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Fish Oil - Omega-3 Exerts Protective Effect in Oxidative Stress and Liver Dysfunctions Resulting from Experimental Sepsis

      Highlights

      • Protective effect, during sepsis, against oxidative stress in the liver.
      • The treatment demonstrated a protective effect against liver injuries and dysfunctions.
      • The treatment showed good results in the evaluations of the liver tissue.
      • Observed improvement, based on the results of the creatinine and urea tests.
      • Omega 3 has potential for the treatment of liver oxidative stress resulting from sepsis.

      Background

      Sepsis is a severe global health problem, with high morbidity and mortality. In sepsis, one of the main affected organs is the liver. Hepatic alterations characterize a negative prognostic. Omega-3 fatty acids (ω3), eicosapentaenoic acid, and docosahexaenoic acid, are part of the main families of polyunsaturated fatty acids. ω3 has been used in studies as sepsis treatment and as a treatment for non-alcoholic liver disease.

      Aim

      We aimed to evaluate the effects of treatment with fish oil (FO) rich in ω3 on liver changes and damage resulting from experimental sepsis.

      Methodology

      A model of severe sepsis in Wistar rats was used. Oxidative stress in the liver tissue was evaluated by means of tests of thiobarbituric acid reactive substances, 2,7-dihydrodichlorofluorescein diacetate , catalase, and glutathione peroxidase, in the serum TBARS, DCF, thiols and, to assess liver dysfunction, alanine aminotransferase and aspartate aminotransferase. Hepatic tissue damage was evaluated using H&E histology.

      Results

      In assessments of oxidative stress in liver tissue, a protective effect was observed in the tests of TBARS, DCF, CAT, and GPx, when compared the sepsis versus sepsis+ω3 groups. Regarding the oxidative stress in serum, a protective effect of treatment with ω3 was observed in the TBARS, DCF, and thiols assays, in the comparison between the sepsis and sepsis+ω3 groups. ω3 had also a beneficial effect on biochemical parameters in serum in the analysis of ALT, creatinine, urea, and lactate, observed in the comparison between the sepsis and sepsis+ω3 groups.

      Conclusion

      The results suggest ω3 as a liver protector during sepsis with an antioxidant effect, alleviating injuries and dysfunctions.

      Graphical abstract

      Keywords

      Abbreviations:

      ALT (alanine aminotransferase), AST (aspartate aminotransferase), CAT (catalase), DCF (2,7-dihydrodichlorofluorescein diacetate), DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), FO (fish oil), GPx (glutathione peroxidase), GTO (oxaloacetic transaminase), GTP (pyruvic transaminase), HE (Hematoxylin and Eosin), ICON (Intensive Care Over Nations), ICU (intensive care unit), IFN- γ (interferon gamma), RNS (reactive nitrogen species), ROS (reactive oxygen species), TBARS (Thiobarbituric Acid Reactive Substances), TGF-β (transforming growth factor beta), TNF-α (tumor necrosis factor alpha), ω3 (omega-3)
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       this study aims to observe beneficial effects of the treatment with fish oil rich in ω3 against oxidative stress, lesions and liver dysfunctions during experimental sepsis.

      Material and Methods

      Ethics Approval

      The experimental protocol was approved by the Ethics Committee on the Use of Animals (ECUA) of UFCSPA (protocol number 244/18).

      Animals

      Male Wistar Rats (20–24 weeks old), weighting between 350 and 450 g, (n = 40), from the bioterium of Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). Animals were kept in cages, in air-conditioned room, relative humidity ranging between 55 and 65%, a 12 h light–dark cycle, temperature of 22 ± 2 °C, with free access to food and water. The animals were accommodated in accordance with the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society. The experimental protocol was approved by the Ethics Committee on the Use of Animals (ECUA) of UFCSPA (protocol number 244/18).

