may promote or maintain the function of the immune system, and its mechanism has not been clarified clearly.Macrophages play important roles in inflammation and the immune response. The present experiment selected macrophages as experimental cells to explore the effects and possible mechanisms of GCs on superoxide anion production of macrophages in different concentrations and different time.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. So do GCs exert rapid effect through nongenomic mechanism on immune cell in regulation of immune reaction?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. We explored the effects of GCs (corticosterone, corticosterone 21-sulfate, HL, hydrocortisone, et al) on superoxide anion production of macrophages in different concentrations and different time.2. We performed the blocking experiments with RU486, CHX and BSA-CORT. We studied the membrane fluidity and activation of PKC in macrophages to get more information about the molecular mechanism involved in.3. To synthesize and identify the GCs'derivative: hydrocortisone as raw materials and lysine or phenylalanine as a carrier, we synthesized hydrocortisone-21-lysine ester hydrochloride (HL) and hydrocortisone-21- phenylalanine ester hydrochloride (HP). Then we used nuclear magnetic resonance and mass spectrometry to identify its structure.The main findings were as follows:1. Corticosterone, corticosterone 21-sulfate, HL, HG, hydrocortisone increased the production of superoxide anion in PMA-stimulated macrophage within 30 min.2. RU486, CHX and BSA-CORT could not block the rapid effect of CORT. Corticosterone rapidly inhibited PKC phosphorylation of PMA-stimulated macrophages and had no significant effect on the macrophages membrane fluidity.3. We synthesized HL and HP and the water-solubility of HL was markedly improved. 1H-NMR and mass spectrometry results showed that the synthetic GCs'derivative structure was correct.Previously, people think that glucocorticoids inhibit the immune function, while our study confirmed the glucocorticoids to promote macrophage respiratory outbreak quickly. Therefore, we speculate that on the state of acute stress, the body increases the secretion of glucocorticoids, which may enhance immune function. Since glucocorticoids reacted within 30 minutes in the experiment, 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.