Evaluation of the modulation of nitric oxide synthase expression in the cerebellum of diabetic albino rats and the possible protective effect of ferulic acid.

INTRODUCTION: Diabetes mellitus is a multisystem disease. Oxidative stress and nitric oxide isoforms are involved in diabetic pathogenesis. Ferulic acid is a natural substance that is distributed broadly in plants with strong potent properties. THE AIM OF THE RESEARCH: This research was designed to study the possible protective role of ferulic acid on oxidative stress and different Nitric oxide synthase isoforms (NOS) in the cerebellum of streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Twenty-four albino male rats were randomly divided into equal four groups: control group, group 2 received ferulic acid orally (10 mg/kg), group 3 diabetic group, group 4 diabetic rats received ferulic acid. After 8 weeks, the left cerebellar hemisphere was taken for tissue homogenate for oxidative markers and real-time PCR for NOS isoforms. Paraffin sections of the right cerebellar hemisphere were stained with cresyl violet, Luxol fast blue and immnunohistochemically stained for neuronal NOS, inducible NOS and endothelial NOS. RESULTS: Degenerative changes were seen in the cerebella of the diabetic rats with significant elevation of Malondialdehyde, Nitric Oxide, and decrease of Superoxide dismutase levels. nNOS expression decreased and iNOS expression increased significantly. The ferulic acid-treated group showed a reduction of the degenerative changes in the cerebellum with significant improvement in oxidative stress marker, an increase of nNOS expression, and a decrease of iNOS expression. CONCLUSIONS: Ferulic acid improves cerebellar functional and histopathological changes induced by diabetes which can be attributed mainly to its anti-oxidative effect and its ability to modulate NOS isoforms.

Modulation of the Endothelin System in Colorectal Cancer Liver Metastasis: Influence of Epigenetic Mechanisms?

Targeting of endothelin system genes is a promising strategy in cancer therapy. The modulation of these genes was explored in a model of colorectal cancer (CRC) liver metastasis and in a panel of CRC tumor cell lines that were exposed to the demethylating agent decitabine. The CC531 rat model mimicking CRC liver metastasis was used for tumor cell re-isolation and analysis of the endothelin system genes and DNA methyltransferases (DNMTs) by microarray. To mimic the effects caused by methylation changes, a panel of seven CRC cell lines was treated with the demethylating agent decitabine. Three genes of the endothelin system were potently modulated at messenger RNA (mRNA) level in rat CC531 cells during liver colonization. The concomitant decrease of two DNMTs suggested an influence from altered methylation. Changes in gene expression were also accomplished by exposure of CRC cells to the demethylating agent decitabine, when using daily low concentrations for 3 days, with minimal cytotoxic effects. Sensitive human SW480 cells showed an almost 100fold upregulation of endothelin-1 mRNA compared to untreated cells. This, however, was different in LS174T cells, which showed no significant increase in gene expression although the methylation levels were significantly decreased at a variety of corresponding loci. We suggest that the mechanism induced by methylation on gene expression in metastatic CRC cells can be compromised. The results question the overall success of treating metastatic CRC by methylation inhibitors.

Effect of human umbilical cord blood derived CD34+ hematopoietic stem cell on the expression of Wnt4 and P53 genes in a rat model of hepatocellular carcinoma.

BACKGROUND AND AIM OF THE WORK: Hepatocellular carcinoma (HCC) is the most frequent primary liver malignancy. Chronic liver injuries as chronic hepatitis C and hepatitis B viruses, aflatoxins consumption and nonalcoholic fatty liver disease are well-established causes of HCC. HCC is associated with a series of molecular changes, as alternation in glypican-3, P53 expression and Wnt/ß-catenin pathway. Hepatic cancer progenitor cells could contribute to HCC development. This research aimed to study the effectiveness of human CD34+ hematopoietic stem cell on Wnt4 and P53 genes expression, histopathological grading and hepatic progenitor cells percentage in HCC rat model. MATERIALS AND METHODS: HCC was induced in the experimental group of outbred Sprague Dawley rats by administration of 50 mg/L N-nitroso-Di-Ethylamine (DEN) in drinking water for 15 weeks. Forty-six animals were used in total, they were initially subdivided into two groups; control (n = 6) and experimental (n = 40), the latter consisting of 4 DEN-unaffected, 6 DEN-lethalities and 30 surviving DEN-animals with elevated AFP. These 30 animals with elevated AFP were then subdivided into a new HCC control group (n = 15) and the stem cell treated group (n = 15). The latter group was injected with CD34+ human hematopoietic stem cell (1 × 106 cells/rat) in the rat's tail vein. Cyclosporine A (10 mg/kg) was injected intraperitoneal, starting 24 h before human stem cell transplantation. After 20 weeks passing since the beginning of the experiment, all rats were sacrificed and liver specimens were subjected to histopathological examination, RT-PCR in order to examine Wnt4 and P53 gene expression and flow cytometry to measure hepatic progenitor OV6 positive cells percentage. RESULTS: The saline-treated HCC group (with prior 15 week DEN exposure) showed higher levels of wnt4 and p53 gene expression (1.59 and 1.36 fold, respectively) and increased percentage in OV6+ progenitor cells (+4.9% in absolute terms) compared to saline-treated controls (p < 0.01, ANOVA). Compared with the saline HCC-group, transplantation with CD34+ human hematopoietic stem cells produced a further increase in the levels of wnt4 (+19.4%) and p53 gene expression (+53%), a 2-fold increase in the percentage of cancer progenitor cells and increased HCC pathology grading (all p < 0.01). The positive correlation between p53 and HCC grade (Spearman rho +0.73, p < 0.05) and negative correlation between wnt and OV6+% levels (rho -0.65, p < 0.05) in the saline-HCC group were not observed in the CD34+ HCC group. CONCLUSIONS: Human CD34+ cells transplantation has a deteriorating effect on HCC.

Effect of human umbilical cord blood stem cell transplantation on oval cell response in 2-AAF/CCL4 liver injury model: experimental immunohistochemical study.

BACKGROUND: Oval cells, specific liver progenitors, are activated in response to injury. The human umbilical cord blood (hUCB) is a possible source of transplantable hepatic progenitors and can be used in cases of severe liver injury. We detected the effect of hUCB stem cell transplantation on natural response of oval cells to injury. METHODS: Twenty-four female albino rats were randomly divided into three groups: (A) control, (B) liver injury with hepatocyte block, and (C) hUCB transplanted group. Hepatocyte block was performed by administration of 2-acetylaminofluorene (2-AAF) for 12 days. CCL4 was administrated at day 5 from experiment start. Animals were sacrificed at 9 days post CCL4 administration, and samples were collected for biochemical and histopathological analysis. Oval cell response to injury was evaluated by the percentage of oval cells in the liver tissue and frequency of cells incorporated into new ducts. RESULTS: Immunohistochemical analysis of oval cell response to injury was performed. There was significant deviation in the hUCB-transplanted (4.9 ± 1.4) and liver injury groups (2.4 ± 0.9) as compared to control (0.89 ± 0.4) 9 days post injury. Detection of oval cell response was dependant on OV-6 immunoreactivity. For mere localization of cells with human origin, CD34 antihuman immunoreactivity was performed. There was no significant difference in endogenous OV-6 immunoreactivity following stem cell transplantation as compared to the liver injury group. CONCLUSIONS: In vivo transplantation of cord blood stem cells (hUCB) does not interfere with natural oval cell response to liver injury.