Vincamine alleviates intrahepatic cholestasis in rats through modulation of NF-kB/PDGF/klf6/PPARγ and PI3K/Akt pathways.

The defect in the hepatobiliary transport system results in an impairment of bile flow, leading to accumulation of toxic compounds with subsequent liver disorders. Vincamine, a plant indole alkaloid that is utilized as a dietary supplement, has been known for its promising pharmacological activities. For the first time, the present study was planned to estimate, at the molecular level, the potentiality of vincamine against alfa-naphthyl isothiocyanate (ANIT)-induced hepatic cholestasis. Liver function tests were analyzed. Hepatic activity of SOD and levels of GSH and MDA were assessed. Hepatic contents of bax, bcl2, NF-kB, PPARγ, catalase, heme-oxygenase-1, NTCP, and BSEP were evaluated using ELISA. mRNA levels of NF-kB, IL-1ß, IL-6, TNFα, PDGF, klf6, PPARγ, and P53 were examined using qRT-PCR. PI3K, Akt and cleaved caspase-3 proteins were assessed using western blotting. Histopathological analyses were performed using hematoxylin & eosin staining. ANIT-induced hepatic cholestasis elevated liver function tests, including AST, ALT, GGT, ALP, and total bilirubin. ANIT reduced the protein expression of NTCP and BSEP hepatic transporters. It induced the expression of the inflammatory genes, TNFα, IL-6, IL-1ß, and PDGF, and the expression of NF-kB at the genetic and protein level and suppressed the anti-inflammatory genes, klf6 and PPARγ. Also, antioxidant markers were reduced during ANIT induction such as GSH, SOD, catalase, heme-oxygenase-1 and PI3K/Akt pathway, while MDA levels were elevated. Furthermore, the expression of P53 gene, bax and cleaved caspase 3 proteins were activated, while bcl2 was inhibited. Also, the histopathological analysis showed degeneration of hepatocytes and inflammatory cellular infiltrates. However, vincamine treatment modulated all these markers. It improved liver function tests. It inhibited the expression of NF-kB, TNFα, IL-6, IL-1ß and PDGF and activated the expression of klf6 and PPARγ. Furthermore, vincamine reduced MDA levels and induced GSH, SOD, catalase, heme-oxygenase-1 and PI3K/Akt pathway. Additionally, it inhibited expression of P53 gene, bax and cleaved caspase 3 proteins. More interestingly, vincamine showed better outcomes on the hepatic histopathological analysis and improved the alterations induced by ANIT. Vincamine alleviated hepatic dysfunction during ANIT-induced intrahepatic cholestasis through its anti-inflammatory and antioxidant efficacies by the modulation of NF-kB/PDGF/klf6/PPARγ and PI3K/Akt pathways.

Vincamine Modulates the Effect of Pantoprazole in Renal Ischemia/Reperfusion Injury by Attenuating MAPK and Apoptosis Signaling Pathways.

Pantoprazole has an antioxidant function against reactive oxygen species (ROS). Vincamine, a herbal candidate, is an indole alkaloid of clinical use against brain sclerosis. The aim of the present experiment is to evaluate, on a molecular level for the first time, the value of vincamine in addition to pantoprazole in treating experimentally induced renal ischemia/reperfusion injury (IRI). One-hundred-and-twenty-eight healthy male Wistar albino rats were included. Serum creatinine, blood urea nitrogen, and malondialdehyde levels were assessed. ELISA was used to estimate the pro-inflammatory cytokines. The expression of Bcl-2 and Bax genes was assessed by quantitative real-time PCR. ERK1/2, JNK1/2, p38, cleaved caspase-3, and NF-κB proteins expressions were estimated using western blot assay. The kidneys were also histopathologically studied. The IRI resulted in impaired cellular functions with increased creatinine, urea nitrogen, malondialdehyde, TNF-α, IL-6, and IL-1ß serum levels, and up-regulated NF-ĸB, JNK1/2, ERK1/2, p38, and cleaved caspase-3 proteins. Furthermore, it down-regulated the expression of the Bcl-2 gene and upregulated the Bax gene. The treatment with vincamine, in addition to pantoprazole multiple doses, significantly alleviated the biochemical and histopathological changes more than pantoprazole or vincamine alone, whether the dose is single or multiple, declaring their synergistic effect. In conclusion, vincamine with pantoprazole multiple doses mitigated the renal IRI through the inhibition of apoptosis, attenuation of the extracellular signaling pathways through proinflammatory cytokines' levels, and suppression of the MAPK (ERK1/2, JNK, p38)-NF-κB intracellular signaling pathway.

