Amro El-Karef

Hepatoprotective effects of glycyrrhizin and omega-3 fatty acids on Nuclear Factor-kappa B pathway in thioacetamide-induced fibrosis in rats

Abstract Nuclear Factor kappa B (NF-κB) is a key transcriptional regulator that plays important roles in the pathogenesis of hepatic inflammation and fibrosis in chronic liver diseases. NF-κB activation leads to production of pro-inflammatory and fibrogenic cytokines. Glycyrrhizin (GL) is reported to suppress liver fibrosis and cirrhosis. Omega-3 fatty acids (ω-3) play an anti-inflammatory role and they are reported to decrease hepatic injury in Thioacetamide (TAA) fibrotic model. We investigated the effects of GL and ω-3 on liver inflammation and fibrosis in rats and clarified the effects of these natural compounds on NF-κB level. 50 male Wistar rats randomized to 5 groups: Control group and 4 groups received TAA 200 mg/kg i.p. twice weekly for 8 weeks: TAA group, GL group (received GL 25 mg/kg daily by oral tube), ω-3 group (received ω-3150 mg/kg daily by oral tube), (GL + ω-3) group (received similar combined doses of both natural compounds). GL and ω-3 alone or in combination protected the liver from TAA hepatotoxic effects as they significantly decreased serum AST activity and serum total bilirubin level, they also significantly increased serum albumin and total protein levels. The hepatoprotective effects of GL and ω-3 were confirmed by the histopathological analysis as they significantly reduced the necroinflammatory scores and the extent of fibrosis. GL and ω-3 significantly decreased liver malondialdehyde level (P < 0.005), liver NF-ƙB level (P < 0.005) and its tissue expression as detected by immunohistochemistry. In conclusion, glycyrrhizin and omega-3 fatty acids alone or in combination have potent anti-inflammatory, anti-oxidant and anti-fibrotic effects.

Protective effects of L-carnosine on CCl4 -induced hepatic injury in rats

The present study was undertaken to investigate the possible protective effect of L-carnosine (CAR), an endogenous dipeptide of alanine and histidine, on carbon tetrachloride (CCl4)-induced hepatic injury. Liver injury was induced in male Sprague-Dawley rats by intraperitoneal (i.p.) injections of CCl4, twice weekly for six weeks. CAR was administered to rats daily, at dose of 250 mg/kg, i.p. At the end of six weeks, blood and liver tissue specimens were collected. Results show that CAR treatment attenuated the hepatic morphological changes, necroinflammation and fibrosis induced by CCl4, as indicated by hepatic histopathology scoring. In addition, CAR treatment significantly reduced the CCl4-induced elevation of liver-injury parameters in serum. CAR treatment also combated oxidative stress; possibly by restoring hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) levels. Moreover, CAR treatment prevented the activation of hepatic stellate cells (HSCs), as indicated by reduced α-smooth muscle actin (α-SMA) expression in the liver, and decreased hepatic inflammation as demonstrated by a reduction in hepatic tumor necrosis factor-α (TNF-α) and restoration of interleukin-10 (IL-10) levels. In conclusion, CCl4-induced hepatic injury was alleviated by CAR treatment. The results suggest that these beneficial, protective effects are due, at least in part, to its anti-oxidant, anti-inflammatory and anti-fibrotic activities.

Naringin attenuates thioacetamide-induced liver fibrosis in rats through modulation of the PI3K/Akt pathway

Aims: Naringin (NR) is a flavanone glycoside extracted from grapefruits and citrus fruits. The aim of this study is to investigate the antifibrotic efficacy of NR in thioacetamide (TAA)‐induced hepatic fibrosis in rats through evaluating NR effect on the PI3K/Akt pathway. Main methods: Hepatic fibrosis was induced in rats by intraperitoneal injection of TAA (200 mg/kg) twice per week for 6 weeks. Simultaneously, NR (40 mg/kg/day, p.o.) was given along with TAA injection. The ratio of P‐Akt/Akt was assessed in hepatic homogenate as well as antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx)) and lipid peroxidation marker, malondialdehyde (MDA). Serum level of interleukin (IL)‐6 were measured using ELISA. Hepatic tissues were examined histopathologically using hematoxylin and eosin (H&E) and Masson trichome staining. Tissue expression of alpha smooth muscle actin (&agr;‐SMA), transforming growth factor &bgr;1 (TGF‐&bgr;1), caspase‐3 and fibronectin were scored immunohistochemically. Finally, the mRNA level of cytokine genes (IL‐1&bgr;, IL‐6, IL‐10, interferon gamma (IFN‐&ggr;)), caspase‐3, TGF‐&bgr;1 and fibronectin were quantified using qPCR. Key findings: NR significantly suppressed Akt phosphorylation associated with increased number of caspase‐3 positive cells especially in the fibrotic areas. Liver tissues of treated rats showed restoration of normal liver histology and decrease in collagen and fibronectin deposition. Furthermore, NR treatment ameliorated oxidative stress and inflammatory cytokine production. Significance: NR alleviated experimental liver fibrosis through inhibition of PI3K/Akt pathway beside its anti‐inflammatory and antioxidant effects. Therefore, NR is a promising therapeutic candidate for hepatic fibrosis. Graphical abstract Figure. No caption available.

