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1.
Int J Mol Sci ; 24(23)2023 Nov 26.
Article in English | MEDLINE | ID: mdl-38069092

ABSTRACT

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4-11 and 37-60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bß, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague-Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases.


Subject(s)
Carica , Cysteine Proteases , Hemostatics , Thrombosis , Rats , Animals , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/chemistry , Latex/chemistry , Papain , Anticoagulants , Carrageenan , Rats, Sprague-Dawley , Thrombosis/drug therapy , Fibrinogen , Fibrin/chemistry
2.
Nutrients ; 14(17)2022 Aug 29.
Article in English | MEDLINE | ID: mdl-36079810

ABSTRACT

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bß, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague-Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.


Subject(s)
Cysteine Proteases , Thrombosis , Animals , Anticoagulants/pharmacology , Carrageenan , Cysteine Proteases/therapeutic use , Estrone/analogs & derivatives , Fibrin/therapeutic use , Fibrinogen , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/therapeutic use , Ficain , Rats , Rats, Sprague-Dawley , Thrombosis/drug therapy
3.
Int J Mol Sci ; 21(5)2020 Mar 03.
Article in English | MEDLINE | ID: mdl-32138302

ABSTRACT

Acne, also known as acne vulgaris, is a common disorder of human skin involving the sebaceous gland and Propionibacterium acnes (P. acnes). Although there are a number of treatments suggested for acne, many of them have limitations in their safety and have efficacy issues. Therefore, there is a high demand to develop safe and effective novel acne treatments. In the present study, we demonstrate the protective effects of Rosa davurica Pall. leaves (RDL) extract against P. acnes-induced inflammatory responses in vitro and in vivo. The results showed that RDL dose-dependently inhibited the growth of skin bacteria, including P. acnes (KCTC3314) and aerobic Staphylococcus aureus (KCTC1621) or Staphylococcus epidermidis (KCTC1917). The downregulation of proinflammatory cytokines by RDL appears to be mediated by blocking the phosphorylations of mitogen-activated protein kinase (MAPK) and subsequent nuclear factor-kappa B (NF-κB) pathways in P. acnes-stimulated HaCaT cells. In a mouse model of acne vulgaris, histopathological changes were examined in the P. acnes-induced mouse ear edema. The concomitant intradermal injection of RDL resulted in the reduction of ear swelling in mice along with microabscess but exerted no cytotoxic effects for skin cells. Instrumental analysis demonstrated there were seven major components in the RDL extract, and they seemed to have important roles in the anti-inflammatory and antimicrobial effects of RDL. Conclusively, our present work showed for the first time that RDL has anti-inflammatory and antimicrobial effects against P. acnes, suggesting RDL as a promising novel strategy for the treatment of acne, including natural additives in anti-acne cosmetics or pharmaceutical products.


Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Edema/immunology , Gram-Positive Bacterial Infections/drug therapy , Gram-Positive Bacterial Infections/immunology , Propionibacterium acnes/pathogenicity , Rosa/chemistry , Animals , Cell Line, Tumor , Disease Models, Animal , Edema/drug therapy , Gram-Positive Bacterial Infections/microbiology , Humans , Mice , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Propionibacterium acnes/immunology
4.
Nutrients ; 11(3)2019 Mar 07.
Article in English | MEDLINE | ID: mdl-30866501

ABSTRACT

Rumex japonicus Houtt. (RJ) is traditionally used in folk medicines to treat patients suffering from skin disease in Korea and other parts of East Asia. However, the beneficial effect of RJ extract on atopic dermatitis (AD) has not been thoroughly examined. Therefore, this study aimed to investigate the anti-inflammatory effects of RJ on AD in vitro and in vivo. Treatment with RJ inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) as well as the activation of nuclear factor-kappa B (NF-κB) in tumor necrosis factor-α (TNF-α) stimulated in HaCaT cells. The five-week-old Balb/c mice were used as an AD-like mouse model by treating them with 1-chloro-2, 4-dinitrobenzene (DNCB). Topical administration of RJ to DNCB-treated mice significantly reduced clinical dermatitis severity, epidermal thickness, and decreased mast cell and eosinophil infiltration into skin and ear tissue. These results suggest that RJ inhibits the development of AD-like skin lesions by regulating the skin inflammation responses in HaCaT cells and Balb/c mice. Thus, RJ may be a potential therapeutic agent for AD.


Subject(s)
Dermatitis, Atopic , Keratinocytes , Plant Extracts/pharmacology , Protective Agents/pharmacology , Rumex , Animals , Cell Line , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/metabolism , Dinitrochlorobenzene/adverse effects , Disease Models, Animal , Female , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Signal Transduction/drug effects , Skin/drug effects , Tumor Necrosis Factor-alpha/metabolism
5.
Toxins (Basel) ; 8(7)2016 07 05.
Article in English | MEDLINE | ID: mdl-27399771

ABSTRACT

An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.


Subject(s)
Chymases/genetics , Cloning, Molecular , DNA, Complementary , Scyphozoa/enzymology , Scyphozoa/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Catalytic Domain , Chymases/antagonists & inhibitors , Chymases/chemistry , Chymases/metabolism , Chymotrypsin/metabolism , Evolution, Molecular , Kinetics , Phenylmethylsulfonyl Fluoride/pharmacology , Phylogeny , Protein Binding , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Substrate Specificity
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