Uticaj vodenog, metanolnog i etil-acetatnog ekstrakta grčkog origana Origanum vulgare ssp. hirtum na dijabetes tip 1 indukovan višestrukim malim dozama streptozotocina kod C57BL/6 miševa
Effect of aqueous, methanolic and ethyl acetate extract of Origanum vulgare ssp. hirtum on type 1 diabetes induced by multiple low doses of streptozotocin in C57BL/6 mice
Doctoral thesis (Published version)
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Type 1 diabetes (T1D) is autoimmune disorder which develops as a consequence of destruction of insulin-producing pancreatic beta cells which is induced by pro-inflammatory mediators. Beta cell destruction leads to impaired insulin production and loss of glucose homeostasis. The hallmark of T1D is formation of insulitis- immune cells infiltration in pancreatic islets. Macrophages are the first cells that infiltrate pancreatic islets. It is considered that proinflammatory M1 macrophages contribute to T1D pathogenesis, while antiinflammatory M2 macrophages play a protective roll in T1D development. Macrophages can activate autoreactive CD4+ and CD8+ T lymphocites that are considered to be key players in T1D pathogenesis. Cytokines produced by immune cells lead to differentiation of specific T helper cells (Th) subsets, that can be destructive (Th1 and Th17) or protective (Th2 i Treg) in T1D development. Immune cells, by vast effector mechanisms, lead to formation of inflammatory milleu around pancreatic islets which ultimately stimulates beta cell apopotosis. Treatment for T1D includes lifetime intake of insulin which can potentially cause a number of short- and long term complications. This is why scientists invest significant efforts in developing new therapeutic approaches for T1D treatment. Plants and their extracts are promising candidates for treatment of T1D, due to their rich content of phenolic compounds which are proven to be beneficial for human health. Origanum vulgare ssp. hirtum (Greek oregano) is native plant rich in phenolic and ester compounds. It is used in traditional medicine as antiseptic, as well as in the treatment of respiratory and stomach ailments. Main known activities of oregano are antibacterial and antioxidant. Also, recently is reported anti-hyperglycaemic activity of oregano in the model of toxic diabetes. These facts make oregano a promising candidate for the treatment of T1D. This study for the first time investigates the effect of different oregano extracts (methanolic, aqueous and ethyl acetate) on multiple low doses streptozotocin (MLDS)-induced T1D in C57BL/6 mice. Also, cellular and molecular mechanisms of oregano extracts action on immune cells (macrophages and lymphocytes) and pancreatic beta cells were investigated both in vitro and in vivo. Additionaly, the effects of specific predominant substances found in oregano extracts on T1D pathogenesis were explored. Oregano extracts, in non-cytotoxic concentrations, showed immunomodulatory effects in vitro. Aqueous and ethyl acetate oregano extracts decreased inflammatory macrophage potential by reducing secretion of nitric oxide (NO) (aqueous oregano extract) and proinflammatory citokine IL-1β (aqueous and ethyl acetate oregano extracts). On the other hand, methanolic oregano extract decreased secretion of NO, while increasing secretion of IL-1β and TNF. Also, methanolic and ethyl acetate oregano extracts reduced in vitro lymphocite secretion of cytokines IL-2 (methanolic oregano extract), IFN-γ and IL-17 (methanolic and ethyl acetate oregano extracts). Besides the effect on immune cells in vitro, oregano extracts exhibited cytoprotective effects on pancreatic beta cells in vitro, protecting them from death induced by cytotoxic cytokines. MEO inhibited beta cell apoptosis by reducing acitivity of caspase 3. Prophylactic treatment with methanolic and ethyl acetate, but not aqueous oregano extract, reduced incidence of MLDS induced T1D in C57BL/6 mice. This was accompanied by preservation of beta cell number and function- methanolic and ethyl acetate oregano extracts inhibited infiltration of immune cells in pancreatic islets, while methanolic oregano extract also augmented production of oxidative mediators and kept insulin levels in the physiological range. Therapeutic treatment of T1D with methanolic oregano extract showed only transient effects. Detailed investigation of oregano extracts mechanisms of action showed that methanolic oregano extract ameliorated T1D by direct effect on Th17 cells and by shifting the balance from proinflammatory Th1/Th17 immune response towards protective Th2/Treg immune response. Ethyl acetate oregano extract, on the other hand, inhibited T1D by attenuating inflammatory M1/Th1/Th17 immune response, and by shifting macrophages towards protective M2 functional phenotype. Rosmarinic and salvianolic acid, as well as carvacrol, are most prevalent substances in methanolic and ethyl acetate oregano extracts. Although they showed immunomodulatory effects in vitro, they failed to protect mice from T1D development in vivo. Determination of one or more active principles of methanolic and ethyl acetate oregano extracts could be the subject of future studies. Results of this dissertation implicate methanolicic and ethyl-acetate oregano extracts as new potential candidates in the treatment of T1D, both in the preventive or combined therapeutic strategies.
Keywords:Type 1 diabetes; Greek oregano; Streptozotocin; Th cell differentiation; Cytokines; Beta cells apoptosis
Source:University of Belgrade, Faculty of Biology, 2016, 1-123
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