Glycyrrhizin ameliorates high fat diet-induced obesity in rats by activating NrF2 pathway

Aim: Obesity based on insulin resistance is a state of chronic oxidative stress and inflammation that are highly regulated through nuclear factor Erythroid 2‐related factor 2 (NrF2) pathway. Materials and methods: 70 male Wistar rats were randomized into two models. The prophylactic model was 10 weeks and rats were grouped into: normal group, GL group (received glycyrrhizin 50 mg/kg/day orally along with normal pellet diet), HFD group and HFD+ GL group (received glycyrrhizin along with HFD). The treatment model was 14 weeks and rats were grouped into: normal group, HFD group and HFD + GL group (received glycyrrhizin from the week 10). Key findings: Glycyrrhizin decreased significantly rat weights and insulin resistance, normalized lipid profile and reduced significantly the adipocytes size in adipose tissue and lipid deposition in the liver tissue through histopathologic examination. Furthermore, glycyrrhizin ameliorated obesity‐induced oxidative stress which indicated by significant decrease in liver malondialdehyde level (P < 0.001) and increase in the total antioxidant capacity (P < 0.001). Interestingly, molecular mechanism of glycyrrhizin was explored, that included significant reduction of liver gluconeogenic enzymes mRNA expression (P < 0.001), a significant increase of liver insulin receptor, NrF2 and homooxygenase‐1 mRNA expressions (P < 0.001) and significant increase and nuclear translocation of NrF2 in liver tissue. Significance: Glycyrrhizin ameliorates HFD‐induced obesity in rats that may be attributed to its ability to increase insulin receptor expression and to activate NrF2 and subsequent homooxygenase‐1 pathway. Thus, this work represents a safe natural compound (glycyrrhizin) that has a great role either as prophylaxis or treatment for insulin resistance related to obesity. Graphical abstract: The effect of high fat diet (HFD) feeding either for 10 or 14 weeks on rats (A), the proposed mechanism of glycyrrhizin either as prophylaxis or treatment in amelioration of obesity‐associated with insulin resistance induced by high fat diet (HFD) feeding in rats (B). G6Pase: glucose‐6‐phosphatase, HDL‐C: high density lipoprotein‐cholesterol, HO‐1: homooxygenase‐1, HOMA IR: homeostatic model assessment of insulin resistance, LDL‐C: low density lipoprotein‐cholesterol, MDA: malondialdehyde, NrF2: nuclear factor erythroid‐derived factor 2‐related factor 2, PEPCK: phosphoenolpyruvate carboxykinase, TAC: total antioxidant capacity. Figure. No Caption available.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression

AIMS Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. MAIN METHODS Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Massons trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. KEY FINDINGS Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. SIGNIFICANCE Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF.

Hepatoprotective Effect of Curcumin on Hepatocellular Carcinoma Through Autophagic and Apoptic Pathways

