Aleksandra Uskoković

Hepatoprotective effects of Gentiana asclepiadea L. extracts against carbon tetrachloride induced liver injury in rats

This study is an attempt to evaluate the hepatoprotective activity of Gentiana asclepiadea L. against carbon tetrachloride-induced liver injury in rats. Methanol extracts of aerial parts (GAA) and roots (GAR) of G. asclepiadea at doses of 100, 200, and 400mg/ kg b.w. were orally administered to Wistar rats once daily for 7 days before they were treated with CCl(4). The hepatoprotective activity of the extracts in this study was compared with the reference drug silymarin. In CCl(4) treated animals, GAA and GAR significantly decreased levels of serum transaminases, alkaline phosphatase and total bilirubin, and increased the level of total protein. Treatment with the extracts resulted in a significant increase in the levels of catalase, superoxide dismutase and reduced glutathione, accompanied with a marked reduction in the levels of malondialdehyde, as compared to CCl(4) treated group. The histopathological studies confirmed protective effects of extracts against CCl(4)-induced liver injuries. No genotoxicity was observed in liver cells after GAA treatment, while GAR showed only slight genotoxic effects by comet assay. Phytochemical analysis revealed the presence of sweroside, swertiamarin and gentiopicrin in high concentrations in both extracts. It could be concluded that the use of G. asclepiadea extracts in the treatment of chemical-induced hepatotoxicity.

Decreased O -GlcNAcylation of the key proteins in kinase and redox signalling pathways is a novel mechanism of the beneficial effect of α-lipoic acid in diabetic liver

The present study aimed to investigate the effects of the treatment with a-lipoic acid (LA), a naturally occurring compound possessing antioxidant activity, on liver oxidant stress in a rat model of streptozotocin (STZ)-induced diabetes by examining potential mechanistic points that influence changes in the expression of antioxidant enzymes such as catalase (CAT) and CuZn/Mn superoxide dismutase(s) (SOD). LA was administered for 4 weeks by daily intraperitoneal injections (10 mg/kg) to STZ-induced diabetic rats, starting from the last STZ treatment. LA administration practically normalised the activities of the indicators of hepatocellular injury, alanine and aspartate aminotransferases, and lowered oxidative stress, as observed by the thiobarbituric acid-reactive substance assay, restored the reduced glutathione:glutathione disulphide ratio and increased the protein sulfhydryl group content. The lower level of DNA damage detected by the comet assay revealed that LA reduced cytotoxic signalling, exerting a hepatoprotective effect. The LA-treated diabetic rats displayed restored specific enzymatic activities of CAT, CuZnSOD and MnSOD. Quantitative real-time PCR analysis showed that LA restored CAT gene expression to its physiological level and increased CuZnSOD gene expression, but the gene expression of MnSOD remained at the diabetic level. Although the amounts of CAT and CuZnSOD protein expression returned to the control levels, the protein expression of MnSOD was elevated. These results suggested that LA administration affected CAT and CuZnSOD expression mainly at the transcriptional level, and MnSOD expression at the post-transcriptional level. The observed LA-promoted decrease in the O-GlcNAcylation of extracellular signal-regulated kinase, protein 38 kinase, NF-kB, CCAAT/enhancer-binding protein and the antioxidative enzymes themselves in diabetic rats suggests that the regulatory mechanisms that supported the changes in antioxidative enzyme expression were also influenced by post-translational mechanisms.

