The radioprotective efficacy of the rat acute-phase protein alpha2-macroglobulin on bone marrow cells

The rat acute phase protein α2-macroglobulin (α2M) plays an important role in the restoration of disrupted homeostasis by inhibiting different types of non-specific proteases and facilitating the transport of cytokines, growth factors and hormones. Previously, we observed that administration of α2M to experimental animals prior to the infliction of life- threatening trauma in the form of scalding or total-body irradiation, significantly improved their survival rates. The aim of the present work was to evaluate the radioprotective effect on blood cells of α2M that, when administered 30 min before irradiation with 6.7 Gy (LD50/30), provides 100% survival of experimental animals where in unprotected irradiated rats the said dose results in 50% lethality. We observed that rats pretreated with α2M, after an initial decline, exhibited complete recovery of the leukocyte count due to the preservation of bone marrow cells, observed as a stable mitotic index. In untreated irradiated rats the decrease of the mitotic index reflected the significant destruction of bone marrow cells that resulted in a protracted decline in the leukocyte count. We conclude that the radioprotection provided by α2M was in part mediated through cytoprotection of new blood cells produced in the bone marrow.Pacovski alfa2-makroglobulin (α2M) ima važnu ulogu u uspostavljanju narušene homeostaze inhibicijom različitih tipova nespecifičnih proteaza i olakšavajući transport citokina, hormona rasta i hormona. Naša ranija istraživanja su pokazala da administracija α2M eksperimentalnim životinjama u traumama tipa opekotine ili ozračivanja celog organizma značajno povećava njihovu stopu preživljavanja. Cilj ove studije je bio izučavanje radioprotektivne uloge α2M na ćelije kosne srži. α2M je apliciran 30 minuta pre ozračivanja pacova dozom od 6.7 Gy (LD50/30) X-zraka i omogućio je 100% preživljavanje pacova za razliku od ozračenih pacova bez tretmana kod kojih je smrtnost bila 50%. Rezultati su pokazali da tretiranje životinja sa α2M, nakon inicijalnog pada, omogućavaju potpuni oporavak broja leukocita kao posledica očuvanja ćelija kosne srži, što se uočava preko stabilnog mitotskog indeksa. Kod ozračenih pacova bez tretmana signifikantno smanjenje mitotskog indeksa, kao posledica narušavanja ćelija kosne srži, rezultuje i u prolongiranom padu broja leukocita. Na osnovu ovih rezultata može se zaključiti da se radioprotektivna uloga α2M delom odvija putem citoprotekcije novih krvnih ćelija u kosnoj srži.Projekat ministarstva br. 143002

Transcription factor p53 exhibits increased binding to the A2-macroglobulin gene promoter and decreased glycosylation in fetal and adult rat liver during the acute-phase response

The binding affinity of p53 for the MG promoter was assessed by DNA-affinity chromatography with the extended α2-macroglobulin (MG) gene promoter (-852/+12) and immunoblot analysis. During the increased MG gene transcription observed in the fetus and the acute-phase (AP) response in both the fetus and the adult, p53 exhibited increased binding to the MG promoter. This increase was accompanied by decreased O-linked N-acetyl glucosamine glycosylation of p53. We suggest that the enzymatic removal of sugar moieties in vivo serves to activate the MG gene promoter binding potential of p53 and its participation in upregulated MG gene transcription.DNK afinitetna hromatografija, sa promotorskim regionom gena za alfa2-makroglobulin (MG) (-852/+12), i imunoblot analiza su pokazale povećan afinitet vezivanja p53 za promotorski region gena za MG u kontrolnoj jetri fetusa, kao i ulogup53 u transkripcionoj regulaciji gena za MG i u fetalnoj i adultnoj jetri tokom akutno-faznog odgovora (AFO). Povećanje vezivanja p53 za promotorski region gena za MG je praćeno smanjenjem stepena glikozilacije p53. Rezultati sugerišu da u in vivo uslovima uklanjanje šećernih ostataka sa p53 omogućava njegovo vezivanje za promotorski region MG gena, kao i ulogu u transkripcionoj aktivaciji ispitivanog gena tokom AFO.Projekat ministarstva br. 143002

Korelacija između koncentracije receptora 2 faktora nekroze tumora u serumu i destrukcije parodoncijuma kod bolesnika sa dijabetes melitusom tip 2 - studija preseka

