Synergistic antioxidant and anti-inflammatory action of N-acetylcysteine in portal hypertensive gastropathy in rats.

Background and Aim: Portal hypertension (PH) is a syndrome associated with cirrhosis and characterized by a progressive increase in portal pressure, with consequent compensatory vascular dilation. Gastric vascular changes associated with oxidative and nitrosative stress characterize the clinical presentation of portal hypertensive gastropathy (PHG). In addition, the inflammatory process is considered an aggravating factor for severity by contributing to gastric tissue injury. The aim of this study was to investigate the synergistic anti-inflammatory and antioxidant action of N-acetylcysteine (NAC) in the stomach of rats with PH. Materials and Methods: Eighteen Wistar male rats were used in this experimental protocol and were divided into three groups with six in each group: sham-operated (SO), partial portal vein ligation (PPVL), and PPVL + NAC. Treatment with NAC at a dose of 10 mg/kg (i.p.) was initiated on day 8 after surgery and continued for 7 days. We evaluated the expression of iNOS, NQO-1, HSP-90, and SOD by Western blot, as well as nuclear factor-kappa B (NF-κB) and tumor necrosis factor (TNF)-α staining by immunohistochemistry, in the rat stomach. Results: The PPVL group exhibited increased expression of HSP-90, iNOS, SOD, and NQO-1 when compared with controls. NAC reduced the expression of all studied proteins. Similarly, NF-κB and TNF-α staining was increased in PPVL animals versus controls and reduced in PPVL + NAC versus PPVL animals, respectively. Conclusion: These results suggest the effectiveness of NAC as a dual anti-inflammatory and antioxidant in animals with experimental PHG induced by partial ligation of the portal vein.

Protective action of glutamine in rats with severe acute liver failure.

BACKGROUND: Severe acute liver failure (SALF) is a rare, but high-mortality, rapidly evolving syndrome that leads to hepatocyte degeneration with impaired liver function. Thioacetamide (TAA) is a known xenobiotic, which promotes the increase of the formation of reactive oxygen species. Erythroid 2-related factor 2 (Nrf2) activates the antioxidant protection of cells. Studies have evidenced the involvement of inflammatory mediators in conditions of oxidative stress. AIM: To evaluate the antioxidant effects of glutamine on Nrf2 activation and NFκB-mediated inflammation in rats with TAA-induced IHAG. METHODS: Male Wistar rats (n = 28) were divided into four groups: control, control+glutamine, TAA, and TAA + glutamine. Two TAA doses (400 mg/kg) were administered intraperitoneally, 8 h apart. Glutamine (25 mg/kg) was administered at 30 min, 24 h, and 36 h. At 48 h, blood was collected for liver integrity analysis [aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP)]. The liver was harvested for histology and assessment of oxidative stress [thiobarbituric acid-reactive substances (TBARS), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione (GSH), Nrf2, Kelch-like ECH-associated protein 1 (Keap1), NADPH quinone oxidoreductase1 (NQO1), superoxide dismutase (SOD)] and inflammatory process. RESULTS: TAA caused disruption of the hepatic parenchyma, with inflammatory infiltration, massive necrosis, and ballooning degeneration. Glutamine mitigated this tissue damage, with visible regeneration of hepatic parenchyma; decreased TBARS (P < 0.001), GSH (P < 0.01), IL-1ß, IL6, and TNFα levels (P <0.01) in hepatic tissue; and decreased blood levels of AST, ALT, and ALP (P <0.05). In addition, CAT, GPx, and GST activities were restored in the glutamine group (P <0.01, P <0.01, and P <0.001, respectively vs TAA alone). Glutamine increased expression of Nrf2 (P < 0.05), NQO1, and SOD (P < 0.01), as well as levels of IL-10 (P <0.001), while decreasing expression of Keap1, TLR4, NFκB (P < 0.001), COX-2 and iNOS, (P < 0.01), and reducing NO2 and NO3 levels (P < 0.05). CONCLUSION: In the TAA experimental model of IHAG, glutamine activated the Nrf2 pathway, thus promoting antioxidant protection, and blunted the NFκB-mediated pathway, reducing inflammation.

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.

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.

Protective effect of glutamine on the main and adjacent organs damaged by ischemia-reperfusion in rats.