      Experimental Sepsis Induction and Treatment

      The animals were weighed and then anesthetized with a solution of ketamine (80 mg/kg) and xylazine (20 mg/kg) intraperitoneally (i.p). The abdomen of each animal was shaved and cleaned with povidone–iodine solution. A 2 cm midline abdominal incision was made to expose the linea alba. The peritoneum was opened by blunt dissection. Sepsis was induced by introducing a sterile gelatin capsule size “1” in the peritoneal cavity containing another sterile capsule size “2” containing the Escherichia coli (200 μL, ATCC 25922) suspension and a non-sterile fecal content (30 mg), a well-consolidated experimental methodology used in previous studies.
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      Physiopathological studies in septic rats and the use of fructose 1,6-bisphosphate as cellular protection.
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      The animals were then divided into four groups: control group (Naive) (i), in which the animals did not undergo any type of surgical or drug intervention; (ii) sham (rats were implanted with an empty capsule and received 600 μL/kg of saline solution via gavage, 1 h before and 4 h after the procedure); (iii) sepsis (induction of sepsis and administration of 600 μL/kg of saline solution via gavage, 1 h before and 4 h after sepsis induction); (iv) sepsis+ω3 (the induction of sepsis and administration of ω3 (EPA/DHA) (600 μL/kg of FO),
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      ,
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      Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease.
      via gavage, 1 h before induction and 4 h after induction). We used fish oil (Catarinense pharma), composed of 54% EPA and 36% docosahexaenoic (DHA), enclosed in gelatin capsules. Each 1000 mg of oil contains 540 mg of EPA and 360 mg of DHA).
      Blood and liver tissue were collected 12 h after sepsis induction, when all animals were euthanized.

      Biochemical Tests

      Biochemical tests were evaluated on samples of 1 mL of serum from each animal of all groups, using 100 μL of serum for each analysis performed.
      The parameters of lactate, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total proteins, urea, and creatinine were evaluated using a commercial kit (Bioclin), according to the manufacturer’s protocol.

      Oxidative Stress Assay

      Oxidative stress assays were performed with 1 g cell lysate of standard liver tissue from each animal, from all groups. Thiobarbituric aid reactive substances (TBARS) and dihydrodichlorofluorescein diacetate (DCF) analyses were also performed in serum samples. Thiols’ analysis was performed only in serum samples.
      Catalase (CAT) activity was measured in liver lysate supernatant (0.25 mL) in potassium phosphate buffer (50 mM, pH 7.0) with H2O2 (2 mM, Sigma–Aldrich, EUA). The decomposition of H2O2 by CAT was noted by the change in absorbance at 240 nm (ΔE) during 1 min (Spectra Max 190, Molecular Devices, Sunnyvale, CA, USA).
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      Enthalpy of decomposition of hydrogen peroxide by catalase at 25° C (with molar extinction coefficients of H2O2 solutions in the UV).
      Total thiols were measured by the reaction of 25 μL of serum with 450 μL of phosphate buffer (0.2 M, pH 8.0) and 25 μL of 5,5′-dithiobis- (2-nitrobenzoic acid) (10 mM, Sigma–Aldrich®, St. Louis, MO, USA), for 30 min in the dark.
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      Tissue sulfhydryl groups.
      Afterward, 200 μL were transferred to a microplate and the spectrophotometric reading was performed at wavelength of 412 nm in a Biochrom microplate reader EZ Read 400.
      Glutathione peroxidase (GPx) activity was measured using tert-butyl-hydroperoxide as a substrate. NADPH disappearance was monitored at 340 nm. The medium contained 2 mM glutathione, 0.15 U/mL glutathione reductase, 0.4 mM azide, 0.5 mM tert-butyl-hydroperoxide, and 0.1 mM NADPH. One GPx unit is defined as 1 μmol of NADPH consumed per minute; the specific activity is represented as GPx units/mg protein.
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      Tissue sulfhydryl groups.
      Reactive oxygen species (ROS) production was evaluated in the liver lysate samples and in serum samples by the fluorescence intensity of the redox-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (100 μM, Sigma–Aldrich)—(excitation and emission wavelengths of 480 and 535 nm, respectively) using SpectraMax M2e (Molecular Devices, USA).
      TBARS concentration was determined as described by Ohkawa, et al.
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      • Ohishi N.
      • Yagi K.
      Assay for lipid peroxides in animal tissues thiobarbituric acid reaction.
      Briefly, 0.2 mL of liver lysate was added to sodium dodecyl sulfate (8.1%, 0.05 mL, acetic acid (0.2 mL, 2.5 M, pH 3.4), and 0.8% thiobarbituric acid (0.25 mL, Sigma, USA) and heated in boiling water (90 °C) for 60 min. After supernatants were transferred to 96 wells microplate and the absorbance was read at 532 nm (EZReader, EUA). The same procedure was performed with 100 μL of serum samples from the same animals in all groups.

