Upregulation Of Glucocorticoid Receptor And CRH Expression In The HPA Axis Induced By Leukotriene B₄ Via MAPK Signal Pathway

Background and objective:Although the pathological aspects of many types of inflammation are well appreciated, their physiological functions and the role in the stress state are mostly unknown. Asthma is a result of pathological airway inflammatory. Many patients with mild asthma have the phenomenon of self-relief after an asthmatic attack. This phenomenon remains to be explained. Our previous studies showed corresponding changes of cytokines and leukotriene B4 (LTB4) between brain and lung tissues in antigen-challenged asthmatic rats. Leukotriene B4 by intracerebroventricular injection (icv) inhibits bronchoconstriction and airway inflammation. This is because expression of corticotrophin-releasing hormone (CRH) mRNA and protein in hypothalamus were significantly increased by LTB4 via i.c.v in sensitized rats after antigen challenge, then markedly increased corticosterone (CORT) and adrenocorticotropic hormone (ACTH) levels in plasma. These effects were blocked by pre-treatment with LTB4 receptor antagonist. These results indicate that leukotriene B4 in central nervous system may have negative feedback regulatory effects on asthma-like reaction in rats. Here, we further investigated the expression of Glucocorticoid receptors (GR) and CRH in the hypothalamic, pituitary, adrenal and lung induced by LTB4 via hypothalamic injection in regulating antigen-induced asthmatic response in sensitized rats. Futhermore, by means of upregulation of GR and CRH mRNA and protein expression in PC12 cells stimulated by LTB4, we studied the signaling pathways.Methods:In vivo study, ovalbumin (OVA)-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB4 and U75302 (a selective LTB4 BLT1 receptor inhibitor) or LY255283 (a selective LTB4 BLT2 receptor inhibitor) was given via hypothalamus injection 30 min before challenge. Rats were euthanized and then decapitated 30min after the final antigen challenge, Hypothalamus, pituitary gland, adrenal gland and lung were dissected.In vitro study, hypothalamus, pituitary gland, adrenal gland were dissected from the rat and cultured 6h in artificial cerebrospinal fluid (ACSF), and treatment with LTB4, U75302 and LY255283.Expression of CRH and GR mRNA and protein in hypothalamus, pituitary gland, adrenal gland and lung were evaluated by RT-PCR, Real-time PCR and Western blot.Then we used rat PC12 cell line as a research model.PC12 cells were pretreated with LTB4 receptor inhibitor, Erkl/2, JNK and p38 inhibitor for 30 min, then incubated with LTB4 (30ng/ml). We studied the activation of LTB4 on MAPK, PI3K-AKT and JAK-STAT signaling pathways. Total mRNA and protein of the cells were extracted and used for determining GR and CRH mRNA and protein expression by RT-PCR and western-blot.Results:(1) In vitro study, treatment with LTB4 for 6h, expression of GR and CRH in hypothalamus, pituitary and adrenal were up-regulated by LTB4 (30ng/ml) both in nomal and asthmatic rats. These effects were completely blocked by pre-treatment with BLT1 receptor antagonist U75302 (10-/mol/L), and partly inhibited by BLT2 receptor antagonist LY255283 (10-/mol/L).(2)In vivo study, expression of GR and CRH mRNA and protein in hypothalamus-pituitary-adrenal axis (HPA axis) were evaluated in this study. We found that OVA challenge alone markedly increased GR and CRH mRNA and protein expression in sensitized rats. Additionally, LTB4 via the hypothalamus injection further increased OVA challenge-induced GR and CRH mRNA and protein expression in HPA axis in sensitized rats. Pretreatment with U75302 at 100ng via the hypothalamus injection significantly suppressed LTB4-induced increase of GR and CRH mRNA and protein expression in antigen challenged sensitized rats. On contrast, LY255283 at 100ng did not significantly block the effects of LTB4. In addition, there is no significantly difference in the expression of GR mRNA in the lung.(3) LTB4 increases expression of GR and CRH in PC 12 cells concentration-dependently. Pretreatment with U75302 (10-7mol/L) significantly suppressed LTB4-induced increase of GR and CRH mRNA and protein expression. On contrast, LY255283 (10-7mol/L) did not significantly block the effects of LTB4.(4)To examine the mechanism behind the LTB4-mediated upregulation of GR, we used inhibitors to block the MAP kinase and NF-κB pathways. Upregulation of GR and CRH mRNA induced by LTB4 was decreased by JNK inhibitors SP600125 and ERK inhibitors U0126. NF-κB inhibitor PDTC can aslo down-regulate the expression of GR mRNA induced by LTB4, however, it has no effect on the upregulation of CRH mRNA induced by LTB4.SB202190 as an inhibitor of P38 MAP kinase can down-regulate the expression of GR protein induced by LTB4. LTB4-mediated expression of GR and CRH were accompanied by activation of Erk1/2, JNK and P38. These findings suggest that P38, JNK and ERK MAPK pathways were involved in the upregulation of GR and CRH induced by LTB4.Conclusions:These results indicate that LTB4 induced upregulation of GR and CRH mRNA and protein expression in the HPA axis through activation of MAPK signal pathway via its BLT1 receptor.