The Regulation Of Alpha-melanocyte Stimulating Hormone And Its Analogues On The High Mobility Group Box-1 And Its Receptor

High mobility group 1 protein (HMGB1), named for its rapid migration properties on electrophoretic gels, is a member of the nonhistone chromatin-associated proteins, and represents a novel group of intracellular proteins that function as inflammatory cytokines when released into the extracellular milieu. The HMGB1 has tripartite Structure, and it possesses an N-terminal A domain and a central B domain, called HMG-boxes. These boxes constitute non-specific DNA-binding regions. An additional C-terminal segment contains 30 highly acidic residues and is involved in interactions with other proteins. HMGB1 localizes to the nucleus and cytoplasm of most cells, to the cell membrane of neuronal (neuroblastoma) cells, and to the filopodia of the advancing plasma membrane of neurites, and increased levels of HMGB1 have been linked to acute and chronic inflammatory conditions. HMGB1 persists at elevated levels for a longer period than other major pro-inflammatory cytokines, such as TNF, IL-1, has been identified as a late mediator of endotoxin lethality. The release of HMGB1 is not only due to cell death, but also depends on the LPS-stimulated macrophages. More and more reports suggest that HMGB1 is now implicated as a mediator of delayed endotoxin lethality and systemic inflammation, and it may be possible to develop anti-inflammatory therapeutics that inhibit HMGB1 release and prevent death in sepsis. The insights gained from these studies of HMGB1 may widen the therapeutic window for treatment of endotoxemia, septic shock, and sepsis, if this newly recognized cytokine proves to be a clinically accessible target.The Receptor for Advanced Glycation End Products (RAGE) first found on vascular smooth muscle cells, is a multiligand member of the immunoglobulin superfamily, comprising an extracellular domain consisting of a single V-type immunoglobulin domain and two C-type immunoglobulin domains. Molecular studies identified that the ligand binding site existed within the V-domain by performing binding assays with truncations of the extracellular domain. After the extracellular domain, it follows a single transmembrane domain and a short 43 amino acid cytosolic tail. RAGE is expressed in a variety of cell types, including endothelial cells, vascular smooth muscle cells, macrophages, mesangial cells, and neurons. HMGB1 can bind to the receptor for advanced glycation end products (RAGE) in a dose-dependent manner. The interaction between HMGB1 and RAGE can amplify the cytokine cascade during systemic inflammation. The aim of this study is to find some evidence about the variation of HMGB1, according to examine the contribution of HMGB-RAGE interactions to the pathophysiological events. α-melanocyte stimulating hormone (α-MSH), a tridecapeptide derived from pro-opiomelanocortin, found in the pituitary, central nervous system, and peripheral tissues, has been shown to influence various inflammatory or immunological conditions by downregulating either the production or the action of the proinflammatory cytokines and upregulating the production of IL-10.α-MSH functions specifically via melanocortin receptors (MCRs), which include five subtypes (MCR-1 to MCR-5) coupled to adenylate cyclase to generate cAMP.α-MSH exerts the actions of anti-inflammation and immunregulation mainly through MC-1R and MC-5R. Scientists found that theα-MSH level decrease in the serum of endotoxic shork patients and increased during convalescence. However, because ofα-MSH is MC1 and MC3 receptor-selective, and has low affinities for MC4 and MC5 receptors, it is not persistent, stable ,effective. [Nle4,D-Phe7]α-MSH(NDP-MSH), is an analogue ofα-MSH and a strongly agonistic peptide, which has more powerful anti-inflammatory ability thanα-MSH and is not susceptible to degradation.Nevertheless it does not show receptor-selective. Moreover, it has a strong effect on skin pigmentation. In my experiment, peptide 39 designed by InsightⅡsoftware is not only high receptor-selective, but also not susceptible to degradation. It significantly decreased HMGB1 and RAGE expressions of cells. Furthermore, the