      Histology—Hematoxylin and Eosin

      The liver tissue was perfused in 10% buffered formaldehyde, then embedded in paraffin, and sliced in microtome in 4 μm sections, which were fixed on slides and stained with Hematoxylin and Eosin (HE). The slides were analyzed, with image formation, under a Leica microscope (DM6 B) and captured by a Leica camera (DFC7000 T).

      Statistical Analysis

      All results are presented as mean ± standard deviation (SD) of values of each group of analysis. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-test was used for comparisons between groups in the assays. A P value < 0.05 was considered statistically significant for the analyzes. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA).

      Results

      Effects of ω3 in Lactate Levels in Experimental Sepsis

      There was a significant elevation in lactate levels in the sepsis group when compared to the control groups naive (∗∗∗P < 0.001; Figure 1) and sham (###P < 0.001; Figure 1), confirming anaerobic respiration due to reduction in the oxygen supply, usually resulting from inflammatory process in sepsis, leading to organic failure. ω3 treatment was able to decrease the lactate levels, when compared to sepsis group ($$$P < 0.001; Figure 1), but they were still higher than naive group (∗P < 0.05; Figure 1).
      Figure 1
      Figure 1Assessment of serum lactate levels. Lactate levels were evaluated in serum from Wistar rats in the experimental groups. Data are presented as mean ± standard deviation for each group. ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups, $$$P < 0.001 between sepsis+ω3 and sepsis groups, and ∗P < 0.05 between the naive and sepsis+ω3 groups. N = 9 in the naive group; N = 9 in the sham group; N = 11 in the sepsis group; N = 10 in the sepsis+ω3 group.

      Effects of ω3 on the Levels of Liver Injury Biomarkers During Experimental Sepsis

      There was a significant elevation of ALT levels in the sepsis group when compared to the control group naive (∗∗P < 0.01; Figure 2A), indicating liver injury and alterations in experimental sepsis. Treatment with ω3 was able to maintain low ALT levels during sepsis, with a significant difference between the sepsis+ω3 and the sepsis groups ($$P < 0.01; Figure 2A).
      Figure 2
      Figure 2Evaluation of markers of liver injury alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The levels of ALT (a) and AST (b) transaminases were evaluated in Wistar rats' serum in the experimental groups. Data are presented as mean ± standard deviation for each group (a): ∗∗P < 0.01 between the naive and the sepsis groups and $$P < 0.01 between the sepsis+ω3 and in the sepsis groups (b): ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups, ∗∗∗P < 0.001 between naive and sepsis+ω3 groups and ###P < 0.001 between sham and sepsis+ω3 groups. N = in the naive group; N = 9 in the sham group; N = 11 in the sepsis group; N = 10 in the sepsis+ω3 group.
      There was a significant increase in AST levels in the sepsis group when compared to the control groups naive (∗∗∗P < 0.001; Figure 2B) and sham (###P < 0.001; Figure 2B). There was also a significant increase in AST levels in the sepsis+ω3 group compared to the control groups naive (∗∗∗P < 0.001; Figure 2B) and sham (###P < 0.001; Figure 2B).

      Effects of ω3 in Liver Tissue in Experimental Sepsis

      In the histological evaluation, in liver tissue stained with HE, it was not possible to obtain quantitative data. Images showed minimum liver congestion in naive and sham groups (Figure 3A and B). It was also observed tissue injury with necrosis, liver congestion, and edema in the sepsis group (Figure 3C) and mild congestion and inflammatory cell infiltrate in the sepsis+ω3 group (Figure 3D).
      Figure 3
      Figure 3Liver tissue stained with HE. The figure depicts HE liver images for the four groups studied. Images A (naive) and B (sham) present minimal congestion. In image C (sepsis), it is possible to see congestion, edema, and necrosis: necrosis (blue arrows) and cellular debris (yellow arrows) (arrows - 20x). Image D (sepsis + ω3) shows vascular and sinusoidal congestion (blue arrows) and mild inflammatory cells infiltrate (yellow arrows) (arrows - 20x). HE, hematoxylin and eosin.