Introduction: The role of tumor necrosis factor-α (TNFα) is well documented in pathogenesis of chronic periodontitis (CP) and type 2 diabetes (T2D). Considering short half-life of TNFα, tumor necrosis factor receptor-2 (TNFR2) is used as prosperous surrogate marker of TNFα activity. Objective The aim was to detect TNFR2 serum concentration and correlate it with periodontal destruction in patients with diagnosed T2D and nondiabetics. Methods The study included 85 patients divided into three groups: T2D + CP (group T2D, n = 34); nondiabetics + CP (Group PD, n = 27); and healthy controls (group HC, n = 24). T2D was diagnosed according to WHO criteria (2013) and periodontitis was diagnosed using International Workshop for a Classification of Periodontal Diseases and Conditions criteria (1999). TNFR2 level was measured by enzyme-linked immunosorbent assay (ELISA). Results There was no difference in TNFR2 level among the groups (Kruskal-Wallis, p = 0.482). Significant correlation (Pearson's correlation coefficient) was observed between clinical attachment loss (CAL) and TNFR2 concentration in PD group (rp = -0.460, p = 0.016). In T2D group, correlations were observed between TNFR2 concentration and CaL (rp = 0.363, p = 0.005) and periodontal inflamed surface area (PISA) (rp = 0.345, p = 0.046) and periodontalepithelial surface area (PESA) (rp = 0.578, p = 0.000). Conclusion Higher concentration of TNFR2 was associated with higher CAL, PESA, and PISA scores in T2D group. Contrary to that, nondiabetics with higher values of CAL exhibited lower concentration of TNFR2, presenting potential protective effect on periodontal destruction. These results imply that diabetes may alter TNFR2 secretion originated from periodontium.Uvod: Uloga faktora nekroze tumora-alfa (TNFα) dokazana je u patogenezi hronične parodontopatije (HP) i dijabetesa melitusa tipa 2 (DM tip 2). S obzirom na to da je poluživot TNFα veoma kratak, receptor 2 faktora nekroze tumora (TNFR2) koristi se kao marker aktivnosti TNFα. Cilj rada Cilj ovog rada je određivanje koncentracije TNFR2 u serumu i koreliranje sa parametrima destrukcije parodoncijuma kod zdravih i ispitanika sa dijagnostikovanim DM tip 2. Metode rada U studiju je uključeno 85 pacijenata podeljenih u tri grupe: DM tip 2 + HP (DM grupa, n = 34), zdravi ispitanici + HP (PD grupa, n = 27) i zdrave kontrole (ZK grupa, n = 24). Dijagnoza DM tip 2 postavljena je na osnovu kriterijuma SZO (2013), dok je dijagnoza HP postavljena na osnovu kriterijuma Internacionalne radionice za klasifikaciju stanja i oboljenja parodoncijuma (1999). Koncentracija TNFR2 merena je ELISA metodom. Rezultati Koncentracija serumskog TNFR2 nije se razlikovala među grupama (Kraskal-Volis, p = 0,482). Postoji značajna korelacija (Pirson) između nivoa pripojnog epitela (NPE) i koncentracije TNFR2 u PD grupi (rp = -0,460, p = 0,016). U DM tip 2 grupi, statistički značajna korelacija uočena je između koncentracije TNFR2 i NPE (rp = 0,363, p = 0,005), kao i parametara uticaja inflamacije iz parodoncijuma na sistemsko zdravlje - PISA (rp = 0,345, p = 0,046) i PESA (rp = 0,578, p = 0,000). Zaključak Kod pacijenata sa dijabetesom veće koncentracije TNFR2 odgovaraju većim vrednostima NPE, PESA i PISA. Nasuprot tome, kod sistemski zdravih ispitanika sa HP veće vrednosti NPE su povezane sa manjim koncentracijama TNFR2, što bi moglo govoriti o potencijalnoj zaštitnoj ulozi ovog citokina na destrukciju parodoncijuma. Rezultati govore da dijabetes može uticati na sekreciju TNFR2 iz parodoncijuma

Dynamic associations of transcription factors with the rat liver nuclear matrix are functionally related to differential alpha-2-macroglobulin gene expression

