Francielli Licks

Antioxidant properties of glutamine and its role in VEGF-Akt pathways in portal hypertension gastropathy

AIM To investigate the effects of glutamine on oxidative/nitrosative stress and the vascular endothelial growth factor (VEGF)-Akt-endothelial nitric oxide synthase (eNOS) signaling pathway in an experimental model of portal hypertension induced by partial portal vein ligation (PPVL). METHODS Portal hypertension was induced by PPVL. The PPVL model consists of a partial obstruction of the portal vein, performed using a 20 G blunt needle as a guide, which is gently removed after the procedure. PPVL model was performed for 14 d beginning treatment with glutamine on the seventh day. On the fifteenth day, the mesenteric vein pressure was checked and the stomach was removed to test immunoreactivity and oxidative stress markers. We evaluated the expression and the immunoreactivity of proteins involved in the VEGF-Akt-eNOS pathway by Western blotting and immunohistochemical analysis. Oxidative stress was measured by quantification of the cytosolic concentration of thiobarbituric acid reactive substances (TBARS) as well as the levels of total glutathione (GSH), superoxide dismutase (SOD) activity, nitric oxide (NO) production and nitrotyrosine immunoreactivity. RESULTS All data are presented as the mean ± SE. The production of TBARS and NO was significantly increased in PPVL animals. A reduction of SOD activity was detected in PPVL + G group. In the immunohistochemical analyses of nitrotyrosine, Akt and eNOS, the PPVL group exhibited significant increases, whereas decreases were observed in the PPVL + G group, but no difference in VEGF was detected between these groups. Western blotting analysis detected increased expression of phosphatidylinositol-3-kinase (PI3K), P-Akt and eNOS in the PPVL group compared with the PPVL + G group, which was not observed for the expression of VEGF when comparing these groups. Glutamine administration markedly alleviated oxidative/nitrosative stress, normalized SOD activity, increased levels of total GSH and blocked NO overproduction as well as the formation of peroxynitrite. CONCLUSION Glutamine treatment demonstrated to reduce oxidative damage but does not reduce angiogenesis induced by PH in gastric tissue, demonstrating a beneficial role for the PI3K-Akt-eNOS pathway.

Antioxidant and anti-inflammatory action of melatonin in an experimental model of secondary biliary cirrhosis induced by bile duct ligation

AIM To evaluate the effects of melatonin (Mel) on oxidative stress in an experimental model of bile duct ligation (BDL). METHODS Male Wistar rats (n = 32, weight ± 300 g) were allocated across four groups: CO (sham BDL), BDL (BDL surgery), CO + Mel (sham BDL and Mel administration) and BDL + Mel (BDL surgery and Mel administration). Mel was administered intraperitoneally for 2 wk, starting on postoperative day 15, at a dose of 20 mg/kg. RESULTS Mel was effective at the different standards, reestablishing normal liver enzyme levels, reducing the hepatosomatic and splenosomatic indices, restoring lipoperoxidation and antioxidant enzyme concentrations, reducing fibrosis and inflammation, and thereby reducing liver tissue injury in the treated animals. CONCLUSION The results of this study suggest a protective effect of Mel when administered to rats with secondary biliary cirrhosis induced by BDL.

N-acetylcysteine modulates angiogenesis and vasodilation in stomach such as DNA damage in blood of portal hypertensive rats

AIM To evaluate the antioxidant effect of N-acetylcysteine (NAC) on the stomach of rats with portal hypertension. METHODS Twenty-four male Wistar rats weighing ± 250 g were divided into four experimental groups (n = 6 each): Sham-operated (SO), SO + NAC, partial portal vein ligation (PPVL), and PPVL + NAC. Treatment with NAC in a dose of 10 mg/kg (i.p.) diluted in 0.6 mL of saline solution was administered daily for 7 d starting 8 d after the surgery. Animals from the PPVL and SO group received saline solution (0.6 mL) for the same period of time as the PPVL + NAC and SO + NAC group. On the 15(th) day the animals were anesthetized and we evaluated portal pressure by cannulating mesenteric artery. After, we removed the stomach for further analysis. We performed immunohistochemical analysis for endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), and nitrotirosine (NTT) proteins in stomach. We also evaluated eNOS and VEGF by Western blot analysis and assessed DNA damage in blood samples by the comet assay. RESULTS The portal hypertension group exhibited increases in portal pressure when compared to SO group (29.8 ± 1.8 vs 12.0 ± 0.3 mmHg) (P < 0.001). The same was observed when we compared the eNOS (56.8 ± 3.7 vs 13.46 ± 2.8 pixels) (P < 0.001), VEGF (34.9 ± 4.7 vs 17.46 ± 2.6 pixels) (P < 0.05), and NTT (39.01 ± 4.0 vs 12.77 ± 2.3 pixels) (P < 0.05) expression by immunohistochemistry of the PPVL animals with the SO group. The expression of eNOS (0.39 ± 0.03 vs 0.25 ± 0.03 a.μ) (P < 0.01) and VEGF (0.38 ± 0.04 vs 0.26 ± 0.04 a.μ) (P < 0.01) were also evaluated by Western blot analysis, and we observed an increase of both proteins on PPVL animals. We also evaluated the DNA damage by comet assay, and observed an increase on damage index and damage frequency on those animals. NAC decreased portal pressure values in PPVL + NAC animals (16.46 ± 2 vs 29.8 ± 1.8 mmHg) (P < 0.001) when compared to PPVL. The expression of eNOS (14.60 ± 4.1 vs 56.8 ± 3.7 pixels) (P < 0.001), VEGF (19.53 ± 3.2 vs 34.9 ± 4.7 pixels) (P < 0.05) and NTT (21.84 ± 0.7 vs 39.01 ± 4.0 pixels) (P < 0.05) evaluated by immunohistochemistry were also reduced in PPVL + NAC animals. Also, when evaluated by Western blot eNOS expression (0.32 ± 0.03 vs 0.39 ± 0.03 a.μ) (P < 0.05) and VEGF expression (0.31 ± 0.09 vs 0.38 ± 0.04 a.μ) (P < 0.01). Furthermore, NAC modulated DNA damage in PPVL + NAC animals. CONCLUSION In view of these results, we believe NAC is able to protect the stomach from the alterations induced by the PPVL procedure.