      Effects of ω3 in Oxidative Stress and in the Antioxidant Process in Experimental Sepsis

      TBARS in Serum

      TBARS levels were higher in sepsis group than control groups naive (∗∗∗P < 0.001; Figure 4A) and sham (#P < 0.05; Figure 4A). The ω3 treatment was able to restore the TBARS levels to the same as the control groups naive and sham ($$$P < 0.001 when compared to sepsis group; Figure 4A).
      Figure 4
      Figure 4Assessments of ROS production. The markers of thiobarbituric acid reactive substances (TBARS) (a) and (b) and 2,7-dihydrodichlorofluorescein diacetate (DCF) (c) and (d) were evaluated in liver tissue and in serum of Wistar rats in the experimental groups. Data are presented as mean ± standard deviation for each group (a): TBARS in liver tissue, ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups and $$$P < 0.001 between the sepsis+ω3 and sepsis groups (b): TBARS in serum, ∗∗∗P < 0.001 between naive and sepsis groups, #P < 0.05 between sham and sepsis groups and $$$P < 0.001 between sepsis+ω3 and sepsis groups (c): DCF in liver tissue, ∗∗∗P < 0.001 between the naive and sepsis groups, ###P < 0.001 between the sham and sepsis groups, $$$P < 0.001 between the sepsis+ω3 and sepsis groups and ∗∗P < 0.01 between naive and sepsis+ω3 groups (d): DCF in serum, ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups and $$$P < 0.001 between sepsis+ω3 and sepsis groups. N = 9 in the naive group; N = 9 in the sham group; N = 11 in the sepsis group; N = 10 in the sepsis+ω3 group. ROS, reactive oxygen species.

      TBARS in Liver Tissue

      As observed in serum, there was a significant increase in the TBARS levels in the sepsis group when compared to the control groups naive (∗∗∗P < 0.001; Figure 4B) and sham (###P < 0.001; Figure 4B). In the same way, ω3 treatment decreased the TBARS levels to the control levels ($$$P < 0.001 compared to sepsis group Figure 4B).

      DCF in Serum

      Experimental sepsis induced an increase in the DCF levels compared to control groups naive (∗∗∗P < 0.001; Figure 4C) and sham (###P < 0.001; Figure 4C), and the treatment with ω3 restored the levels to the similar values of control groups ($$$P < 0.001 compared to sepsis group; Figure 4C).

      DCF in Liver Tissue

      In the same way as we observed in the serum, DCF levels in liver tissue were higher in sepsis group than control groups naive (∗∗P < 0.01; Figure 4D) and sham (##P < 0.01; Figure 4D). ω3 treatment reduced DCF levels during experimental sepsis, when compared to sepsis group ($$$P < 0.001; Figure 4D), but the levels were still slightly higher than naive group (∗∗P < 0.01; Figure 4D).

      CAT and GPx in Liver Tissue

      CAT activity in liver tissue was reduced in the sepsis group when compared to the control groups naive (∗∗∗P < 0.001; Figure 5A) and sham (###P < 0.001; Figure 5A). ω3 treatment during experimental sepsis increased CAT activity when compared to sepsis group ($P < 0.05; Figure 5A), but this activity was lower than control groups naive (∗∗∗P < 0.001; Figure 5A) and sham (###P < 0.001; Figure 5A).
      Figure 5
      Figure 5Assessments of the antioxidant activity of catalase (CAT), glutathione peroxidase (GPx), and thiols. Activities of CAT (a) and GPx (b) were evaluated in liver tissue and activity of thiols (c) were evaluated in Wistar rats' serum in the experimental groups. Data are presented as mean ± standard deviation for each group (a): ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups, $P < 0.05 between sepsis+ω3 and sepsis groups, ∗∗∗P < 0.001 between naive and sepsis+ω3 groups and ###P < 0.001 between sham and sepsis+ω3 groups (b): ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups, $$$P < 0.001 between sepsis+ω3 and sepsis, ∗∗P < 0.01 between naive and sham groups and ∗P < 0.05 between naive and sepsis+ω3 groups (c): ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups, $$$P < 0.001 between sepsis+ω3 and sepsis groups and ∗P < 0.05 between naive and sepsis+ω3 groups. N = 9 in the naive group; N = 9 in the sham group; N = 11 in the sepsis group; N = 10 in the sepsis+ω3 group.
      Likewise, the activity of glutathione peroxidase in liver tissue was significantly reduced during experimental sepsis when compared to control groups naive (∗∗∗P < 0.001; Figure 5B) and sham (###P < 0.001; Figure 5B). Treatment with ω3 increased the enzymatic activity of GPx when compared to sepsis group ($$$P < 0.001; Figure 5B), but the levels were still lower than the naive group (∗P < 0.05; Figure 5B). And also observed lower levels of GPx in the sham group than naive group (∗∗P < 0.01; Figure 5B).

