Linda Elsa Muñoz-Espinosa

Temporal relationship of serum markers and tissue damage during acute intestinal ischemia/reperfusion.

OBJECTIVE: It is essential to identify a serological marker of injury in order to study the pathophysiology of intestinal ischemia reperfusion. In this work, we studied the evolution of several serological markers after intestinal ischemia reperfusion injury in rats. The markers of non-specific cell damage were aspartate aminotransferase, alanine aminotransaminase, and lactic dehydrogenase, the markers of inflammation were tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta, and the markers of intestinal mucosal damage were intestinal fatty acid binding protein and D-lactate. We used Chiús classification to grade the histopathological damage. METHODS: We studied 35 Wistar rats divided into groups according to reperfusion time. The superior mesenteric artery was clamped for 30 minutes, and blood and biopsies were collected at 1, 3, 6, 12, 24, and 48 hours after reperfusion. We plotted the mean ± standard deviation and compared the baseline and maximum values for each marker using Students t-test. RESULTS: The maximum values of interleukin-1 beta and lactic dehydrogenase were present before the maximal histopathological damage. The maximum tumor necrosis factor alpha and D-lactate expressions coincided with histopathological damage. Alanine aminotransaminase and aspartate aminotransferase had a maximum expression level that increased following the histopathological damage. The maximum expressions of interluken-6 and intestinal fatty acid binding protein were not significantly different from the Sham treated group. CONCLUSION: For the evaluation of injury secondary to acute intestinal ischemia reperfusion with a 30 minute ischemia period, we recommend performing histopathological grading, quantification of D-lactate, which is synthesized by intestinal bacteria and is considered an indicator of mucosal injury, and quantification of tumor necrosis factor alpha as indicators of acute inflammation three hours after reperfusion.

Performance of the international classifications criteria for autoimmune hepatitis diagnosis in Mexican patients

The revised score of the International Autoimmune Hepatitis Group (R-IAIHG) and the simplified criteria (SC) are used for diagnosis of autoimmune hepatitis (AIH). Our aim is to evaluate the performance of these classifications to differentiate AIH from other autoimmune liver diseases. The frequency of diagnosis of definite AIH was similar both by the R-IAIHG and the SC systems (41% versus 40%), whereas diagnosis of probable AIH was made more commonly by the R-IAIHG than the SC (59% versus 29%), and 23 patients that have been graded as definite (n = 7) or probable (n = 16) AIH by the R-IAIHG had non-diagnostic scores by the SC system. The scoring systems rendered concordant diagnosis of definite (n = 15) and probable (n = 13) AIH in 28/73 patients (38%). Discordant diagnoses of AIH were rendered in 45/73 patients (62%). The R-IAIHG exhibited a sensitivity of 95%, specificity of 90%, and positive predictive value (PPV) and negative predictive value (NPV) of 93% for both. On the other hand, the SC had a lower sensitivity (65%) but a higher specificity (100%), PPV of 100%, and NPV of 68%. In conclusion, both international scoring systems diagnosed the same number of cases as definite AIH. The R-IAIHG showed a higher sensitivity in diagnosing AIH, whereas the SC showed a higher specificity. SC are easier to apply at the bedside and exclude more patients that could have a different etiology.

Effects of Thalidomide and Pentoxyphylline Over Local and Remote Organ Injury After Intestinal Ischemia/Reperfusion

OBJECTIVE We investigated the effects of thalidomide alone or in combination with pentoxyphylline upon intestinal ischemia/reperfusion (I/R) injury in the rat. MATERIALS AND METHODS Twenty male Wistar rats were randomized into 5 groups: sham-operated (SHAM), control (CTL), thalidomide (400 mg/kg) treatment (THAL), pentoxyphylline (50 mg/kg) treatment and a combination group (THAL + POX). I/R was induced by clamping the superior mesenteric artery for 45 minutes, followed by 120 minutes of reperfusion. We measured serum concentrations of aspartate-aminotransferase (AST), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-alpha as well as lipid peroxidation and antioxidant status. Intestinal samples were morphologically analyzed, and dry to wet (W/D) ratios calculated in intestinal, lung and liver samples, as a measurement of tissue edema. RESULTS Serum concentrations of AST, LDH, and TNF-alpha were increased after I/R in the CTL compared with the SHAM group (P < .05). Lipid peroxidation was also increased, and antioxidant capacity in serum, decreased (P < .05). The W/D ratio was elevated in all tissue samples as well (P < .05). Both thalidomide and pentoxyphylline effectively reduced AST, LDH, TNF-alpha, and lipid peroxidation levels, as well as attenuated tissue edema and intestinal injury induced by I/R (P < .05). Combination treatment showed only modest additive effects on lung W/D ratio and TNF-alpha levels. CONCLUSION Both drugs protected the intestine, lungs, and liver against intestinal I/R injury, probably by inhibition of TNF-alpha and lipid peroxidation. However, combination treatment showed small, additive effects.