Intestinal ischemia and reperfusion (I/R) causes cellular and tissue damage to the intestine and remote organs such as the liver. Increased production of ROS and nitric oxide and dysregulation of cytoprotective enzymes may be involved in intestinal I/R. The aim was to evaluate the protective effects of glutamine on the intestine and liver of rats with intestinal I/R injury. Twenty male Wistar rats (300 g) were divided into four groups: sham-operated (SO), glutamine + SO (G + SO), I/R, and glutamine + I/R (G + I/R). Occlusion of the SMA for 30 min was followed by 15-min reperfusion. Glutamine (25 mg/kg/day) was administered once daily 24 and 48 h before I/R induction. Blood and tissue of were collected for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, histopathological analysis, immunohistochemistry of IL-1ß and TNF-α, thiobarbituric acid reactive substance (TBARS) and nitric oxide, Nrf2/keap1, superoxide dismutase (SOD), NADPH quinone oxidoreductase1 (NQO1), inducible nitric oxide synthase (iNOS), heat shock protein (HSP70), glucose-regulated protein 78 (GRP78), and activating transcription factor 6 (ATF-6) by western blot. Statistic analysis by ANOVA-Student-Newman-Keuls test (mean ± SE) significantly was p < 0.05. Tissue damage, AST, ALT, IL-1ß, TNF-α, TBARS, NO, Keap1, iNOS, GRP78, and ATF-6 expression were significantly lower in the G + I/R group as compared to the I/R group. Expression of Nrf2, SOD, NQO1, and HSP70, was significantly higher in the G + I/R group as compared to I/R group. Pre-treatment with glutamine provided protection against oxidative damage in the intestine and liver in an experimental model of intestinal I/R.

Inflammatory bowel and oxidative stress changes in an experimental model of portal hypertension: action of N-acetylcysteine / Alterações intestinais inflamatórias e de estresse oxidativo em modelo experimental de hipertensão portal: ação da N-acetilcisteína

Introduction: Portal hypertension (PH) is characterized by vasodilatation in the portal system and the bowel is one of the severely affected organs. N-acetylcysteine (NAC) is a molecule with important properties and widely used in clinical practice. Objective: To evaluate NAC action in the bowel of animals submitted to the animal model of partial portal vein ligation (PPVL). Methods: 18 male Wistar rats were divided into three experimental groups (n = 6): sham-operated (SO), PPVL, and PPVL + NAC. On the 8th day after surgery, N-acetylcysteine (10 mg/kg, ip) was administered daily for 7 days. On the 15th day the animals' bowel was collected for oxidative stress analysis, immunohistochemistry and Western blot. We evaluated the expression of NF-KB and TNF-α by immunohistochemistry and of iNOS by Western blot. Lipid peroxidation was assessed by TBARS technique, and the activities of antioxidant enzymes superoxide dismutase (SOD) and glutation peroxidase (GPx) were checked. Results: We observed an increased expression of NF-KB and TNF-α in PPVL group, and an increased iNOS expression assessed by Western blot. NAC reduced the expression of all proteins evaluated. We also observed an increase in oxidative stress in the bowel of mice PPVL group compared to controls (SO), and NAC was effective in reducing these values in PPVL + NAC group. Also, a reduction in the activity of SOD and GPx enzymes was observed in the diseased group, and NAC was able to restore the activity of the enzymes assessed. Conclusion: We suggest the anti-inflammatory and antioxidant action of NAC in the bowel of animals submitted to PPVL model.

Introdução: A Hipertensão Portal (HP) é caracterizada por uma vasodilatação no sistema portal, e o intestino é um dos órgãos gravemente acometidos. A N-acetilcisteína (NAC) é uma molécula com importantes propriedades, amplamente utilizada na clínica. Objetivo: Avaliar a ação da NAC no intestino de animais submetidos ao modelo animal de ligadura parcial da veia porta (LPVP). Métodos: Foram utilizados 18 ratos machos Wistar divididos em três grupos experimentais (n = 6): Sham-operated (SO), LPVP, LPVP + NAC. No 8° dia após a cirurgia, a N-acetilcisteína (10 mg/kg,ip) foi administrada diariamente durante 7 dias. No 15° dia foi coletado o intestino dos animais para análises de estresse oxidativo, imunohistoquímica e Western blot. Nós avaliamos a expressão do NF-kb e TNF-α; por imunohistoquímica e da iNOS por Western blot. A lipoperoxidação foi avaliada pela técnica de TBARS, e as atividades das enzimas antioxidantes Superóxido Dismutase (SOD) e GlutationaPeroxidase (GPx) foram verificadas. Resultados: Observamos um aumento da expressão do NF-kb e TNF-α;; no grupo LPVP, e aumento na expressão da iNOS avaliada por Western blot. A NAC reduziu a expressão de todas as proteínas avaliadas. Observamos um aumento do estresse oxidativo no intestino dos ratos do grupo LPVP com relação aos controles (SO), sendo a NAC eficaz na redução desses valores no grupo LPVP + NAC. Ainda, uma redução na atividade das enzimas SOD e GPx no grupo doente, sendo a NAC capaz de restaurar a atividade das enzimas avaliadas. Conclusão: Sugerimos a ação anti-inflamatória e antioxidante da NAC no intestino de animais submetidos ao modelo LPVP.