BACKGROUND AND RATIONALE Microtubule-associated protein light chain 3-II (LC3-II), and Sequestosome-1 (SQSTM1) are proteins that can be used as markers for autophagic pathway. Bcl-2 protein is reported to be inversely correlated with apoptosis. We aimed to investigate the effects of curcumin on liver inflammation and fibrosis up to the first dysplastic stage of Hepatocellular carcinoma (HCC) induced by Thioacetamide (TAA) in rats and to clarify the effects of curcumin on LC3-II, SQSTM1, and Bcl-2. Male Sprague-Dawley rats were randomized into four groups: Control group, TAA group, Curcumin low-dose group, and Curcumin highdose group. The last three groups received TAA 200 mg/kg i.p. twice weekly for 18 weeks. Oxidative stress markers as hepatic malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity were measured by colorimetric methods. Hepatic SQSTM1 concentration was measured by ELISA, and gene expression levels of Bcl-2, and LC3-II were measured by RT-PCR.We also investigated the in vitro effect of curcumin on HepG2 cells viability through MTT assay, and the involvement of autophagy in this effect. RESULTS Curcumin increased the survival percent in rats, decreased -fetoprotein (AFP) concentration, and serum aspartate aminotransferase (AST) activity, and increased serum albumin concentration. Curcumin also significantly reduced oxidative stress in liver, inhibited apoptosis, and induced autophagy. In vitro, curcumin (50 µM) decreased HepG2 cells viabilityand the concentration of SQSTM1. CONCLUSIONS Curcumin leads to protection against TAA induced HCC up to the first dysplastic stage through activating autophagic pathway and inhibiting apoptosis. Also, the antioxidant activity of curcumin almost prevents liver fibrosis.BACKGROUND AND RATIONALE Microtubule-associated protein light chain 3-II (LC3-II), and Sequestosome-1 (SQSTM1) are proteins that can be used as markers for autophagic pathway. Bcl-2 protein is reported to be inversely correlated with apoptosis. We aimed to investigate the effects of curcumin on liver inflammation and fibrosis up to the first dysplastic stage of Hepatocellular carcinoma (HCC) induced by Thioacetamide (TAA) in rats and to clarify the effects of curcumin on LC3-II, SQSTM1, and Bcl-2. Male Sprague-Dawley rats were randomized into four groups: Control group, TAA group, Curcumin low-dose group, and Curcumin highdose group. The last three groups received TAA 200 mg/kg i.p. twice weekly for 18 weeks. Oxidative stress markers as hepatic malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity were measured by colorimetric methods. Hepatic SQSTM1 concentration was measured by ELISA, and gene expression levels of Bcl-2, and LC3-II were measured by RT-PCR.We also investigated the in vitro effect of curcumin on HepG2 cells viability through MTT assay, and the involvement of autophagy in this effect. RESULTS Curcumin increased the survival percent in rats, decreased -fetoprotein (AFP) concentration, and serum aspartate aminotransferase (AST) activity, and increased serum albumin concentration. Curcumin also significantly reduced oxidative stress in liver, inhibited apoptosis, and induced autophagy. In vitro, curcumin (50 μM) decreased HepG2 cells viabilityand the concentration of SQSTM1. CONCLUSIONS Curcumin leads to protection against TAA induced HCC up to the first dysplastic stage through activating autophagic pathway and inhibiting apoptosis. Also, the antioxidant activity of curcumin almost prevents liver fibrosis.

Calretinin expression as a reliable prognostic marker in different molecular subtypes of breast carcinoma

Background: Calretinin (CR), a known mesothelial marker, is expressed in both epithelial and mesenchymal malignancies including breast cancer. Aims: We aimed to measure the frequency of CR expression in correlation with other clinicopathological parameters of different molecular subtypes of invasive breast carcinoma and to study its prognostic implications in this common cancer.Study Design: Tissue microarrays were constructed from 225 tissue samples of breast carcinoma cases. Subjects and Methods: Immunostaining for CR in addition to estrogen receptors, progesterone receptors, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor, CK5/6, and Ki-67 for molecular subtyping. Statistical Analysis Used: Chi-square and Fishers exact tests were done using SPSS 18.0 software (IBM Inc.). Survival data were analyzed using Kaplan–Meier test, Log-rank test, and Cox proportional hazard models. Results: Cases of invasive breast carcinomas with different grades were classified into 84 luminal A, 45 luminal B, 27 HER2 positive, 40 basal-like, and 29 unclassified. High CR expression was associated with tumors of high grade (P < 0.0001), high locoregional recurrence (P = 0.005), hormonal receptors negative, and high Ki-67 indices. They frequently display a basal-like phenotype (70%, P < 0.0001), HER2 (59.3%), and luminal B (33.3%) tumors compared to luminal A (9.5%) and unclassified subtypes (17.2%). Moreover, it is associated with poor overall patient survival (P = 0.034), but it does not affect disease-free survival. Conclusions: Calretinin could be a reliable predictor marker of adverse prognosis in breast cancer.