      Thiols in Serum

      Thiols levels in serum were significantly reduced in the sepsis group when compared to the control groups naive (∗∗∗P < 0.001; Figure 5C) and sham (###P < 0.001; Figure 5C). ω3 treatment during experimental sepsis significantly increased the levels of thiols when compared to sepsis group ($$$P < 0.001; Figure 5C), with levels slightly lower than those observed in naive group (∗P < 0.05; Figure 5C).

      Effects of ω3 in Creatinine, Urea, and Total Proteins Levels

      Creatinine levels were significantly higher in sepsis animal than control groups naive (∗∗∗P < 0.001; Figure 6A) and sham (###P < 0.001; Figure 6A). ω3 treatment reduced creatinine levels, compared to sepsis group ($$$P < 0.001; Figure 6A), being able to restore the levels to the same values of the control groups.
      Figure 6
      Figure 6Assessments of markers of kidney and liver injuries: creatinine, urea, and total proteins. Evaluations of creatinine (a), urea (b), and total proteins (c) were performed in Wistar rats' serum in the experimental groups. Data are presented as mean ± standard deviation for each group (a): ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups and $$$P < 0.001 between sepsis+ω3 and sepsis groups (b): ∗∗∗P < 0.001 between naive and sepsis groups, ###P < 0.001 between sham and sepsis groups, $$$P < 0.001 between sepsis+ω3 and sepsis groups, ∗∗P < 0.01 between naive and sepsis+ω3 groups and ##P < 0.01 between sham and sepsis+ω3 groups (c): ∗∗P < 0.01 between naive and sepsis groups, ##P < 0.01 between sham and sepsis groups, ∗∗P < 0.01 between naive and sepsis+ω3 groups and ##P < 0.01 between sham and sepsis+ω3 groups. N = 9 in the naive group; N = 9 in the sham group; N = 11 in the sepsis group; N = 10 in the sepsis+ω3 group.
      In the same way, the levels of urea were significantly increased in sepsis group, compared to control groups naive (∗∗∗P < 0.001; Figure 6B) and sham (###P < 0.001; Figure 6B). Treatment with ω3 in experimental sepsis reduced urea levels, compared to sepsis group ($$$P < 0.001; Figure 6B); however, the levels were not equal to those of control naive (∗∗∗P < 0.001; Figure 6B) and sham (###P < 0.001; Figure 6B).
      The levels of total proteins in serum of sepsis animals were significantly lower than those observed in control groups naive (∗∗P < 0.01; Figure 6C) and sham (##P < 0.01; Figure 6C). ω3 treatment was not able to increase the total protein levels to the same values of control groups naive (∗∗P < 0.01; Figure 6C) and sham (##P < 0.01; Figure 6C).

      DISCUSSION

      The clinical condition presented by septic patients is due to, among other factors, the exaggerated inflammatory response and can progress quickly to septic shock.
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      which can attenuate the condition of organic dysfunction resulting from septic shock.
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      The liver is one of the main organs affected during sepsis. Hepatocellular injuries are characterized and diagnosed, primarily, by an increase in liver enzymes, such as ALT and AST,

      Rocha L. Evaluation of liver markers in alcoholic students at the college anhanguera de Anápolis-goiás. Rev. Acad. Oswaldo Cruz. oswaldocruz.br/revista_academica/content/pdf/Edicao_12_Silva_Leandro_Rocha.pdf, 1, 130-145.

