Mechanisms Of Protection Against Acute Lung Injury By A Novel Alpha-melanocyte-stimulating Hormone Analogue

Alpha-melanocyte stimulating hormone (α-MSH), an endogenous neuropeptide, is derived from a larger precursor molecule pro-opiomelanocortin (POMC). POMC is a 29-kDa polypeptide that yields the biologically active peptides adrenocorticotropin (ACTH), endorphins (α,β,γ) and melanotropins (α-,β-, andγ-MSH).α-MSH was originally isolated and characterized from the intermediate lobe of the pituitary and it was first recognized by its effect on skin melanophores in lower vertebrates. Although necessary for its influence on pigmentation,α-MSH mediates other biologic functions. It has antipyretic, anti-inflammatory and antimicrobial effects. It is also involved in the control of food intake and body weight.α-MSH is produced by pituitary cells and astrocytes, as well as the cells of skin and other peripheral tissues. Studies revealed that endogenousα-MSH level increases in various clinical and experimental pathologic conditions such as rheumatoid arthritis, myocardial infarction, endotoxemia and HIV infection. Moreover, endotoxin injection to normal subjects causes a marked increase in plasma peptide concentration. In addition to plasmaα-MSH that reaches the tissues, local peptide production at inflammatory sites also increases. These observations suggest that increase inα-MSH concentration during inflammation is a compensatory mechanism and if the peptide is not sufficient to counteract the action of inflammatory mediators, the disease becomes more severe.The roles ofα-MSH were supported by the gene cloning of a family of specific receptors. To date five melanocortin receptors have been described (MC1R to MC5R). All of them belong to the heterodimeric guanine nucleotide-binding protein (G-protein) coupled family of receptors, characterized by the presence of seven transmembrane repeat spanning domains. Five melanocortin receptor subtypes (MC1-MC5) are recognized. MC1 is expressed in melanocytes, melanoma cells and cells involved in the immune/inflammatory response including monocytes, dendritic cells and lymphocytes. MC2, the ACTH receptor, is mainly expressed in the adrenal glands but also in white adipose tissue and in the skin. MC3 is expressed in the brain, placenta, gut and heart. MC4 occurs in various brain areas but has been recently recognized also in peripheral organs in the rat. The MC5 receptor, initially recognized in the brain, was subsequently found to be ubiquitous, but mainly in the periphery. Antipyretic and anti-inflammatory activities ofα-MSH have been traced to a "message sequence" contained within the COOH-terminal region, the tripeptide Lys-Pro-Val. Although the mechanism of the antimicrobial action is poorly understood, it is conceivable thatα-MSH could be useful in the treatment of various infectious conditions.However,α-MSH has its limitation as a novel melanogenic drugs, such as higher selective to MC1R and MC3R but lower to MC4R and MC5R. Therefore, a novel alpha-melanocyte stimulating hormone analogue was developmented by our laboratory. This new peptide has been proved to be more selective to MC1R and MC5R. thanα-MSH, two key receptors which the peptide is depended on for its anti-inflammatory activity. Besides, the novel peptide is also an agonist to MC1R, MC3R, MC4R and MC5R. The result shows that our peptide has a good applicative prospect.The ALI/ARDS may occur as a consequence of critical illness of diverse etiologies, including direct injury to lung, such as pneumonia, aspiration, toxic inhalation, near-drowning, or lung contusion, as well as indirect mechanisms, such as sepsis, burn, pancreatitis, gynecological insults (abruption of placenta, amniotic embolism, eclampsia), or massive blood transfusion. The mortality rate associated with ARDS has declined from 90% about twenty years ago to 30%～40% at present. However, it is still one of the major causes of pulmonary and nonpulmonary morbidity in patients after discharge. Animal studies that have attempted to mimic human ALI/ARDS have been useful and will likely continue to provide valuable observations regarding both the mechanisms underlying the pathogenesis, progression and resolution of this syndrome and ways in which its course can be modulated therapeutically. However, the lack of an animal model that unequivocally mimics key aspects of human ALI/ARDS has been limiting in mechanistic studies and in providing meaningful and rapid extrapolations to the clinical syndrome. Furthermore, there is uncertainty as to which of the many available animal models best reflects the human clinical syndrome. Animal models are usually monitored over a shorter term than the human syndrome, which requires hours or days to develop. A two-hit theory suggests that an initial insult, such as hemorrhage and resuscitation (H/R), primes the host for an exaggerated or abnormal response to a second insult, often infection. Hemorrhagic shock is the most important cause of early death after major trauma. There is increasing evidence that lipopolysaccharide (LPS) induces an inflammatory response in the lung. Therefore, in the present studies, a two-hit model of resuscitated hemorrhagic shock followed by intratracheal LPS in the rodent was used to evaluate the protective mechanisms ofα-MSH and our novel peptide to the