Participation of the nuclear matrix in regulation of alpha-2-macroglobulin (α2M) gene transcription during rat liver development and the acute-phase (AP) response are examined. DNA affinity chromatography of fetal and adult liver internal nuclear matrix proteins under basal and AP conditions with the α2M gene promoter (-852/+12) and immunoblot analysis revealed diverse patterns of association of transcription factors with the nuclear matrix. HNF-6, C/EBPα, and STAT5b were involved in basal and C/EBPβ, STAT1, and STAT3 in AP-stimulated α2M expression. These findings support the assumption that transcription factor-nuclear matrix interactions serve to channel gene regulatory proteins to DNA sequences.Cilj rada je ispitivanje učešća jedarnog matriksa u regulaciji transkripcije gena za alfa-2-makroglobulin tokom razvića jetre pacova i akutno faznog odgovora (AFO). Nakon DNK afinitetne hromatografije proteina unutrašnje mreže jedarnog matriksa fetalne i adultne jetre, u bazalnim i AFO uslovima, sa promotorskim elementom gena za α2M (-852/+12) i imunoblot analize, identifikovane su dinamičke asocijacije transkripcionih faktora uključenih u regulaciju ekspresije gena za α2M sa jedarnim matriksom. HNF-6, C/EBPα, STAT5b su uključeni u regulaciju bazalne ekspresije gena za α2M, dok C/EBPβ, STAT1, STAT3 posreduju u regulaciji ekspresije ovog gena tokom AFO. Opisane interakcije doprinose razumevanju predloženih mehanizama kojima se transkripcioni faktori usmeravaju ka ciljnim regulatornim elementima DNK.Projekat ministarstva br. 143002

Ameliorating effects of antioxidative compounds from four plant extracts in experimental models of diabetes

Given that oxidative stress plays a major role in pancreatic β-cell dysfunction and ultimate destruction, as well as in different complications of diabetes, therapy with antioxidants has assumed an important place in the management of diabetes. The relatively limited effects of established antioxidant compounds have stimulated efforts to develop new therapeutic strategies, e.g. to increase the endogenous antioxidant defences through pharmacological modulation of key antioxidant enzymes. Plant extracts are gaining popularity in treating diabetes because many substances synthesized by higher plants and fungi possess antioxidant activities and can prevent or protect tissues against the damaging effects of free radicals. This review summarizes experimental models of diabetes and possible mechanisms that lie behind the antioxidative effects of α-lipoic acid (LA), a powerful antioxidant and compound that stimulates cellular glucose uptake, as well as of plant extracts from sweet chestnut (Castanea sativa), edible mushroom (Lactarius deterrimus) and natural products containing β-glucans in the treatment of diabetes. Their roles in preventing pancreatic β-cell death and in ameliorating the effects of severe diabetic complications are discussed.Terapija antioksidansima zauzima značajno mesto u lečenju dijabetesa s obzirom da oksidativni stres u velikoj meri doprinosi narušavanju funkcije i strukture β-ćelija pankreasa kao i razvoju komplikacija u dijabetesu. Zbog ograničenog dejstva postojećih antioksidativnih jedinjenja traga se za novim terapijskim rešenjima u tretmanu dijabetesa, kao što je povećanje endogene antioksidativne zaštite organizma putem farmakološke modulacije ključnih antioksidativnih enzima. Primena biljnih ekstrakata u lečenju dijabetesa postaje sve popularnija. Mnoge supstance koje se nalaze u sastavu viših biljaka i gljiva poseduju antioksidativna svojstva koja mogu da zaštite tkiva od štetnih uticaja slobodnih radikala. U ovom revijalnom radu opisani su eksperimentalni modeli dijabetesa kao i mogući mehanizmi koji leže u osnovi antioksidativnog dejstva α-liponske kiseline (LA), snažnog antioksidansa i jedinjenja koje stimuliše ćelijsku apsorpciju glukoze, kao i biljnih ekstrakata izolovanih iz slatkog kestena (Castanea sativa), jestivih pečuraka (Lactarius deterrimus) i prirodnih proizvoda koji sadrže β-glukan u lečenju dijabetesa. Opisani su njihova uloga u sprečavanju smrti β-ćelija pankreasa kao i blagotvorno dejstvo na komplikacije u dijabetesu.Projekat ministarstva br. 17302

Lymphocytes' 'Last Stand' on the Nuclear Matrix After Whole Body Exposure of Rats To Low-Let Ionizing Radiation