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      being ALT, the most specific marker for liver injury and dysfunction.
      • Neto A.S.
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      Marcadores hepáticos, prevalência de alterações da síndrome metabólica e efeito do bypass gástrico com reconstrução em Y-de-Roux em pacientes obesos mórbidos.
      ,
      • Penn R.
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      Transaminase levels were significantly elevated in the sepsis group, confirming the hepatic injury resulting from sepsis (Figure 2A).

      Rocha L. Evaluation of liver markers in alcoholic students at the college anhanguera de Anápolis-goiás. Rev. Acad. Oswaldo Cruz. oswaldocruz.br/revista_academica/content/pdf/Edicao_12_Silva_Leandro_Rocha.pdf, 1, 130-145.

      • Neto A.S.
      • Rossi F.M.B.
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      • Rossi M.
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      • McGill M.R.
      The past and present of serum aminotransferases and the future of liver injury biomarkers.
      Treatment with ω3 was able to maintain normal ALT levels in septic animals (Figure 2A), confirming the beneficial effects of ω3 to the liver
      • Yang J.
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      and suggesting that ω3 treatment can also play a protective role against liver tissue injuries resulting from sepsis. On the other hand, the high levels of AST transaminase observed in sepsis + ω3 might be due to the fact that AST is twenty times more concentrated than ALT in the striated cardiac muscle.

      Rocha L. Evaluation of liver markers in alcoholic students at the college anhanguera de Anápolis-goiás. Rev. Acad. Oswaldo Cruz. oswaldocruz.br/revista_academica/content/pdf/Edicao_12_Silva_Leandro_Rocha.pdf, 1, 130-145.