Effect of glutamine on liver injuries induced by intestinal ischemia-reperfusion in rats

INTRODUCTION Intestinal ischemia-reperfusion (I/R) injury may cause cell and tissue damage, reaching also other organs such as the liver. Because of the involvement of free radicals in I/R injury, treatment options with antioxidants have been studied and tested. OBJECTIVE To evaluate the effect of glutamine (Gln) in the liver of animals with intestinal I/R injury. METHODS We used 20 male Wistar rats divided into four groups: sham-operated (SO); glutamine + sham-operated (G+SO); intestinal ischemia-reperfusion (I/R); glutamine + intestinal ischemia-reperfusion (G+I/R). The superior mesenteric artery was clamped for 30 minutes and reperfused for 15 minutes. Gln (25 mg/kg/day) diluted in 1 ml of saline was administered intraperitoneally on the two days before I/R induction. RESULTS Levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), lipid peroxidation (LPO) and expressions of interleukin-6 (IL-6) and nuclear factor kappa B (NF-kB) showed a significant reduction in the G+I/R group as compared with the I/R group. The activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) showed an increase in the G+I/R group as compared with the I/R group. CONCLUSION Pretreatment with Gln reduced oxidative, tissue damage and showed a decrease expression of inflammatory mediators.

Glutamine prevents oxidative stress in a model of portal hypertension

AIM To evaluate the protective effects of glutamine in a model of portal hypertension (PH) induced by partial portal vein ligation (PPVL). METHODS Male Wistar rats were housed in a controlled environment and were allowed access to food and water ad libitum. Twenty-four male Wistar rats were divided into four experimental groups: (1) control group (SO) - rats underwent exploratory laparotomy; (2) control + glutamine group (SO + G) - rats were subjected to laparotomy and were treated intraperitoneally with glutamine; (3) portal hypertension group (PPVL) - rats were subjected to PPVL; and (4) PPVL + glutamine group (PPVL + G) - rats were treated intraperitoneally with glutamine for seven days. Local injuries were determined by evaluating intestinal segments for oxidative stress using lipid peroxidation and the activities of glutathione peroxidase (GPx), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) after PPVL. RESULTS Lipid peroxidation of the membrane was increased in the animals subjected to PH (P < 0.01). However, the group that received glutamine for seven days after the PPVL procedure showed levels of lipid peroxidation similar to those of the control groups (P > 0.05). The activity of the antioxidant enzyme GTx was decreased in the gut of animals subjected to PH compared with that in the control group of animals not subjected to PH (P < 0.01). However, the group that received glutamine for seven days after the PPVL showed similar GTx activity to both the control groups not subjected to PH (P > 0.05). At least 10 random, non-overlapping images of each histological slide with 200 × magnification (44 pixel = 1 μm) were captured. The sum means of all areas, of each group were calculated. The mean areas of eNOS staining for both of the control groups were similar. The PPVL group showed the largest area of staining for eNOS. The PPVL + G group had the second highest amount of staining, but the mean value was much lower than that of the PPVL group (P < 0.01). For iNOS, the control (SO) and control + G (SO + G) groups showed similar areas of staining. The PPVL group contained the largest area of iNOS staining, followed by the PPVL + G group; however, this area was significantly smaller than that of the group that underwent PH without glutamine (P < 0.01). CONCLUSION Treatment with glutamine prevents gut mucosal injury after PH in rats.