STAT3/NFκB Interplay in the Regulation of α2‐Macroglobulin Gene Expression During Rat Liver Development and the Acute Phase Response

The synthesis of alpha‐2‐macroglobulin (α2M) is low in adult rat liver and elevated in fetal liver. During the acute‐phase (AP) response it becomes significantly increased in both adult and fetal liver. In this work, the cross talk of STAT3 and NF‐κB transcription factors during α2M gene expression was analysed. Using immunoblotting, their cellular compartmentalization was examined by comparing the cytoplasmic levels of STAT3 and NF‐κB with their active equivalents, the 86 and 91 kDa isoforms and p65‐subunit, respectively, in the nuclear extract and nuclear matrix. Different partitioning dynamics of the transcription factors were observed. At the level of protein‐DNA interactions, studied by α2M promoter affinity chromatography, it was established that different ratios of promoter‐binding STAT3 isoforms participated in elevated hepatic transcription in the basal state fetus and the AP‐adult, but only the 91 kDa isoform in the AP‐fetus. Unchanged levels of DNA‐bound p65 in the control and AP‐fetus suggest that it participated in constitutive transcription. The promoter‐binding of p65 observed in the AP‐adult suggests that it was involved in transcriptional stimulation of α2M expression. The selective enrichment of the AP‐adult nuclear matrix with promoter‐binding STAT3 disclosed the importance of this association in the induction of transcription. Protein‐protein interactions were examined by co‐immunoprecipitation. Interactions between the 86 kDa STAT3 isoform and p65 that were observed in the control and AP‐fetus and of both the 86 and 91 kDa STAT3 isoforms with p65 in the AP‐adult, suggest that protein‐protein interactions were functionally connected to increased transcription. We concluded that α2M gene expression is driven by developmental‐ and AP‐related mechanisms that rely on STAT3/NF‐κB interplay. IUBMB Life, 59: 170‐178, 2007

Association of the glucocorticoid receptor with STAT3, C/EBPβ, and the hormone-responsive element within the rat haptoglobin gene promoter during the acute phase response

Upregulation of haptoglobin (Hp) expression in the rat during the acute phase (AP) response is the result of synergistic effects of IL‐6–, IL‐1β–, and corticosterone‐activated signaling pathways. IL‐6 signaling terminates in cis–trans interactions of the Hp gene hormone‐responsive element (HRE) with transcription factors STAT3 and C/EBPβ. The aim of this study was to examine the unresolved molecular mechanism of glucocorticoid action. A 3‐fold rise in serum corticosterone at 2 and 4 h of the AP response induced by turpentine administration preceded a 2.3‐fold increase in the rate of Hp gene transcription at 12 h that was accompanied by a 4.8‐fold increase in glucocorticoid receptor (GR), the appearance of an 86‐kDa STAT3 isoform and 3.9‐, 1.9‐, and 1.7‐fold increased amounts of 91‐kDa STAT3, 35‐ and 42‐kDa C/EBPβ isoforms in the nucleus. These events resulted in 4.6‐ and 2.5‐fold increased Hp levels in the liver and serum at 24 h. HRE affinity chromatography and immunoblot analysis revealed that maximal occupancy of the HRE with GR, STAT3, and C/EBPβ at 12 h correlated with increased transcriptional activity of the Hp gene. Coimmunoprecipitation experiments showed that activated GR established de novo interaction with STAT3 isoforms while GR–C/EBPβ interactions observed during basal transcription increased during the AP response. Computer analysis of the HRE disclosed two potential GR‐binding sites: one overlapping STAT3, another adjacent to a C/EBPβ‐binding site. This finding and the experimental results suggest that activated GR through direct interactions with STAT3 and C/EBPβ, participates in Hp gene upregulation as a transcriptional coactivator.

Proteolytic events in cryonecrotic cell death: Proteolytic activation of endonuclease P23 ☆