Hepatic Ischemia/Reperfusion Injury Is Diminished by Atorvastatin in Wistar Rats

BACKGROUND AND AIMS Temporal occlusion of the hepatoduodenal ligament (HDL) is often used during liver surgeries in order to reduce blood loss, resulting in ischemia/reperfusion injury (I/R). The aim of the study was to investigate the effects of atorvastatin (ATOR) on hepatic I/R injury and on serum levels of tumor necrosis factor-alpha (TNF-α), endothelin-1 (ET-1), antithrombin III (ATIII) and intracellular adhesion molecule-1 (ICAM-1). METHODS Liver ischemia was induced in Wistar rats by clamping the HDL for 60 min, followed by either 60 or 180 min reperfusion. Rats received either vehicle or 10 mg/kg ATOR before hepatic I/R. Control group received sham surgery. Livers were examined for histological damage and serum AST, ALT, TNF-α, ET-1, ATIII and ICAM-1 concentrations were measured. RESULTS After I/R, AST and ALT were significantly elevated, ATIII levels were significantly depleted, both TNF-α and ICAM-1 levels increased and ET-1 was significantly elevated (at 180 min). ATOR pretreatment attenuated these alterations and diminished histological injury scores. CONCLUSIONS Our results show that ATOR protects the liver from I/R injury.

Behavior of Oxidative Stress Markers in Alcoholic Liver Cirrhosis Patients

Alcohol is the most socially accepted addictive substance worldwide, and its metabolism is related with oxidative stress generation. The aim of this work was to evaluate the role of oxidative stress in alcoholic liver cirrhosis (ALC). This study included 187 patients divided into two groups: ALC, classified according to Child-Pugh score, and a control group. We determined the levels of reduced and oxidized glutathione (GSH and GSSG) and the GSH/GSSG ratio by an enzymatic method in blood. Also, protein carbonyl and malondialdehyde (MDA) content were estimated in serum. MDA levels increased in proportion to the severity of damage, whereas the GSH and GSSG levels decreased and increased, respectively, at different stages of cirrhosis. There were no differences in the GSH/GSSG ratio and carbonylated protein content between groups. We also evaluated whether the active consumption of or abstinence from alcoholic beverages affected the behavior of these oxidative markers and only found differences in the MDA, GSH, and GSSG determination and the GSH/GSSG ratio. Our results suggest that alcoholic cirrhotic subjects have an increase in oxidative stress in the early stages of disease severity and that abstinence from alcohol consumption favors the major antioxidant endogen: GSH in patients with advanced disease severity.

Gemfibrozil attenuates the inflammatory response and protects rats from abdominal sepsis

Sepsis is a serious condition characterized by an infectious process that induces a severe systemic inflammatory response. In this study, the effects of gemfibrozil (GFZ) on the inflammatory response associated with abdominal sepsis were investigated using a rat model of cecal-ligation and puncture (CLP). Male Wistar rats were randomly divided into three groups: Sham-operated group (sham), where laparotomy was performed, the intestines were manipulated, and the cecum was ligated but not punctured; control group, subjected to CLP; and GFZ group, which received GFZ prior to undergoing CLP. The groups were then subdivided into three different time-points: 2, 4 and 24 h, indicating the time at which blood samples were obtained for analysis. Serum concentrations of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were determined. The LDH, AST and ALT values were significantly elevated following CLP compared with those in the sham group, and GFZ treatment was able to reduce these elevations. GFZ also reduced the sepsis-induced elevations of TNF-α and IL-1. In conclusion, GFZ treatment was able to attenuate the inflammatory response associated with CLP-induced sepsis, by diminishing the release of inflammatory cytokines, thereby reducing tissue injury and oxidative stress.