Pantoprazole Attenuates MAPK (ERK1/2, JNK, p38)-NF-κB and Apoptosis Signaling Pathways after Renal Ischemia/Reperfusion Injury in Rats.

Ischemia/reperfusion injury (IRI) in the kidney is the most common cause of acute renal dysfunction through different cell damage mechanisms. This study aimed to investigate, on molecular basics for the first time, the effect of pantoprazole on renal IRI in rats. Different biochemical parameters and oxidative stress markers were assessed. ELISA was used to estimate proinflammatory cytokines. qRT-PCR and western blot were used to investigate the gene and protein expression. Renal histopathological examination was also performed. IRI resulted in tissue damage, elevation of serum levels of creatinine, urea nitrogen, malondialdehyde, TNF-α, IL-6, IL-1ß, up-regulation of NF-κB, JNK1/2, ERK1/2, p38, and cleaved caspase-3 proteins. Furthermore, it up-regulated the expression of the Bax gene and down-regulated the expression of the Bcl-2 gene. Treatment of the injured rats with pantoprazole, either single dose or multiple doses, significantly alleviated IRI-induced biochemical and histopathological changes, attenuated the levels of proinflammatory cytokines, down-regulated the expression of NF-κB, JNK1/2, ERK1/2, p38, and cleaved caspase-3 proteins, and the Bax gene, and up-regulated Bcl-2 gene expression. Moreover, treatment with pantoprazole multiple doses has an ameliorative effect that is greater than pantoprazole single-dose. In conclusion, pantoprazole diminished renal IRI via suppression of apoptosis, attenuation of the pro-inflammatory cytokines' levels, and inhibition of the intracellular signaling pathway MAPK (ERK1/2, JNK, p38)-NF-κB.

Mutant matrix metalloproteinase-9 reduces postoperative peritoneal adhesions in rats.

INTRODUCTION: Postoperative peritoneal adhesions continue to be a major source of morbidity and occasional mortality. Studies have shown that matrix metalloproteinase-9 (MMP-9) levels are decreased postoperatively which may limits matrix degradation and participate in the development of peritoneal adhesions. In this proof-of-principle study, we evaluated the effect of gene therapy with catalytically inactive mutant MMP-9 on postoperative peritoneal adhesions in rats. METHODS: Adenovirus encoding mutant MMP-9 (Ad-mMMP-9) or saline was instilled in the peritoneal cavity after cecal and parietal peritoneal injury in rats. Expression of mutant MMP-9 transcript was verified by sequencing. Adenovirus E4 gene expression, adhesion scores, MMP-9, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-ß1 (TGF-ß1) expression were evaluated at sacrifice one week after treatment. RESULTS: Both mutant MMP-9 transcripts and adenovirus E4 gene were expressed in Ad-mMMP-9 treated adhesions. Adhesions severity decreased significantly (p = 0.036) in the Ad-mMMP-9-treated compared with saline-treated adhesions. Expression of MMP-9 mRNA and protein were elevated (p = 0.001 and p = 0.029, respectively) in the Ad-mMMP-9-treated adhesions compared with saline-treated adhesions. While tPA levels were increased (p = 0.02) in Ad-mMMP-9 treated adhesions compared with saline-treated adhesions, TGF-ß1 and PAI-1 levels were decreased (p = 0.017 and p = 0.042, respectively). No difference in mortality were found between groups (p = 0.64). CONCLUSIONS: Mutant MMP-9 gene therapy effectively transduced peritoneal adhesions resulting in reduction of severity of primary peritoneal adhesions.

