Regulation Of Expression Of Corticotropin-releasing Hormone And Urocortin By LPS In Rat Peritoneal Macrophages And Its Mechanism

Background and objectives:Corticotropin-releasing hormone(CRH) is one of the key neurohormones and regulators in the hypothalamus-pituitary-adrenal axis which regulates the releasing of arenocorticotrophic hormone(ACTH) to control the secreting of glucocorticoid(GC) in adrenal gland. Urocortin is one of CRH structurally related peptide and it could bind to both of main CRH receptors(CRH-R1 and CRH-R2).Some studies indicate that there are relationships between CRH and UCN and the immune system.CRH and UCN may regulate the immune cells via their receptors on these cells.Macrophages are among the initiator cells during an inflammatory response and involve the cellular and humoral immune process.They are important to kill microorganisms and play a key role in the immune and inflammatory response.LPS is a main component of G~- bacteria and is an important proinflammatory mediator.LPS can induce macrophages secreting a large number of cytokines such as TNF-αand exogenous CRH and UCN can augment the effect of LPS.LPS also can up-regulate Toll-like receptor 4 on macrophages and exogenous CRH and UCN also enhance the action of LPS.Therefore,CRH and UCN may play an important role in the inflammation induced by LPS,but we don't know still that expression of CRH and UCN in macrophages activated by LPS.Thus,the present study aims to investigate expression of CRH and UCN in macrophages activated by LPS and the signal transduction pathway by which they are regulated by LPS.Materils and methods:We cultured rat peritoneal macrophages from healthy and adult Wistar rats and used RT-PCR and ELISA to study as follows:(1) We detected CRH and UCN mRNA and peptides in cultured rat peritoneal macrophages without stimulation to observe basic expression of CRH and UCN in terminal macrophages.(2) We used 5ng/ml or 25ng/ml TPA to stimulate cultured rat petitoneal macrophages,and detected CRH and UCN mRNA and peptides in cells at 1h,2h,4h,8h after stimulation to observe the rule of CRH and UCN expression in activated terminal macrophages.(3) We used 10ng/ml or 100ng/ml LPS to stimulate cultured rat petitoneal macrophages,and detected CRH and UCN mRNA and peptides in cells at 1h,2h,4h,8h after stimulation to observe the rule of CRH and UCN expression in early stage in terminal macrophages activated by endotoxin.(4) We pre-treated cells with 10μmol/L,50μmol/L,100μmol/L PD98059 to block ERK1/2,10μmol/L,50μmol/L SB203580 to block JNK and 10μmol/L,50μmol/L SP600125 to block p38MAPK at 30min before stimulated by LPS and detected CRH and UCN mRNA and peptides at 4h after stimulated by LPS to observe the action of the MAPK transduction pathway in the process in which LPS regulated the expression of CRH.The main results are as follows:1.Normally in cultured rat peritoneal macrophages,there are CRH and UCN mRNA and peptides detected,intracellular CRH peptide is about 0.07±0.01ng/10~7cells and intracellular UCN peptide is about 1.0±0.50ng/10~7cells.2.TPA up-regulate CRH and UCN mRNA transcription and their peptides synthesis in a time-dependent manner.CRH mRNA reach the peak at 8h after stimulated by TPA (p＜0.01,n=8) and was about 2.1 folds of the control.There is no notable difference between different concentration groups.CRH peptides reach the peak at 8h after stimulated by 25ng/ml TPA(p＜0.01,n=6),about 0.14±0.03 ng/10~7 cells.UCN mRNA reach the peak at 4h after stimulated by TPA(p＜0.01,n=8) and was 2.1-2.3 folds of the control.There is no notable difference between different concentration groups.UCN peptides reach the peak at 8h after stimulated by 25ng/ml TPA(p＜0.01,n=6),about 7.01±0.74 ng/10~7 cells.3.LPS up-regulate CRH and UCN mRNA transcription and their peptides synthesis in a time-dependent manner.CRH mRNA reach the peak at 4h after stimulated by LPS (p＜0.01,n=8) and was 1.9-2.1 folds of control.There is no notable difference between different concentration groups.CRH peptides reach the peak at 2h after stimulated by 100ng/ml LPS(p＜0.01,n=6),about 0.30±0.07ng/10~7 cells and maintained 0.15±0.07 ng/10~7 cells at 8h,about 2.1 folds of control.UCN mRNA reach the peak at 4h after stimulated by LPS(p＜0.01,n=8) and was 1.8-2.0 folds of control.There is no notable difference between different concentration groups.UCN peptides reach the peak at 8h after stimulated by 100ng/ml LPS(p＜0.01,n=6),about 8.68±1.25ng/10~7 cells.4.The specific blocker of ERK1/2 PD98059 blocks the up-regulation of CRH induced by LPS depending on the concentration.CRH mRNA is inhibited by 95%and peptide is inhibited by 90%when PD98059 is 100μmol/L.The specific blockers of JNK and p38MAPK could also block notably the up-regulation of CRH induced by LPS depending on the concentration.CRH mRNA is inhibited by 41%and 49%respectively when their concentration is 50μmol/L.The conclusions are as follows:1.Tthere are CRH and UCN normally in cultured peritoneal macrophages and this indicates they may regulate the immune cells including macrophages.2.TPA may enhance CRH and UCN expression in macrophages.CRH and UCN can also be up-regulated by LPS in the early stage and maintain at a high level for a long time, which indicates activated macrophages can express a large amount of CRH and UCN to regulate the immune and inflammatory response.3.The up-regulation of CRH and UCN by LPS depends on MAPK pathway particially and the ERK1/2 signal transduction pathway may be the key.4.Both in normal macrophages or cells stimulated by LPS,UCN peptide is further more than CRH.We suppose that UCN would play a more important role in regulating the immune cells than CRH in autocrine and/or paracrine manner.