الفهرس 
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Abstract
Summary and conclusion
The current work was designed to investigate the potential hepatoprotective effect of liraglutide in methotrexate-induced liver injury model as well as its underlying molecular mechanisms by studying its effects on some key events such as oxidative stress, inflammation and apoptosis signaling pathway. To fulfill these goals, this study was divided into two parts:
I- Dose screening for the optimum hepatoprotective dose of liraglutide After acclimatization for 2 weeks, 48 rats were randomly divided into six groups of eight rats each and treated for 10 consecutive days as follows:
group 1: served as a negative control, receiving saline only during the entire experimental period.
group 2: given a single intraperitoneal injection of methotrexate at a dose of 20 mg/kg BW on the eighth day.
group 3, 4, and 5: received liraglutide at a dose of 0.3, 0.6 and 1.2 mg/kg respectively i.p twice daily and on the eighth day was given single i.p MTX injection at a dose of 20 mg/kg. The dose range of liraglutide is selected based on previous reports
The assessed parameters for phase I to select the optimum liraglutide dose were:
1. Assessment of liver enzymes: Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT)
2. Histopathological examination H & E
II- Studying the hepatoprotective mechanism of liraglutide against MTX induced liver injury
Based on serum ALT & AST and histopathological examination results, liraglutide 1.2 mg/kg dose was selected to be the optimum hepatoprotective dose and used for assessment of oxidative stress and inflammatory markers and studying the signaling hepato-protection pathway.
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The following parameters were investigated:
1-Hepatic oxidative stress markers
Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances (TBARS) measured as malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD)
2- Inflammatory markers: Liver contents of Tumor necrosis factor-alpha (TNF- α), interleukin-6 (IL-6), nuclear factor-kappa b NF-κB (p65) subunit expression and carboxy-oxygenase-2 expression (COX-2).
3- Apoptotic markers: Bax, Bcl-2, Caspase 3 expression, and Caspase-3 activity
4- Nrf-2/HO-1/pCREB Pathway: phosphorylated cAMP response element-binding protein (pCREB), Nuclear factor erythroid 2-related factor2 (Nrf2), and Heme oxygenase-1 (HO-1)
5- Cytotoxicity Assay: HepG-2 cells (human Hepatocellular cancer cell line)
The results of the present study can be summarized as follows:
I-Dose screening for the optimum hepatoprotective dose of liraglutide
1. Hepatotoxicity markers-Serum alanine aminotransferase and aspartate aminotransferase levels:
As compared to the control group, rats received MTX showed a significant rise in serum ALT and AST. Concurrent administration of all the tested doses of liraglutide showed a significant decrease in serum aminotransferases as compared to the MTX group.
2. Histopathological Examination:
Microscopic examination of H&E-stained liver sections revealed that 1.2 mg/kg dose of liraglutide almost preserved the normal architecture of the liver.
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Collectively, the results of the acute study proved that liraglutide at a dose of 1.2 mg/kg is more protective than 0.3 & 0.6 mg/kg. Therefore the molecular study was carried out using the 1.2 mg/kg dose.
The results of the present study can be summarized as follows:
1-The ameliorative outcome of liraglutide on MTX-induced oxidative stress was examined via the determination of GSH, SOD, and MDA. MTX-induced rats showed significant deterioration in hepatic GSH content and abolished the activity of SOD by 85.7% and 128.7% respectively when compared with the control group. MTX-induced rats pre-treated with 1.2 mg/kg liraglutide produced a significant elevation in GSH and SOD by 5.7 folds and 1.5 folds respectively, when compared with the MTX-induced group. Contrarily, MDA is increased by 71.5% in MTX-induce rats as compared to the control group. Pre-treatment with liraglutide halted MDA content by 45.2% as compared to the MTX-induced group. Accordingly, liraglutide pre-treatment protects against MTX induced oxidative stress.
2- Immunohistochemical detection of COX-2 and NF-kB showed almost negative expression in the control group. In contrast, the MTX group revealed a significant increase in the expression of COX-2 and NF-kB as shown by intense brown staining as compared to the control group. Conversely, Pre-treatment with 1.2 mg/kg liraglutide significantly reduced the expressions of COX-2 and NF-kB as compared to the MTX group. In addition, the liraglutide administered group showed negative levels of expression for COX-2 and NF-kB as compared to the control group.
