Petra Turčić

Interaction of α-melanocortin and its pentapeptide antisense LVKAT: effects on hepatoprotection in male CBA mice.

The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of discontinuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of α-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of α-MSH in 3’→5’ direction was used to design antisense peptide to the central motif that serves as α-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of α-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of α-MSH in vivo.

Effects of α-Melanocortin Enantiomers on Acetaminophen- Induced Hepatotoxicity in CBA Mice

Proteins and peptides in mammals are based exclusively on l-amino acids. Recent investigations show that d-amino acids exhibit physiological effects in vivo, despite of their very small quantities. We have investigated the hepatoprotective effects of the l- and d-enantiomers of α-melanocortin peptide (α-MSH). The results showed that peptide-enantiomerism is related to the protective effects of melanocortin peptides in vivo. l-α-MSH exhibited potent hepatoprotective effect in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice, while its d-mirror image was inefficient. Furthermore, the antibody to the l-peptide did not recognize the d-structure. The results indicate that the opposite peptide configuration may be used to modulate its function and metabolism in vivo and in vitro.

The Influence of α-, β-, and γ-Melanocyte Stimulating Hormone on Acetaminophen Induced Liver Lesions in Male CBA Mice

Research over the past decade has indicated that melanocortin peptides are potent inhibitors of inflammation and a promising source of new anti-inflammatory and cytoprotective therapies. The purpose of the present paper is to compare protective effects of α-, β-, and γ-melanocyte stimulating hormone on acetaminophen induced liver lesions in male CBA mice. Acetaminophen was applied intragastrically in a dose of 150 mg/kg, and tested substances were applied intraperitoneally 1 hour before acetaminophen. Mice were sacrificed after 24 hours and intensity of liver injury was estimated by measurement of plasma transaminase activity (AST and ALT) and histopathological grading of lesions. It was found that α-, β-, and γ-MSH decrease intensity of lesions by both criteria in a dose-dependent manner.

Hepatoprotective Effects of Met-enkephalin on Acetaminophen- Induced Liver Lesions in Male CBA Mice

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p > 0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Modulation of γ2-MSH Hepatoprotection by Antisense Peptides and Melanocortin Subtype 3 and 4 Receptor Antagonists

Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

Cytoprotective Effects of β-Melanocortin in the Rat Gastrointestinal Tract

Recently discovered anti-inflammatory and immunomodulatory properties of melanocortin peptides led to the conclusion that they might serve as new anti-inflammatory therapeutics. The purpose of this work was to examine the effectiveness of β-melanocortin (β-MSH) in two experimental models: ethanol-induced gastric lesions and TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced colitis in male Wistar rats. Three progressive doses of β-MSH were used: 0.125, 0.250 and 0.500 mg/kg. Our results suggest that β-MSH acts as a protective substance in the gastric lesions model, which can be seen as a statistically significant reduction of hemorrhagic lesions at all three doses, compared to the control group. The most efficient dose was 0.250 mg/kg. Statistically significant reduction in mucosal surface affected by necrosis and the reduction of overall degree of inflammation in the colitis model indicates an anti-inflammatory effect of β-MSH at a dose of 0.250 mg/kg. The results justify further research on β-MSH peptide and its derivates in the inflammatory gastrointestinal diseases, and point out the possibility of using β-MSH in studies of digestive system pharmacology.

The influence of alpha-melanocortin enantiomers on acetaminophen-induced hepatis in mice

Background L-alpha-Melanocortin is a strong inhibitor of inflammation. It is a promising new anti-inflammatory and hepatoprotective peptide. Consequently, its melanocortin receptors (MC1, MC3, MC4 and MC5) could be possible targets for the development of new antiinflammatory drugs for chronic inflammatory liver disease. For a long time it has been believed that only the L-enantiomers of amino acids are present in higher animals, but recent investigations show that D-amino acids also exhibit physiological effects in vivo, despite their very small quantities. The aim of this study was to compare hepatoprotective effects of L-alpha-melanocortin and D-alpha-melanocortin using the acetaminophen model of chemical liver damage in male CBA mice.