Genetic polymorphism of estrogen receptor alpha gene in Egyptian women with type II diabetes mellitus.

Estrogen might play an important role in type 2 diabetes mellitus pathogenesis. A number of polymorphisms have been reported in the estrogen receptor alpha gene including the XbaI and PvuII restriction enzyme polymorphisms. The aim of this study was to determine if ESRα gene polymorphisms are associated with type 2 diabetes mellitus and correlated with lipid profile. Ninety diabetic Egyptian patients were compared with forty healthy controls. ESRα genotyping of PvuII and XbaI was performed using restriction fragment length polymorphism analysis. Our study showed that there is more significant difference in the frequency of C and G polymorphic allele between patients and control groups in PvuII and XbaI respectively. Also carriers of minor C and G alleles of PvuII and XbaI gene polymorphisms were associated with increased fasting blood glucose and disturbance in lipid profile as there is an increase in total cholesterol, triglycerides and Low density lipoprotein. So findings of present study suggest the possibility that PvuII and XbaI polymorphisms in ERα are related to T2DM and with increased serum lipids among Egyptian population.

Brain-derived neurotrophic factor in sera of breastfed epileptic infants and in breastmilk of their mothers.

BACKGROUND: Human milk contains leukocytes expressing brain-derived neurotrophic factor (BDNF), which may facilitate epileptogenesis. Our study aimed to estimate levels of BDNF in the sera of breastfed infants with idiopathic epilepsy and in breastmilk of their mothers and to assess its value as a marker of epilepsy severity. PATIENTS AND METHODS: Thirty breastfed infants <2 years of age with idiopathic epilepsy and 15 control healthy breastfed infants were recruited for the study. Patients were subjected to thorough medical history, clinical examination, and assessment of disease severity. Routine laboratory and radiological investigations, including, liver, renal, and thyroid screen, brain magnetic resonance imaging, and measurement of serum and breastmilk BDNF levels, were performed. RESULTS: Serum BDNF levels of epileptic infants and milk BDNF levels of their mothers were significantly higher than values for controls (p=0.0001). They were positively correlated with age, weight, length, and head circumference of epileptic children. Also, serum and milk BDNF levels were significantly increased with increased duration of illness and frequency of seizures. There was a significant positive correlation between serum and breastmilk levels of BDNF and significantly higher levels in severe cases of epilepsy. CONCLUSIONS: Serum and milk BDNF levels are higher in epileptic infants than in controls and may be used as a marker of disease severity.

Mutant MMP-9 and HGF gene transfer enhance resolution of CCl4-induced liver fibrosis in rats: role of ASH1 and EZH2 methyltransferases repression.

UNLABELLED: Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl4 induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl4 for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-ß1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. CONCLUSION: Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.

Serum level of ghrelin in umbilical cord in small and appropriate for gestational age newborn infants and its relationship to anthropometric measures.

OBJECTIVE: To compare the cord blood ghrelin level in (SGA) infants with the level in appropriate for gestational age (AGA) infants, and determine its relationship to anthropometric measurements at delivery. MATERIALS AND METHODS: Fifty newborn infants (30 SGA newborns and 20 AGA infants) were included in the study and were subjected to complete clinical examinations, anthropometric measurement, and ghrelin assays. RESULTS: The cord blood ghrelin level in SGA infants was significantly higher than that in AGA infants. Cord ghrelin level correlated negatively with gestational age, weight, length, and body mass index in SGA group. CONCLUSION: Cord ghrelin concentration increased in SGA infants due to state of prolonged undernutrition the source of ghrelin unknown may be from the mother placenta or fetal tissues.