We examined the functions of the rat lymphocyte nuclear matrix after a single exposure to total body irradiation with doses ranging from sublethal to lethal. Irradiation induced systemic oxidative stress, detected as increased activities of serum SOD and catalase, lymphocyte DNA damage, detected by the Comet assay, and apoptosis. After irradiation with lower doses, the recruitment of DNA repair centers on the matrix was observed by Western analysis as increased levels of matrix-associated PARP-1, p53 and PCNA. Augmented partitioning of the pro-survival transcription factor NF-κB on the matrix was also detected after irradiation. Exposure to a lethal dose caused breakdown of the matrix, observed as lamin B cleavage, and of the matrix-associated DNA repair centers, detected as caspase-mediated PARP-1 proteolysis and loss of protein associations with the matrix. These findings suggest that the nuclear matrix establishes functional 2 interactions in a defensive mechanism, integrated in a decision-making process that resolves cell fat

PARP-1 and YY1 Are Important Novel Regulators of CXCL12 Gene Transcription in Rat Pancreatic Beta Cells

Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12) transcription. The roles of poly(ADP-ribose) polymerase-1 (PARP-1) and transcription factor Yin Yang 1 (YY1) in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ)-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the functional interplay of these proteins could finely balance Cxcl12 transcription

Treatment of streptozotocin-induced diabetic rats with Castanea sativa and Lactarius deterrimus extracts decreases liver damage by initiating activation of the Akt prosurvival kinase

Diabetes is the most important non-infectious disease affecting 5% of the general population. Different plant and mushroom extracts with hypoglycemic and antioxidant properties have been used traditionally as antidiabetic herbal medicines. The aim of this study was to study the in vivo effect of extracts obtained from the edible mushroom, Lactarius deterrimus (Ld), and chestnut, Castanea sativa (Cs), on the alleviation of liver damage in streptozotocin (STZ)-induced diabetic rats. The extracts were applied, either alone or in combination, for four weeks, starting from the last day of STZ administration. Diabetic rats treated with the extracts exhibited reduced hyperglycemia and lower hepatic oxidative stress. Extract treatment decreased the level of O-linkage of N-acetylglucosamine modified superoxide dismutase, catalase and NF-κB. Masson trichrome staining showed a decrease in collagen fiber deposition in the liver. Immunoblot analysis revealed the activation of the prosurvival Akt kinase after extract application. The obtained results revealed that the hyperglycemia-reducing and antioxidant effects of the Ld and Cs extracts suppressed cytotoxic signaling pathways, attenuating the negative effects of diabetes on the liver. The examined extracts are beneficial in the prevention of liver damage and could be considered for prediabetes and diabetes management after a definitive phytochemical description of extract constituents and subsequent evaluation in preclinical and clinical studies

CXC Chemokine Ligand 12 Protects Pancreatic β-Cells from Necrosis through Akt Kinase-Mediated Modulation of Poly(ADP-ribose) Polymerase-1 Activity

The diabetes prevention paradigm envisages the application of strategies that support the maintenance of appropriate β-cell numbers. Herein we show that overexpression of CXC chemokine ligand12 (CXCL12) considerably improves the viability of isolated rat Langerhans islet cells and Rin-5F pancreatic β-cells after hydrogen peroxide treatment. In rat islets and wt cells hydrogen peroxide treatment induced necrotic cell death that was mediated by the rapid and extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1). In contrast, CXCL12-overexpressing cells were protected from necrotic cell death as a result of significantly reduced PARP-1 activity. CXCL12 downstream signalling through Akt kinase was responsible for the reduction of PARP-1 activity which switched cell death from necrosis to apoptosis, providing increased protection to cells from oxidative stress. Our results offer a novel aspect of the CXCL12-mediated improvement of β-cell viability which is based on its antinecrotic action through modulation of PARP-1 activity

Beneficial effects of α-lipoic acid in diabetes- and drug- induced liver injury

This review summarizes the effects of α-lipoic acid (LA) on liver damage and complications in diabetes and drug toxicity. LA is a naturally occurring dithiol compound that plays an essential role in mitochondrial metabolism in its protein-bound form. In contrast, free LA in supplements has diverse biological actions, and its antioxidant effect is its most studied and important activity. Due to its strong antioxidant potential, LA could have a promising role in the treatment of pathologies resulting from an imbalance in redox homeostasis. This includes diabetes, which produces deleterious effects on many organs, including the liver. In diabetes specifically, LA prevents β-cell destruction, enhances glucose uptake, and its antioxidant effects may be particularly useful in slowing down the development of diabetic complications. Diabetes-related liver damage is a serious complication in which oxidative stress is the main contributor to tissue injury. Oxidative stress is regarded as one of the main pathological mechanisms underlying liver pathologies provoked by other insults, such as drug toxicity, where LA could also be a useful agent in therapeutic intervention. However, before wider application of LA in a clinical setting, experimental and clinical research needs to be extended