Antioxidant and anti-inflammatory activities of berberine attenuate hepatic fibrosis induced by thioacetamide injection in rats

Berberine (BBR) is an isoquinoline alkaloid extracted from the roots, rhizomes and stems of coptis. Liver fibrosis is a worldwide health problem with no established therapy until now. The aim of our study is to investigate the efficacy of BBR on hepatic fibrosis induced in rats and to uncover other mechanisms. Rats were injected with thioacetamide (TAA) (200 mg/kg, i.p) twice per week for 6 weeks to induce fibrosis. Treated groups were gavaged with BBR (50 mg/kg/day, p.o) simultaneously with TAA injection. Hepatic antioxidant enzymes (catalase, SOD, GPx) were assessed in hepatic homogenate. Their activities were attenuated by TAA injection and elevated by BBR administration. Additionally, serum IL-6 and mRNA levels of IL-1β, IL-6, IL-10 and IFN-γ were evaluated as inflammatory markers. Our results showed that BBR suppressed the inflammation induced by TAA injection. Tissue expression of α-SMA (marker of activated HSCs), TGF-β1 and fibronectin were measured by immunohistochemistry as well as mRNA expressions of TGF-β1 and fibronectin were quantified as fibrotic markers. The collagen deposition in hepatic tissues was assessed by Massons trichome staining. BBR significantly alleviated TGF-β1 production, decreased collagen and fibronectin deposition and consequently attenuated hepatic fibrogenesis. Akt pathway controls cell survival, proliferation, migration and adhesion. The relative phosphorylation of Akt was determined in hepatic homogenates that was increased with TAA injection and decreased by BBR treatment. Inhibition of Akt pathway has been linked to the intrinsic pathway of apoptosis. Caspase-3, caspase-9, Bcl-2 and Bax were quantified as apoptotic markers using qPCR and also caspase-3 by immunohistochemistry. BBR-treated rats showed an increase in the expression of apoptotic markers. Moreover, BBR-treated rats showed restoration of normal liver lobular architecture as shown by H&E staining. In conclusion, BBR is a potential therapeutic candidate for liver fibrosis owing to its antioxidant and anti-inflammatory activities.

Vitamin D potentiates anti-tumor activity of 5-fluorouracil via modulating caspase-3 and TGF-β1 expression in hepatocellular carcinoma-induced in rats

We investigated the role of vitamin D (Vit D) alone and in combination with 5-fluorouracil (5-FU) in thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) in rats. Fifty male Sprague-Dawley rats were randomized into a control group and 4 groups that received TAA (200 mg/kg, i.p.) twice per week for 16 weeks. These 4 groups were further divided as follows: HCC group; 5-FU group (75 mg/kg, i.p., once weekly for 3 weeks starting from the 12th week); Vit D group (200 IU/kg daily by oral tube for 16 weeks); and 5-FU + Vit D group (received the previously mentioned dosage regimens of 5-FU and Vit D). HCC was detected by histopathological changes in liver sections and the elevation of serum α-fetoprotein (AFP). Treatment with 5-FU + Vit D significantly decreased gene expression of nuclear factor erythroid 2-related factor 2 (NrF2) and transforming growth factor β1 (TGF-β1) at both the gene and protein level and serum AFP concentrations in comparison with their corresponding monotherapy. Moreover, 5-FU + Vit D treatment enhanced apoptosis by increasing caspase-3 gene and protein expression. Conclusively, Vit D enhances antitumor activity of 5-FU in an HCC-induced model and improves liver function of treated animals. Combination therapy in a TAA-induced HCC rat model was more effective than 5-FU or Vit D through the modulation of TGF-β1, caspase-3, and NrF2 expressions.

Simvastatin exerts antifibrotic effect and potentiates the antischistosomal effects of praziquantel in a murine model: Role of IL10

Previous studies on simvastatin use in experimental schistosomiasis in mice did not provide a full explanation of its mechanism as antischisome. In this study, we tried to find out the role of IL-10 in the mechanism of action of simvastatin. We used 50 clear mice. Ten were used as normal not treated while 40 were infected with shistosome mansoni then divided into 4 groups; 3 treated groups by praziquantel, simvastatin and combined (praziquantel plus simvastatin) respectively and one group non-treated. The simvastatin treated group showed shortening and loss of the tubercles and disappearance of the spines with swelling and twisted shape of the worms. In addition, it also showed mitigation of ovideposition activity of the worms in the liver and reduction of the fibrous component of the liver granuloma producing a protective effect on the liver. This effect was associated with lowering of IL-10. This may explain the role of IL-10 in the protective effect of simvastatin. Combination of treatment with simvastatin plus praziquantel produced more significant effects in different parameters compared with praziquantel treated group. We recommend using simvastatin as add on therapy to standard antischistosomal therapy, praziquantel. Both drugs affect the worm motility and sucker activity and the ova deposition. Simvastatin has an additional pleiotropic effect halting inflammation and decreasing fibrosis due to increasing IL-10 leading to a hepatoprotective effect. Further clinical studies are needed to further validate these findings.