Subclinical abnormal glucose tolerance is a predictor of death in liver cirrhosis

AIM To determine if subclinical abnormal glucose tolerance (SAGT) has influence on survival of non-diabetic patients with liver cirrhosis. METHODS In total, 100 patients with compensated liver cirrhosis and normal fasting plasma glucose were included. Fasting plasma insulin (FPI) levels were measured, and oral glucose tolerance test (OGTT) was performed. According to OGTT results two groups of patients were formed: those with normal glucose tolerance (NGT) and those with SAGT. Patients were followed every three months. The mean follow-up was 932 d (range of 180-1925). Survival was analyzed by the Kaplan-Meyer method, and predictive factors of death were analyzed using the Cox proportional hazard regression model. RESULTS Of the included patients, 30 showed NGT and 70 SAGT. Groups were significantly different only in age, INR, FPI and HOMA2-IR. Patients with SAGT showed lower 5-year cumulated survival than NGT patients (31.7% vs 71.6%, P = 0.02). Differences in survival were significant only after 3 years of follow-up. SAGT, Child-Pugh B, and high Child-Pugh and Model for End-Stage Liver Disease (MELD) scores were independent predictors of death. The causes of death in 90.3% of cases were due to complications related to liver disease. CONCLUSION SAGT was associated with lower survival. SAGT, Child-Pugh B, and high Child-Pugh and MELD scores were independent negative predictors of survival.

Hepatoprotective effect of commercial herbal extracts on carbon tetrachloride-induced liver damage in Wistar rats

Background: Various hepatoprotective herbal products from plants are available in Mexico, where up to 85% of patients with liver disease use some form of complementary and alternative medicine. However, only few studies have reported on the biological evaluation of these products. Objective: Using a model of carbon tetrachloride (CCl4)-induced hepatotoxicity in rats, we evaluated the effects of commercial herbal extracts used most commonly in the metropolitan area of Monterrey, Mexico. Materials and Methods: The commercial products were identified through surveys in public areas. The effect of these products given with or without CCl4 in rats was evaluated by measuring the serum concentrations of aspartate amino transferase (AST) and alanine amino transferase (ALT), and histopathological analysis. Legalon® was used as the standard drug. Results: The most commonly used herbal products were Hepatisan® capsules, Boldo capsules, Hepavida® capsules, Boldo infusion, and milk thistle herbal supplement (80% silymarin). None of the products tested was hepatotoxic according to transaminase and histological analyses. AST and ALT activities were significantly lower in the Hepavida+CCl4-treated group as compared with the CCl4-only group. AST and ALT activities in the silymarin, Hepatisan, and Boldo tea groups were similar to those in the CCl4 group. The CCl4 group displayed submassive confluent necrosis and mixed inflammatory infiltration. Both the Hepatisan+CCl4 and Boldo tea+CCl4 groups exhibited ballooning degeneration, inflammatory infiltration, and lytic necrosis. The silymarin+CCl4 group exhibited microvesicular steatosis. The Hepavida+CCl4- and Legalon+CCL4-treated groups had lower percentages of necrotic cells as compared with the CCl4-treated group; this treatment was hepatoprotective against necrosis. Conclusion: Only Hepavida had a hepatoprotective effect.

Is Ischemic Preconditioning a Useful Therapeutic Strategy in Liver Transplantation? Results from the First Pilot Study in Mexico.

BACKGROUND AND AIMS The protective effect of ischemic preconditioning (IP) in liver transplantation (LT) has been studied with controversial results. We undertook this study to investigate whether IP of cadaveric donor livers is protective to allografts. METHODS IP (LT + IP, n = 6) was induced by 10-min hilar clamping. These were compared to cadaver donors with no IP (LT, n = 7). Clinical data and blood were obtained in donors and recipients for biochemical and inflammatory mediator (IM) measurements (P-selectin, leukotriene B4, myeloperoxidase, ICAM-1, IL-1, IL-6, and TNF-α). Liver tissue samples were obtained from donors and recipients (90 min after reperfusion). RESULTS No significant differences were found in demographic characteristics between donors and recipients. When comparing both groups (LT + IP vs. LT only), ICU stay was longer in LT + IP group. For biochemical parameters, a significant difference was found only with a higher total bilirubin at postoperative day 3 in LT + IP group. There was no statistical difference in IM between LT and LT + IP groups at different stages of the study. Histological analysis of donor grafts indicated the presence of steatosis (50%) in one graft from the LT + IP group. However, in post-reperfusion biopsies neither neutrophil infiltration nor grade of necrosis showed significant difference between groups. No incidence of primary graft nonfunction (PGNF) was observed and graft and patient survival was similar in the two groups at 24 months. CONCLUSION IP does not seem to protect against I/R injury in cadaveric LT, and no PGNF was seen.

S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells

AIM To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. METHODS We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellmans recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. RESULTS SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level. CONCLUSION A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.