Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro

Alchemilla vulgaris L. (lady’s mantle) was used for centuries in Europe and Balkan countries for treatments of numerous conditions and diseases of the reproductive system, yet some of the biological activities of lady’s mantle have been poorly studied and neglected. The present study aimed to estimate the potential of A. vulgaris ethanolic extract from Southeast Serbia to prevent and suppress tumor development in vitro, validated by antioxidant, genoprotective, and cytotoxic properties. A total of 45 compounds were detected by UHPLC–HRMS analysis in A. vulgaris ethanolic extract. Measurement of antioxidant activity revealed the significant potential of the tested extract to scavenge free radicals. In addition, the analysis of micronuclei showed an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes. A. vulgaris extract strongly suppressed the growth of human cell lines derived from different types of tumors (MCF-7, A375, A549, and HCT116). The observed antitumor effect is realized through the blockade of cell division, caspase-dependent apoptosis, and autophagic cell death. Our study has shown that Alchemilla vulgaris L. is a valuable source of bioactive compounds able to protect the subcellular structure from damage, thus preventing tumorigenesis as well as suppressing tumor cell growth. © 2022 by the authors

The role of indolamine 2,3 deoxygenase in the immunomodulation of ulcerative colitis: parameters of inflammation in the oral cavity and colon

Ulcerozni kolitis je inflamacijska bolest creva koja se karakteriše fazama egzacerbacije i remisije. Zbog sličnosti u pogledu molekulskih mehanizama odgovornih za nastanak i progresiju, mišji kolitis indukovan dekstran natrijum sulfatom (DSS-kolitis) je jedan od najčešće korišćenih eksperimentalnih modela za ispitivanje ulceroznog kolitisa. Analogno ulceroznom kolitisu ljudi, imunske ćelije (makrofagi, dendritske ćelije (DCs), neutrofili, T limfociti) imaju najvažniju ulogu u patogenezi DSS-kolitisa. Regulatorni T limfociti (Treg), produkcijom imunosupresivnih citokina, suprimiraju inflamaciju i podstiču regeneraciju oštećene sluznice creva. Postoji dramatična razlika u toku DSS-kolitisa kod miševa različitih sojeva. Tako, BALBc miševi razvijaju lakši oblik bolesti sa spontanom regeneracijom sluzokože creva dok S57BL/6 miševi razvijaju težu formu bolesti koja progredira u hronicitet. U lamina propri-ji creva se triptofan metaboliše kinureninskim putem, aktivnošću enzima indolamin 2,3 dioksigenaze (IDO). Najveća aktivnost IDO-a je zabeležena u DCs i mezenhimskim matičnim ćelijama (MSCs) koje se zbog svojih imunomodulacijskih karakteristika primenjuju u terapiji inflamacijskih bolesti, uključujući i ulcerozni kolitis. Rezultati ove studije su pokazali da DCs BALBc miševa poseduju znatno veći kapacitet produkcije IDO-a naspram DCs C57BL/6 životinja, što sledstveno indukuje ekspanziju Treg limfocita koji suprimiraju inflamaciju i podstiču regeneraciju sluznice creva. Stepen oštećenja sluznice creva, pa tako i razvoj bolesti, se najpouzdanije utvrđuju kolonoskopijom, ali se ova invazivna dijagnostička metoda ne može često izvoditi. Određivanje fekalnog kalprotektina predstavlja najčešće korišćeni alternativni dijagnostički parametar za praćenje toka bolesti i uspešnosti terapije. Ipak, nedostatak validiranog cut-off-a i nedovoljna specifičnost ovog testa, ukazuje na potrebu za uvođenje novih neinvazivnih dijagnostičkih metoda kojim se može procenjivati uspešnost terapije i stepen regeneracije obolele sluznice. Rezultati ove studije ukazuju da se praćenjem koncentracije kinurenina u gingivalnoj tečnosti, serumu i fecesu može pratiti progresija ulceroznog kolitisa. Zbog činjenice da su MSCs jedan od najznačajnih izvora IDO-a, u ovoj studiji je ispitivan i značaj IDO/kinureninskog puta za terapijske efekte MSCs u imunomodulaciji