      ,
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      Once the hemodynamic dysfunctions are frequent and severe in sepsis,
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      the increase in AST may be related to the potential cardiac injuries resulting from the experimental sepsis model. We emphasize that ALT is the most specific liver injury biomarker
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      • Rossi F.M.B.
      • Amarante R.D.M.
      • Rossi M.
      Marcadores hepáticos, prevalência de alterações da síndrome metabólica e efeito do bypass gástrico com reconstrução em Y-de-Roux em pacientes obesos mórbidos.
      • McGill M.R.
      The past and present of serum aminotransferases and the future of liver injury biomarkers.
      • Penn R.
      • Worthington D.J.
      Is serum gamma-glutamyltransferase a misleading test?.
       and the one most relevant to our study.
      In the histological evaluation, in liver tissue stained with HE, it was possible to observe and describe alterations consistent to the ones related in the literature,
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      indicating minimum liver congestion in the naive and sham groups (Figure 3A and B), tissue injury with necrosis, congestion, and edema in the sepsis group (Figure 3C), and mild congestion and inflammatory cell infiltrate in the sepsis+ω3 group (Figure 3D), suggesting promising results regarding the role of ω3 in the preservation of liver tissue.
      Liver injuries and dysfunctions in sepsis result mainly from the inflammatory process, along with oxidative stress.
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      ,
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      Note on an eruption resembling that of variola in the skin of a mummy of the twentieth dynasty (1200-1100B.C.).
      In the initial sepsis phase, pro-inflammatory mediators are released in high amounts by many different type cells in the immune system. This phenomenon can be amplified by both an increase in the ROS and reactive nitrogen species.
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      It is believed that ROS may be important injury cell mediators, contributing to the severe state of organic injury in the sepsis. The pro-inflammatory effects of ROS include tissue and endothelial injuries and neutrophils recruitment, causing the release of proteolytic enzymes and even more ROS production, increasing, therefore, hepatocytes injuries.
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      One of the most used assays to access the oxidative stress is the measurement of TBARS, produced as a subproduct of lipidic peroxidation, resulting from tissue injuries generated by oxidative stress.
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      It was observed a significant increase in TBARS in serum and liver tissue samples in septic animals in comparison with the other groups, which was reverted by ω3 (Figure 4A,B). Another biomarker used to evaluate ROS in our study was DCF.
      • Lee B.K.
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      As well as TBARS, DCF levels in the sepsis group were significantly increased when compared to the other groups, both in serum and liver tissue, with ω3 being able to reduce these levels similar to values of the ones in the control groups in serum (Figure 4C, D). Taken together, DCF and TBARS results emphasize the potential therapeutical approach of ω3 against oxidative stress in sepsis, indicating an apparent antioxidant effect during experimental sepsis, both in serum and in liver tissue.
      The increase of peroxide production in sepsis and, consequently, of ROS results in oxidative stress and low antioxidant potential.
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      Thus, the activity of antioxidant enzymes and levels of antioxidant agents, such as CAT, GPx, and thiols, are predictors of oxidative stress and can be used in its diagnose.
      • Hsiao S.
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      ,
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      ,
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      ORIGINAL PAPER A simple automated procedure for thiol measurement in human serum samples.
      We observed a reduction in CAT and GPx activity in the liver tissue of the sepsis group, confirming the high levels of ROS in the liver resulting from sepsis (Figure 5A and 5 B), which has been already observed in previous studies.
      • Hsiao S.
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      ,
      • Sidonia B.
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      • Vlad L.
      • et al.
      Hypothermia effects on liver and kidney oxidative stress parameters in an experimental model of sepsis in rats.
      ω3 treatment increased CAT and GPx activity but was not able to restore theirs levels to the ones observed in the naive group, which could be explained by the extremely oxidant environment that occurs in severe sepsis. Previous studies have also described the ω3 capacity of increasing antioxidant enzymes activity, contributing to reduce ROS production.
      • Yang J.
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      ,
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      The significant difference in CAT and GPx activity between sepsis+ω3 and the naive groups may be coherent, when we consider that the natural antioxidant mechanisms are activated and, therefore, are fighting against the high levels of ROS and oxidative stress resulting from sepsis, but are suppressed in the septic animals that have received no treatment, as occurs in the severe sepsis condition.
      • Hsiao S.
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      ,
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      It is noteworthy that the reduced levels of GPx in the sham group (Figure 5B) could be explained by the adhesion of the agar capsule, used in the procedure to induce sepsis, to the liver, as well as the manipulation of the peritoneum in the region which most of the liver is located. We can hypothesize this explanation by the fact that no other experiment has shown differences between naive and sham groups. Likewise, when assessing the levels of thiols in serum, we observed a significant decrease in the sepsis group, which was practically restored to the control levels when we administrated ω3 (Figure 5C). These results are like those observed in the CAT and GPx evaluation in the liver tissue. Taken together, these data can suggest that ω3 has beneficial effects, contribute to re-establish the redox balance that is compromised during sepsis.
      The mechanisms of the protective effect of ômega-3 against oxidative stress are not yet fully understood. Studies have suggested that this effect may be related to its anti-inflammatory and immunomodulator properties.