effects of peptide 39 are appraised in the mouse model for endotoxic shock.PartⅠThe regulation ofα-melanocyte stimulating Hormone and its analogues on the expression of high mobility group box-l and its receptor in endotoxemiaInitially, We sought to study the presence of the receptor for advanced glycation endproducts (RAGE) and its ligands, high mobility group box-l in endotoxemia mice. According to the method reported by Galanos, endotoxemia was modeled by endotoxic injection of 6 to 8-week-old female BALB/c mice. Animals were injected intraperitoneally with D-Gal (20mg) and LPS (0.5μg) in PBS. 36 BALB/c mice were divided randomly into 4 groups: group A, PBS (negative control), and other groups were separately injected with D-Gal (20mg) and LPS (0.5μg) in PBS. After 18h, 24h, 32h, the presence of RAGE and HMGB1 in the lung, liver, kidney, spleen, encephalon of mice were examined by Western Blot and RT-PCR. Representative results were shown that LPS stimulation for 24h resulted in the high presence of RAGE and HMGB1, while the levels of RAGE and HMGB1 were much higher in the lung and the liver. Subsequently, make sure the time-effect relationship of the regulation ofα-melanocyte stimulating hormone and its analogues on HMGB1. 45 BALB/c mice were observed in 5 groups: group A, PBS (negative control); group B, LPS (positive control); group C, 2.5mg/kgα-MSH and its analogue (NDP-MSH, peptide 39) in 1h after LPS administration; group D, 2.5mg/kgα-MSH and its analogues (NDP-MSH, peptide 39) in 2h after LPS administration; group E, 2.5mg/kgα-MSH and its analogue (NDP-MSH, peptide 39) in 3h after LPS administration. The tissue samples (liver and lung) were taken when cytokines release reached the maximum (24h for HMGB-1 and RAGE),and they were tested by Western Blot and RT-PCR. New evidence suggested that the level of HMGB1 and RAGE significantly decreased when the peptide 39 was injected in 2h after LPS administration(P<0.05).PartⅡThe regulation ofα-melanocyte stimulating hormone and its analogue on the expression of high mobility group box-l and its receptor in U937U937 cell line were plated on 24-well plastic flasks in RPMI-1640 with calf serum, at 37℃. In the first place, U937 was incubated with 100μg/ml LPS for periods of 4, 12, 18, and 24 hours, HMGB1 reached the peak level when it was incubated with LPS for 18h, accompanying the up-regulation of RAGE was obviously. In the second place, time-effect relationship ofα-melanocyte stimulating hormone and its analogues were tested in vitro. After U937 was cultured in 24-well plates with LPS at various time points as described above, the cells were divided randomly into 5 groups: group A, negative control; group B, LPS (positive control); group C-E,α-MSH and its analogues (NDP-MSH, peptide 39) were respectively added in 1h,2h,3h after LPS stimulation. The result showed that peptide 39 which was added in 1h after LPS stimulation, down-regulated the expression of HMGB-1 and RAGE significantly(P<0.05). Last but not least, concentration gradient ofα-MSH and its analogues in U937 played an important role. Three concentrations ofα-MSH and its analogues were available ( 10-5M 10-7M 10-9M ) . And several lines of evidence demonstrate that 10-5M peptide 39 was the best .To our knowledge, RAGE signaling activates MAP kinase signaling, leading downstream to increased NF-κB activity . Rac and Cdc42 which have been shown in some cases to act by RAGE signaling, plays key signaling roles in cytoskeletal reorganization, membrane trafficking, transcriptional regulation, cell growth and development. Inhibition by sRAGE can decrease NF-κB, p44/p42, p38, SAP/JNK as well as MAPK activity. There is evidence from several studies thatα-MSH is able to suppress the LPS induced NF-κB activation which is required for most types of inflammation. According to the report, we choose Cdc42-Rac-MKK6-p38 to study the regulation ofα-MSH and its analogue on the HMGB1and RAGE. As investigated by Western Blot suggesting thatα-MSH contributed to down-regulate the expression of p38, NF-κB. These findings provide evidence for the cross-talk between HMGB1, RAGE andα-MSH signaling which may facilitate the future investigation of theα-MSH signaling and its regulation.