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.

Antioxidant effect of mesalazine in the experimental colitis model induced by acetic acid / Efeito antioxidante da mesalazina no modelo de colite experimental induzida por ácido acético

Abstract Introduction Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gastrointestinal tract, without specific cause or pathogen. Objective The effect of mesalazine in a colitis model induced by acetic acid (AA) was evaluated. Methods We used 40 Wistar rats, ±350 g, divided into 4 groups: control (CO); control + mesalazine (CO + M); colitis (CL) and colitis + M (CL + M) at 24 and 48 h of treatment. The animals received the substances by an intracolonic enema of AA 4% and treatment with mesalazine PO 20 mg/kg after colitis induction. Results Mesalazine reduced tissue damage in the gut, normalized sphincter anal pressure levels and decreased lipid peroxidation, metabolites of nitric oxide and iNOS and NF-kB expression in the treated groups in both treatment time points (24 and 48 h), as well as the activity of antioxidant enzymes. Conclusion Mesalazine was effective in reducing tissue damage and oxidative and inflammatory damage, restored antioxidant capacity and increased anal sphincter pressure levels, possibly due to its antioxidant effect.

Resumo Introdução A doença inflamatória intestinal (DII) caracteriza-se por uma inflamação crônica do trato gastrointestinal sem uma causa ou patógeno específico. Objetivo Foi avaliado o efeito da mesalazina no modelo de colite induzida por ácido acético (AA). Material e métodos Foram utilizados 40 ratos wistar, ±350 gramas, divididos em 4 grupos: Controle (CO); Controle + Mesalasina (CO + M); Colite (CL) e Colite + M (CL + M) nos tempos de 24 e 48 horas de tratamento. Os animais foram submetidos à administração intracolônica por enema com solução de AA a 4% e tratamento com mesalazina na dose oral de 20 mg/kg após a indução da colite. Resultados A mesalazina reduziu as lesões teciduais no intestino, normalizou os níveis de pressão anal esfincteriana, reduziu a lipoperoxidação, metabólitos do óxido nítrico e expressão da iNOS e do NF-kB nos grupos tratados em ambos os tempos de tratamento (24 e 48 horas), bem como a atividade das enzimas antioxidantes. Conclusão A mesalazina demonstrou eficácia na redução das lesões teciduais, danos oxidativos e inflamatórios, restabeleceu a capacidade antioxidante e aumentou os níveis de pressão anal esfincteriana, possivelmente pelo seu efeito antioxidante.

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.

Antioxidant effect of N-acetylcysteine on prehepatic portal hypertensive gastropathy in rats.

BACKGROUND: Portal hypertension is a clinical syndrome associated with the development of a hyperdynamic circulation and gastroesophageal varices. Aim. To evaluate the antioxidant effect of N-acetylcysteine on portal hypertensive rats. MATERIAL AND METHODS: Portal hypertension was induced by partial portal vein ligation (PPVL). Oxidative damage in the stomach was measured by lipoperoxidation trough thiobarbituric acid reactive substances (TBARS) and antioxidant enzyme activity; we also evaluated nitrates and nitrites level and histology stained by hematoxylin-eosin. We performed evaluation of portal pressure and measurement of vessels diameter. Liver damage was evaluated by measuring hepatic enzymes. The animals were divided in four experimental groups (n = 6): Sham-operated (SO), SO + NAC, Partial portal vein ligation (PPVL) and PPVL + NAC. N-acetylcysteine (10 mg/kg ip) was administered daily for 7 days and started 8 days after surgery. RESULTS: The portal hypertensive group showed an increase in portal pressure, vessels diameter, levels of TBARS and nitrates and nitrites when compared to SO group. These values were accompanied by a decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) antioxidant enzyme activity. Histology showed dilated vessels in the gastric mucosa in the PPVL group. NAC was able to decrease portal pressure values, vessels diameter, TBARS and also nitrates and nitrites levels when compared to PPVL group. Furthermore, PPVL+NAC group presented an increase in SOD and GPx activity. N-acetylcysteine attenuated damage in gastric mucosa. CONCLUSION: Oxidative stress is associated with portal hypertension and that antioxidant NAC is able to minimize damages of PPVL in rats.

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.