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      ,
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      ,
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      ,
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      EPA and DHA acids may not only increase the production of eicosanoids but also the production of lipoxinis, resolvins, protectins, and maresins that may increase phagocytosis and the resolution of inflammation. They can also inhibit the production of adhesion molecules (ICAM 1, VCAM-1, ELAM-1). In addition to these effects, they can limit the generation and activity of inflammatory mediators, including protein kinase (JNK, MAPK, p38), nuclear factor κB, chemokines, and cytokines (TNFα, TNF-1β, IL-1, IL-1, IL- 6, IL-8, and MCP-1). They are also capable of improving the glucose uptake and hypothalamic regulation.
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      Considering that the oxidative stress in sepsis is due to an inflammatory process,
      • Nesseler N.
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      • Morel F.
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      ,
      • Sidonia B.
      • Horatiu R.
      • Vlad L.
      • et al.
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      ,
      • Léon K.
      • Moisan C.
      • Amérand A.
      • Poupon G.
      • Her E.L.
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       it is possible that the beneficial results of ω3 are related to its anti-inflammatory effect.
      Urea, creatinine, and total proteins have also been evaluated, with the purpose of analyzing organic dysfunction related to kidney and liver metabolism in sepsis.
      • Koca U.L.
      • Olguner Ç.G.
      • Ergür B.U.L.
      • et al.
      The effects of dexmedetomidine on secondary acute lung and kidney injuries in the rat model of intra-abdominal sepsis.
      • Frank L.E.E.
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      • Park J.
      • Hwang I.
      • Ha N.
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      Effects of the anti-sepsis drug, (S)-1-(α-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD-712), on mortality, inflammation, and organ injuries in rodent sepsis models.
      Creatinine levels were restored by treatment with ω3 (Figure 6A). The same phenomenon was observed regarding the urea levels, but the restored levels in the sepsis+ω3 group remained a little higher than the control groups (Figure 6B). Although the ω3 treatment was not able to restore the total proteins levels (Figure 6C), the overall data confirm the kidney and metabolic dysfunction that has already been reported in other studies in murine
      • Koca U.L.
      • Olguner Ç.G.
      • Ergür B.U.L.
      • et al.
      The effects of dexmedetomidine on secondary acute lung and kidney injuries in the rat model of intra-abdominal sepsis.
      • Frank L.E.E.
      • Sosenko I.R.S.
      Failure of premature rabbits to increase antioxidant enzymes during hyperoxic exposure: increased susceptibility to pulmonary oxygen toxicity compared with term rabbits.
      • Park J.
      • Hwang I.
      • Ha N.
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      Effects of the anti-sepsis drug, (S)-1-(α-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD-712), on mortality, inflammation, and organ injuries in rodent sepsis models.
      resulting from sepsis. However, the creatinine and urea results agree with the other data obtained in our study, suggesting a beneficial effect of ω3 on organ dysfunction, not only about the liver but also a possible protective effect against renal dysfunction due to sepsis.
      Considering that organic dysfunctions, such as liver dysfunction, are recurrent and severe during sepsis
      • Nesseler N.
      • Launey Y.
      • Aninat C.
      • Morel F.
      • Mallédant Y.
      • Seguin P.
      Clinical review : the liver in sepsis.
      ,
      • Huang M.
      • Cai S.
      • Su J.
      The pathogenesis of sepsis and potential therapeutic targets.
       and may leave long-term sequelae, affecting the quality of life of patients during and after sepsis,
      • Hsiao S.
      • Kung C.
      • Su C.
      • et al.
      Impact of oxidative stress on treatment outcomes in adult patients with sepsis.
       preclinical studies focusing on specific treatments and therapies for hepatic dysfunction are of great relevance to the advancement of adjuvant therapy in the organic pathophysiology of sepsis.
      • Nesseler N.
      • Launey Y.
      • Aninat C.
      • Morel F.
      • Mallédant Y.
      • Seguin P.
      Clinical review : the liver in sepsis.
       Treatment with fish oil rich in ω3 has shown an apparent protective effect against organic dysfunction due to sepsis, with reduced lactate levels in serum from septic Wistar rats. ω3 has shown a beneficial effect against liver injury, reducing ALT levels. The treatment has also provoked the decrease of ROS and oxidative stress, with the reduction of TBARS and DCF, both in serum and liver tissue. CAT and GPx, antioxidant markers, showed increased activity in liver tissue in the sepsis+ω3 group and, in the same group, thiols levels in serum were higher than in the sepsis group. Creatinine and urea were also reduced due to ω3 treatment. These are optimistic results and have shown the ω3, a promising tool as a treatment for liver injuries and alterations due to sepsis, especially in the oxidative stress, acting as an anti-inflammatory and potent antioxidant.

      Funding

      The authors received no financial support for the research, authorship, and/or publication of this article.

      Author contributions

      Mary JSG Velasque, Fernanda B Nunes, Marilene Porawski, and Gisele Branchini conceived the idea and the formulation of research and the study design. Mary JSG Velasque, Gisele Branchini, and Anderson V Catarina wrote the manuscript. Anderson V Catarina performed the statistical analysis of the data. Lais Bettoni, Renata S Fernandes, Arthur F da Silva, Gilson P Dorneles, Igor M da Silva, Maeli A Santos, Juliana Sumienski, Alessandra Peres, and Maria Beatriz Kohek contribute to the experiments with animals and to the biochemical analyses. Adriana V Roehe performed the histological evaluation of liver slides. Gisele Branchini, Marilene Porawski, and Fernanda B Nunes supervised the whole study development.

      Conflict of interests

      All authors have none to declare.

      Acknowledgments

      The authors would like to acknowledge the multiple contributions of all authors engaged in the study, both in the experimental phase and in the development of the study discussion. The authors also thank the technical team of the several laboratories of UFCSPA for the valuable assistance that allowed the execution of this work.

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