Furthermore, concerning the effect of liraglutide on the pro-inflammatory cytokines, TNFα and IL-6 showed a significant increase in MTX-induced rats by 155 % and 46% respectively, when compared to the control group. Interestingly, Pre-treatment with 1.2 mg/kg liraglutide resolved TNFα and IL-6 levels rise by 78 % and 28.5% decrease respectively, when compared to MTX-induced rats.
3- Caspase-3 and Bax immunoreactivity were increased while Bcl-2 was decreased in livers of MTX-induced rats resulting in apoptotic cell death as compared with the control
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group. Interestingly, pre-treatment with liraglutide significantly suppressed the levels of expression of Caspase-3, and Bax, while increased Bcl-2 expression level when compared with MTX group. In addition, pre-treatment with liraglutide significantly suppressed the Bax/Bcl-2 ratio in comparison with the MTX group. In the liraglutide alone group, non-significant changes in levels of expression were detected for Caspase-3, Bax, and Bcl-2 as compared to the control group.
To assure the effect of liraglutide pre-treatment on the MTX-induced apoptosis, Bax and Bcl-2 protein expressions were assessed by western blot. In addition, Caspase-3 activity was measured. One-way ANOVA showed significant differences among groups on the percent change in liver Bax and Bcl-2 protein expression and in Caspase-3 activity. Rats received MTX showed a sharp increase in liver Bax expression and caspase-3 activity while Bcl-2 expression was decreased as compared to the control group. Pre-treatment with liraglutide showed a significant decrease in Bax expression and Caspase-3 activity and a significant increase in Bcl-2 expression as compared to the MTX-induced group.
4-To explore the mechanism underlying the effect of liraglutide on protecting MTX induced liver damage, the activation of the Nrf2/HO-1/CREB pathway was examined. MTX- induced rats exhibited a significant decline in Nrf2 by 67.4% when compared to the control group. On the other hand, the liraglutide pre-treatment group significantly increased Nrf2 levels by nearly 2 folds when compared to MTX-induced rats. HO-1, a Nrf2-regulated enzyme, showed a significant increase in the MTX group by 23.4% when compared to the control group. In contrast, liraglutide pre-treatment rats showed a significant decrease of 26.7% when compared to MTX-induced rats.
In the same way, ELISA analysis revealed that pCREB tissue levels were significantly decreased by 40.8% in MTX-induced rats when compared to control group. However, a significant increase in pCREB levels was observed in the liraglutide pre-treatment group where its levels reached nearly 79% rise as compared to the MTX-induced group.
5- The IC50 of MTX was 369±7.9 μg/ml after 48hrs. Liraglutide was able to induce growth inhibition after 48hrs of incubation and the IC50 was 50±1.8 μg/ml. The
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simultaneous combination of MTX with 10 μg/ml and 20 μg/ml of liraglutide resulted in a significant reduction of IC 50; where the IC50 of MTX with 10 μg/ml liraglutide was 322 ± 5.8 μg/ml. Moreover, The IC50 of MTX with 20 μg/ml liraglutide was decreased to 242 ± 3.9 μg/ml. To verify the type of interaction between MTX and liraglutide, isobologram analysis was carried out and revealed that the interaction between MTX and liraglutide was additive in HepG-2 cell line (I = 1.07 for 10 μg/ml liraglutide and I= 1.05 for 20 μg/ml liraglutide; respectively).
In conclusion, Pre-treatment of liraglutide in MTX induced rats reversed the toxic effects of MTX by decreasing oxidative stress, inflammation, and apoptosis. Results suggest that liraglutide exerts hepatoprotective effects mainly by regulation of the triggering of the transcription factors Nrf-2 and NF-kB. Such protective effects are mediated through the up-regulation of Nrf2 and pCREB concomitant with down-regulation of NF-kB with subsequent inhibition of inflammatory, oxidative stress and apoptotic pathways. Based on the current findings, liraglutide as a direct antioxidant may be an effective treatment to preserve normal liver function under conditions of oxidative stress and thus improve outcomes in this significant patient population. A controlled clinical trial is needed to explore the potential of these results in patient care, in both cancer and types 2 diabetic patients.