Although cryosurgery is attaining increasing clinical acceptance, our understanding of the mechanisms of cryogenic cell destruction remains incomplete. While it is generally accepted that cryoinjured cells die by necrosis, the involvement of apoptosis was recently shown. Our studies of liver cell death by cryogenic temperature revealed the activation of endonuclease p23 and its de novo association with the nuclear matrix. This finding is strongly suggestive of a programmed-type of cell death process. The presumed order underlying cryonecrotic cell death is addressed here by examining the mechanism of p23 activation. To that end, nuclear proteins that were prepared from fresh liver, which is devoid of p23 activity, were incubated with protein fractions isolated from liver exposed to freezing/thawing that possessed a presumed p23 activation factor. We observed that the activation of p23 was the result of a proteolytic event in which cathepsin D played a major role. Different patterns of proteolytic cleavage of nuclear proteins after in vitro incubation of nuclei and in samples isolated from frozen/thawed liver were observed. Although both processes induced p23 activation, the incubation experiments generated proteolytic hallmarks of apoptosis, while freezing/thawing of whole liver resulted in typical necrotic PARP-1 cleavage products and intact lamin B. As an explanation we offer a hypothesis that after freezing, cells possess the potential to die through necrotic as well as apoptotic mechanisms, based on our finding that the cytosol of cells exposed to cryogenic temperatures contains both necrotic and apoptotic executors of cell death.

The Importance of the CXCL12/CXCR4 Axis in Therapeutic Approaches to Diabetes Mellitus Attenuation

The pleiotropic chemokine (C–X–C motif) ligand 12 (CXCL12) has emerged as a crucial player in several diseases. The role of CXCL12 in diabetes promotion and progression remains elusive due to its multiple functions and the overwhelming complexity of diabetes. Diabetes is a metabolic disorder resulting from a failure in glucose regulation due to β-cell loss and/or dysfunction. In view of its ability to stimulate the regeneration, proliferation, and survival of β-cells, as well as its capacity to sustain local immune-isolation, CXCL12 has been considered in approaches aimed at attenuating type 1 diabetes. However, a note of caution emerges from examinations of the involvement of CXCL12 in the development of diabetes and its complications, as research data indicate that CXCL12 displays effects that range from protective to detrimental. Therefore, as a beneficial effect of CXCL12 in one process could have deleterious consequences in another, a more complete understanding of CXCL12 effects, in particular its functioning in the cellular microenvironment, is essential before CXCL12 can be considered in therapies for diabetes treatment.

CXCL12 protects pancreatic β-cells from oxidative stress by a Nrf2-induced increase in catalase expression and activity

Due to intrinsically low levels of antioxidant enzyme expression and activity, insulin producing pancreatic β-cells are particularly susceptible to free radical attack. In diabetes mellitus, which is accompanied by high levels of oxidative stress, this feature of β-cells significantly contributes to their damage and dysfunction. In light of the documented pro-survival effect of chemokine C-X-C Ligand 12 (CXCL12) on pancreatic β-cells, we examined its potential role in antioxidant protection. We report that CXCL12 overexpression enhanced the resistance of rat insulinoma (Rin-5F) and primary pancreatic islet cells to hydrogen peroxide (H2O2). CXCL12 lowered the levels of DNA damage and lipid peroxidation and preserved insulin expression. This effect was mediated through an increase in catalase (CAT) activity. By activating downstream p38, Akt and ERK kinases, CXCL12 facilitated Nrf2 nuclear translocation and enhanced its binding to the CAT gene promoter, inducing constitutive CAT expression and activity that was essential for protecting β-cells from H2O2.

Synthesis and toxicological studies of in vivo anticoagulant activity of novel 3-(1-aminoethylidene)chroman-2,4-diones and 4-hydroxy- 3-(1-iminoethyl)-2H-chromen-2-ones combined with a structure-based 3-D pharmacophore model