Adenovirus-mediated overexpression of human tissue plasminogen activator prevents peritoneal adhesion formation/reformation in rats.

BACKGROUND: Tissue-plasminogen activator (tPA) demonstrated beneficial effects on peritoneal adhesion formation; however, its short half-life limits its continual fibrinolytic effect. Therefore, we delivered adenovirus encoding tPA to prevent adhesions. METHODS: Rats were subjected to peritoneal injury and assigned to two protocols. In de novo adhesion protocol, adenovirus encoding human tPA gene (Ad-htPA) was instilled after peritoneal injury in group 1 (n = 22), whereas group 2 received phosphate-buffered saline (PBS) (n = 24). In recurrent adhesion protocol, group 1 (n = 15) received the same Ad-htPA dose after adhesiolysis and group 2 (n = 13) received PBS. Adhesion severity was scored 1 week after ad-htPA instillation. Adhesions were analyzed for htPA mRNA by reverse transcription-polymerase chain reaction and levels of htPA, and fibrinolytic inhibitors PAI-1, TIMP-1, and TGF-beta1 were measured using enzyme-linked immunosorbent assay. RESULTS: htPA mRNA and protein were only expressed in adhesions from treated groups. A reduction in adhesion scores (P < .01) and in fibrinolytic inhibitors (P < .001) occurred in the treatment groups. Also, negative correlation was found (r = -.69, P < .01) between adhesion scores and htPA protein, but a positive correlation was found (r = .90, P < .01) between adhesion score and fibrinolytic inhibitors. No bleeding or wound complications were encountered. CONCLUSION: Administration of adenovector encoding htPA is safe and decreased de novo and recurrent peritoneal adhesions.

Colchicine inhibits intimal hyperplasia and leukocyte VEGF expression in dogs.

BACKGROUND: Restenosis due to intimal hyperplasia following percutaneous transluminal angioplasty limits its long-term efficacy. We evaluated the effect of colchicine on the development of intimal hyperplasia following balloon angioplasty and on the vascular endothelial growth factor (VEGF) expression in leukocytes. MATERIAL AND METHODS: Adult dogs underwent balloon angioplasty of the right iliofemoral artery. Group 1 served as control, while groups 2 and 3 (six animals per group) received 0.1 and 0.5 mg/kg/d of colchicine p.o., respectively, starting 2 d before angioplasty and continued for 14 d. Before angioplasty and at day 14, blood samples were collected for drug toxicity analysis and the determination of leukocyte expression of VEGF. Animals were euthanized and iliofemoral arteries were perfusion fixed in situ and processed for histological and morphometric analysis. RESULTS: Balloon angioplasty without colchicine resulted in 446% (P < 0.001), 111% (P = 0.7), and 267% (P < 0.001) increase in intimal and medial thickness and intima/media ratio compared with contralateral uninjured iliofemoral arteries. Low-dose and high-dose colchicine resulted in 32% and 58% reduction in intima/media ratio, respectively (both P < 0.001). VEGF expression in leukocytes of control group was up-regulated (40%), but was down-regulated by 12% and 55%, respectively, in low-dose and high-dose colchicine groups at 2 wk after angioplasty compared with preangioplasty expression. The results of complete blood count and serum transaminases and creatinine were within normal range. CONCLUSION: This study demonstrates that oral colchicine for 2 wk significantly reduces intimal hyperplasia following balloon angioplasty in dogs through down-regulation of leukocyte VEGF expression and without apparent toxicity.

The hepatoprotective effect of carnosine against ischemia/reperfusion liver injury in rats.