lung.Melanocortins are potent modulators of inflammation. Administration of melanocortins is immunoprotective in models of systemic inflammation, peritonitis, myocardial ischemia, renal ischemia, allergic airway inflammation, experimental heart transplantation, and colonic inflammation. In many of these studies, administration of melanocortin peptides results in the suppression of cytokine production, inhibition of leukocyte infiltration, and preservation of tissue histology. But, according to our present comprehension, its protection on ALI has not been researched. In this paper, a two-hit model of ALI is used to test the anti-inflammatory activity of our novel alpha-melanocyte stimulating hormone analogue and traditionalα-MSH. In this model, characteristic hallmarks of ARDS such as lung edema and interstitial neutrophil infiltration were observed by the evaluation of total protein, TNF-α, IL-1β, MPO, SOD, MDA, P-selectin, PAI-1, CINC-1, CC16, HO-1 and ACE2 by means of histopathology, Real-Time PCR, ELISA, and immunohistochemistry. The data demonstrate that both our new peptide andα-MSH have good protective effect on ALI. For example, the alpha-melanocyte stimulating hormone analogue and traditionalα-MSH can significantly inhibit the upregulation of TNF-α, IL-1β, MPO, SOD and MDA. Besides, some new protective mechanisms of them have been explored, including the regulatory effects ofα-MSH and its noval analogue on the expression of P-selectin, PAI-1, CINC-1, CC16, HO-1 and ACE2. CINCs are known to be potent chemotactic factors for rat neutrophils, and to belong to the IL-8 superfamily. CINCs can induce a marked increase in neutrophils adherent to the venular endothelium, and enhance their transendothelial migration across the venular wall. It is well established that neutrophil-mediated tissue injury is a common mechanism underlying the development of organ dysfunction during acute lung injury including ARDS. Our data demonstrate that both the new peptide andα-MSH have significant inhibition to CINC-1. The accumulation of neutrophils in the lung tissue is initially mediated through increased adhesion of neutrophils to endothelial cells of the inflamed pulmonary microvasculature, and this process requires the upregulation of various adhesion molecules expressed on the surface of pulmonary microvessel endothelial cells as well as circulating neutrophils. Extensive research has demonstrated that activation of coagulation and inhibition of fibrinolysis are thought to underlie the events in ALI. Mechanisms that contribute to the changes in the homeostatic balance include both tissue factor expression and increased plasminogen activator inhibitor (PAI-1) synthesis or release. Similar experiments exposing PAI-1 knockout mice to bleomycin showed a decrease in fibrin deposition in the PAI-1 knockout mice, while controls showed PAI-1 to be increased by bleomycin. Clara cells act as stem cells in the repair of the bronchial epithelium, have a high xenobiotic biotransformation capacity and secrete several substances with important biological activities. One of the major proteins secreted by Clara cells is the 16 kDa Clara cell protein (CC16). Even though the exact in vivo function of the CC16 remains to be clarified, there is growing evidence that this protein plays a protective role against pulmonary inflammatory response. During several months of 2002, severe acute respiratory syndrome (SARS) caused by SARS-coronavirus (SARS-CoV) spread rapidly from China throughout the world, causing more than 800 deaths due to the development of acute respiratory distress syndrome (ARDS), which is the severe form of acute lung injury (ALI). Interestingly, a novel homologue of angiotensin-converting enzyme, termed angiotensin-converting enzyme 2 (ACE2), has been identified as a receptor for SARS-CoV. Angiotensin-converting enzyme and ACE2 share homology in their catalytic domain and provide different key functions in the renin-angiotensin system (RAS). Angiotensin-converting enzyme cleaves angiotensin I to generate angiotensin II, which is a key effector peptide of the system and exerts multiple biological functions, whereas ACE2 reduces angiotensin II levels. Importantly, recent studies using ACE2 knockout mice have demonstrated that ACE2 protects murine lungs from ARDS. Furthermore, SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo, which can be attenuated by blocking the renin-angiotensin pathway, suggesting its key role in ALI. Both the new peptide andα-MSH can significantly inhibit the upregulation of PAI-1 and P-selectin and downregulation of and CC16. However, failure to induce the downregulation of ACE2 after hemorrhagic shock and LPS hits underscores an incomplete understanding of the two-hit model. In general,α-MSH and its new analogue could attenuate the progression of the inflammation, thereby preventing the development of chronic lung disease.There are three originality of this experiment: first, the novel alpha-melanocyte stimulating hormone analogue has been proved to be effective in the protection against acute lung injury. Second, some new mechanisms of this protection has been discovered, including inhibition of PAI-1, CINC-1 and P-selectin and the modulation of ACE2, HO-1 and CC16 byα-MSH. The third, the two-hit model has been improved to mimic hemorrhagic shock and endotoxemia better.