Mirjana Mihailović

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.

Methanol extract from the stem of Cotinus coggygria Scop., and its major bioactive phytochemical constituent myricetin modulate pyrogallol-induced DNA damage and liver injury

The present study was undertaken to investigate the hepatoprotective effect of the methanol extract of Cotinus coggygria Scop. in rats exposed to the hepatotoxic compound pyrogallol. Assessed with the alkaline version of the comet assay, 1000 and 2000mg/kg body weight (bw) of the extract showed a low level of genotoxicity, while 500mg/kg bw of the extract showed no genotoxic potential. Quantitative HPLC analysis of phenolic acids and flavonoids in the methanol extract of C. coggygria showed that myricetin was a major component. To test the hepatoprotective effect, a non-genotoxic dose of the C. coggygria extract and an equivalent amount of synthetic myricetin, as present in the extract, were applied either 2 or 12h prior to administration of 100mg/kg bw of pyrogallol. The extract and myricetin promoted restoration of hepatic function by significantly reducing pyrogallol-induced elevation in the serum enzymes AST, ALT, ALP and in total bilirubin. As measured by the decrease in total score and tail moment, the DNA damage in liver was also reduced by the extract and by myricetin. Our results suggest that pro-surviving Akt activity and STAT3 protein expression play important roles in decreasing DNA damage and in mediating hepatic protection by the extract. These results suggest that myricetin, as a major component in the extract, is responsible for the antigenotoxic and hepatoprotective properties of the methanol extract of C. coggygria against pyrogallol-induced toxicity.

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

Biochemical and pharmacological evaluation of 4-hydroxychromen-2-ones bearing polar C-3 substituents as anticoagulants.

The objective of this study was to investigate in vitro and in vivo anticoagulant activity of sixteen 4-hydroxycoumarin derivatives bearing polar C-3 scaffolds. The activity was evaluated by measuring prothrombin time. Enhanced anticoagulant activity in vitro was observed for all tested compounds. Upon successive administration of 0.5 mg/kg of body weight to adult Wistar rats, over a period of five days, four derivatives (2b, 4c, 5c and 9c) presented anticoagulant activity in vivo. The most active compound was 2b, with PT = 30.0 s. Low or non-toxic effects in vivo were determined based on the catalytic activity of liver enzymes and the concentration of bilirubin, iron and proteins. Metabolic pathways of the most active compounds in vivo were determined after GC/MS analysis of collected rat urine samples. The excretion occurs by glucuronidation of 7-hydroxy forms of tested derivatives. In vivo results were described using PLS-based CoMFA and CoMSIA 3D-QSAR studies, which showed CoMFA-SE (q(2) = 0.738) and CoMSIA-SEA (q(2) = 0.763) to be the statistically most relevant models. Furthermore, molecular docking and DFT mechanistic studies performed on the rat VKORC1 homology model revealed interactions between the 4-OH coumarin group in the form of phenolic anion and the Cys135 catalytic site in the transition state.

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.

C/EBPα AND C/EBPβ ARE PERSISTENTLY ASSOCIATED WITH THE RAT LIVER NUCLEAR MATRIX THROUGHOUT DEVELOPMENT AND THE ACUTE PHASE RESPONSE

The partitioning of C/EBPα and C/EBPβ on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPα and C/EBPβ was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage‐ and AP‐related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPα decreased and C/EBPβ increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.

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.

Study of genotoxicity and antigenotoxicity of the Cotinus coggygria Scop. methanol extract by Drosophila melanogaster sex-linked recessive lethal test

The genotoxic and antigenotoxic effects of Cotinus coggygria Scop. methanol extract was investigated using the Drosophila sex-linked recessive lethal (or SLRL) test. The results presented here show that the methanol extract of Cotinus coggygria in a concentration of 5% and artificial chemical agent ethyl methanesulfonate EMS (0.75 ppm) induce recessive lethal mutations on X-chromosome on Drosophila melanogaster in all broods (I, II and III). Post-treatment with lower concentration of the methanol extract of Cotinus coggygria (2%) was effective in reducing genotoxicity of mutagen.