The potential protective effect of the natural antioxidant, carnosine was evaluated against ischemia/reperfusion liver injury in rats. Ischemia was induced by clamping the pedicle supplying the left hepatic lobe for 60 min followed by reperfusion for 2 h. Untreated rats exposed to ischemia/reperfusion showed significant elevation of serum aspartate aminotransferase and alanine aminotransferase levels, and malondialdehyde level and caspase-3 activity in liver homogenates associated with significant reduction in hepatic nitrite level, catalase and glutathione peroxidase activities as compared with sham-operated group. Pre-treatment with a single i.p. dose of carnosine (250 mg/kg), 30 min prior to the ischemic episode significantly attenuated the deterioration in the measured biochemical parameters observed with ischemia/reperfusion-induced liver injury. Also, light and electron microscopic examinations in ischemia/reperfusion untreated group revealed severe hepatic damage, such as cytoplasmic vacuolation, necrotic and apoptotic cell death, which was markedly ameliorated by pre-ischemic treatment with carnosine. These results strongly emphasize that carnosine can be useful as a prophylactic treatment to protect the liver against hypoxia-reoxygenation damage.

Hypovitaminosis D in female patients with chronic low back pain.

Chronic low back pain (LBP) is an extremely common problem in practice, where it is often labeled idiopathic. No sufficient studies have been conducted to analyze the contribution of hypovitaminosis D to the etiology of chronic LBP in populations wherein vitamin D deficiency is endemic. The present study was, therefore, carried out to examine hypovitaminosis D and its determinants in female patients with chronic LBP during the childbearing period. Sixty female patients complaining of LBP lasting more than 3 months were clinically studied rheumatologically and neurologically. Questionnaires and indices quantifying risk factors associated with vitamin D deficiency were utilized. Biochemical assays of serum calcium, phosphorus, alkaline phosphatase (ALP), parathormone (PTH), and 25-hydroxyvitamin D (25 OHD) were performed and compared to those of 20 matched healthy controls. The determinants of vitamin D levels in patients were examined by stepwise regression. Patients with LBP had significantly lower 25 OHD levels (p < 0.05) and significantly higher PTH (p < 0.05) and ALP (p < 0.001) than controls, although there were no significant group differences in calcium and phosphorus. Hypovitaminosis D (25 OHD < 40 ng/ml) was found in 49/60 patients (81%) and 12/20 (60%) of controls, with an odds ratio of 2.97. Although many risk factors related to sun exposure, clothing, diet, and pregnancy were significantly correlated with vitamin D levels in patients, only limited duration of sun exposure, contributing 55% to the variance of 25 OHD, limited areas of skin exposed (13%), and increased number of pregnancies (2%), were significant determinants of vitamin D levels in patients. Despite the sunny climate, hypovitaminosis D is prevalent among Egyptian women in the childbearing period, especially those presenting with chronic LBP, where it is associated with hyperphosphatasia and hyperparathyroidism, without alterations in serum calcium. The major determinant of hypovitaminosis D in our patients is limited sun exposure.

TGF-beta1, TNF-alpha and cytochrome c in human astrocytic tumors: a short-term follow up and correlation with survival.

OBJECTIVES: To evaluate the association of signals of apoptosis namely, TGF-beta1, TNF-alpha and cytochrome c release in cytoplasm with survival rate to determine the potential use of such parameters as predictive markers for patients with astrocytomas. DESIGN AND METHODS: We measured TGF-beta1, TNF-alpha and cytoplasmic cytochrome c in 30 astrocytic tumors Grade II, III and IV. RESULTS: We found that TNF-alpha and cytochrome c release in Grade IV tends to be significantly lower than those in Grade II, whereas TGF-beta1 did not significantly change in the different grades. Patients with astrocytic tumors having elevated cytochrome c showed a better survival rate compared to those with less release. There is neither a correlation shown between TNF-alpha and cytochrome c release nor between TNF-alpha and patient survival. TGF-beta1 was positively correlated with cytochrome c release. Patients showing such correlation had increased survival rate over 18 months follow up period. CONCLUSION: These data suggest that TGF-beta1 and cytochrome c may be useful prognostic markers that help patients' stratification and in adjusting the disciplines of therapy.