DSS-kolitisa. Koncentracija inflamacijskih citokina, posebno 4 IFN-γ i TNF-α, utiču na promenu fenotipa i efektorskih funckija MSCs. MSCs su nakon aplikacije u miševe soja C57BL/6 (u kojima je serumska koncentracija inflamacijskih citokina bila visoka), uzrokovale značajan porast koncentracije kinurenina u serumu što je rezultiralo ekspanzijom Treg limfocita u crevima, smanjenjem inflamacije i ublažavanjem simptoma kolitisa. Sa druge strane, nakon aplikacije u DSS-om tretirane BALBc miševe (kod kojih su bile niske vrednosti inflamacijskih citokina), MSCs nisu uzrokovale IDO/kinurenin-zavisnu ekspanziju Treg limfocita u crevima već su indukovale ekspanziju inflamacijskih ćelija u kolonu što je dovelo do pogoršanja kolitisa.Ulcerative colitis is an entity of inflammatory bowel disease characterized by stages of exacerbation and remission. Due to the similarity with respect to the molecular mechanisms responsible for the onset and progression, murine colitis induced by dextran sodium sulfate (DSS-colitis) is one of the most commonly used experimental models for testing ulcerative colitis. Analogous to human ulcerative colitis, immune cells (macrophages, dendritic cells (DCs), neutrophils, T lymphocytes) play the most important role in the pathogenesis of DSScolitis. Regulatory T lymphocytes (Tregs), by producing immunosuppressive cytokines, suppress inflammation and promote the regeneration of damaged intestinal mucosa. There is a striking difference in the course of DSS-colitis in mice of different strains. Thus, BALBc mice develop a milder form of disease with spontaneous regeneration of intestinal mucosa while C57BL/6 mice develop a more severe form of disease that progresses to chronicity. In the lamina propria of the intestine, tryptophan is been metabolized by kynurenine via the activity of the enzyme indoleamine 2,3 dioxygenase (IDO). The highest activity of IDOs has been reported in DCs and mesenchymal stem cells (MSCs), which, because of their immunomodulatory properties, are used in the treatment of inflammatory diseases, including ulcerative colitis. The results of this study showed that DCs of BALBc mice have a significantly higher IDO production capacity than DCs of C57BL/6 animals, which consequently induces the expansion of Treg cells that suppress inflammation and promote mucosal regeneration. The degree of damage to the intestinal mucosa, as well as the development of the disease, are most reliably determined by colonoscopy, but this invasive diagnostic method cannot be performed frequently. The determination of fecal calprotectin is the most commonly used alternative diagnostic parameter for monitoring the course of the disease and the success of therapy. However, the lack of validated cut-off and the lack of specificity of this test indicate the need for the introduction of new, non-invasive diagnostic methods that can evaluate the success of therapy and the degree of regeneration of diseased mucosa. Our results indicate that the monitoring of local and systemic parameters of ulcerative colitis can be observed by monitoring the concentrations of immunomodulatory factors in gingival fluid, serum and feces. MSCs have been used in the treatment of ulcerative colitis for their immunomodulatory properties, but the results are bidirectional. As one of the most significant sources of IDO, we examined the role of IDO/kynurenine pathway in immunomodulation of mesenchymal stem cells in a model of DSS-induced colitis. After application of MSCs in C57BL/6 strain mice, 6 they polarized the immune response and transferred the medium from proinflammatory to immunosuppressive environment, where Treg lymphocyte expansion suppressed inflammation and allowed regeneration. On the other hand, the application of MSCs in animals of the BALBc strain contributed to the overall exacerbation of the disease followed by an increase in inflammation parameters