Eight synthesized 3-(1-aminoethylidene)chroman-2,4-diones and 4-hydroxy-3-(1-iminoethyl)-2H-chromen-2-ones were evaluated as in vivo anticoagulants by intraperitoneal application to adult male Wistar rats in order to examine their pharmacological potential, evaluate ther toxicity and propose the mechanism of action. Two of them, 2f and 2a, in concentration of 2mg/kg of body weight, presented remarkable activity (PT=130s; PT=90s) upon seven days of continuous application. The results of rat serum and liver biochemical screening, as well those of histopathological studies, proved the compounds to be non-toxic. Activity of the compounds was further examined on the molecular level. Here, molecular docking studies were performed to position the compounds in relation to the active site of VKORC1 and determine the bioactive conformations. Docking results suggested a non-covalent mode of action during which the proton transfer occurs from Cys135 SH towards 4-carbonyl group of anticoagulant. All crucial interactions for anticoagulant activity were confirmed in generated structure-based 3-D pharmacophore model, consisted of hydrogen bond acceptor and hydrophobic aromatic features, and quantified by a best correlation coefficient of 0.97.

The protective effect of a mix of Lactarius deterrimus and Castanea sativa extracts on streptozotocin-induced oxidative stress and pancreatic β-cell death

Pancreatic β-cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus. In the present study, we tested extracts from the edible mushroom Lactarius deterrimus and the chestnut Castanea sativa, as well as their mixture (MIX Ld/Cs), for potential beneficial effects on streptozotocin (STZ)-induced pancreatic β-cell death. Analysis of chelating effects, reducing power and radical-scavenging assays revealed strong antioxidant effects of the C. sativa extract and MIX Ld/Cs, while the L. deterrimus extract displayed a weak to moderate effect. The antioxidative effect of the chestnut extract corresponds with the high content of phenolics and flavonoids identified by HPLC analysis. In contrast, the mushroom extract contains relatively small amounts of phenols and flavonoids. However, both extracts, and especially their combination MIX Ld/Cs, increased cell viability after the STZ treatment as a result of a significant reduction of DNA damage and improved redox status. The chestnut extract and MIX Ld/Cs significantly lowered the STZ-induced increases in superoxide dismutase and catalase activities, while the mushroom extract had no impact on the activities of these antioxidant enzymes. However, the L. deterrimus extract exhibited good NO-scavenging activity. Different mechanisms that underlie antioxidant effects of the mushroom and chestnut extracts were discussed. When combined as in the MIX Ld/Cs, the extracts exhibited diverse but synergistic actions that ultimately exerted beneficial and protective effects against STZ-induced pancreatic β-cell death.

SOLUBILITY PARTITIONING OF C/EBPβ ON THE RAT HEPATOCYTE NUCLEAR MATRIX BY HYDROPHOBIC INTERACTIONS

The greatest part of nuclear C/EBPβ (a major 35kD protein, 30 and 38kD isoforms) was observed to partition with the nuclear matrix. Cross‐linking experiments with formaldehyde suggested that the association reflected the in situ juxtapositioning of C/EBPβ to nuclear matrix proteins in isolated nuclei. The association of C/EBPβ with the nuclear matrix resisted RNase and DNase treatment and extraction with protein sulfhydryl reducing agents combined with high ionic strength salt. C/EBPβ displayed a proclivity to extensively reassemble with the filament‐forming nuclear matrix proteins after a cycle of solubilization with urea, followed by its removal by dialysis. These findings suggest that the C/EBPβ moieties were anchored to the nuclear matrix through hydrophobic protein‐protein interactions with the lamins. Subsequent separation of nuclear matrix‐associated C/EBPβ into insoluble, reassembling, and soluble nuclear matrix protein (SNMP) fractions after a cycle of solubilization/reassembly pointed to the sub‐partitioning of C/EBPβ on the nuclear matrix. DNA affinity chromatography using the rat haptoglobin gene cis ‐element and SNMP revealed the binding of p35 during basal transcription, and p35 and p30 during elevated haptoglobin gene transcription in the course of the acute‐phase (AP) response. It was concluded that the appearance of cis ‐element‐binding p30 in the SNMP fraction resulted from its increased solubility (decreased hydrophobicity) and inability to reassociate with the lamins during urea removal. The observed solubility partitioning of C/EBPβ on the nuclear matrix framework could represent a level of control of the general availability of regulatory proteins for establishing interactions with DNA.