Pyridoxamine and Caloric Restriction Improve Metabolic and Microcirculatory Abnormalities in Rats with Non-Alcoholic Fatty Liver Disease.

INTRODUCTION: This study aims to examine the effect of a diet intervention and pyridoxamine (PM) supplementation on hepatic microcirculatory and metabolic dysfunction in nonalcoholic fatty liver disease (NAFLD). METHODS: NAFLD in Wistar rats was induced with a high-fat diet for 20 weeks (NAFLD 20 weeks), and control animals were fed with a standard diet. The NAFLD diet intervention group received the control diet between weeks 12 and 20 (NAFLD 12 weeks), while the NAFLD 12 weeks + PM group also received PM. Fasting blood glucose (FBG) levels, body weight (BW), visceral adipose tissue (VAT), and hepatic microvascular blood flow (HMBF) were evaluated at the end of the protocol. RESULTS: The NAFLD group exhibited a significant increase in BW and VAT, which was prevented by the diet intervention, irrespective of PM treatment. The FBG was elevated in the NAFLD group, and caloric restriction improved this parameter, although additional improvement was achieved by PM. The NAFLD group displayed a 31% decrease in HMBF, which was partially prevented by caloric restriction and completely prevented when PM was added. HMBF was negatively correlated to BW, FBG, and VAT content. CONCLUSION: PM supplementation in association with lifestyle modifications could be an effective intervention for metabolic and hepatic vascular complications.

Pyridoxamine improves metabolic and microcirculatory complications associated with nonalcoholic fatty liver disease.

OBJECTIVE: We investigated the protective effects of pyridoxamine against metabolic and microcirculatory complications in nonalcoholic fatty liver disease. METHODS: Nonalcoholic fatty liver disease was established by a high-fat diet administration over 28 weeks. Pyridoxamine was administered between weeks 20 and 28. The recruitment of leukocytes and the number of vitamin A-positive hepatic stellate cells were examined by in vivo microscopy. Laser speckle contrast imaging was used to evaluate microcirculatory hepatic perfusion. Thiobarbituric acid reactive substances measurement and RT-PCR were used for oxidative stress and inflammatory parameters. advanced glycation end products were evaluated by fluorescence spectroscopy. RESULTS: The increase in body, liver, and fat weights, together with steatosis and impairment in glucose metabolism observed in the nonalcoholic fatty liver disease group were attenuated by pyridoxamine treatment. Regarding the hepatic microcirculatory parameters, rats with high-fat diet-induced nonalcoholic fatty liver disease showed increased rolling and adhesion of leukocytes, increased hepatic stellate cells activation, and decreased tissue perfusion, which were reverted by pyridoxamine. Pyridoxamine protected against the increased hepatic lipid peroxidation observed in the nonalcoholic fatty liver disease group. Pyridoxamine treatment was associated with increased levels of tumor necrosis factor alpha (TNF-α) mRNA transcripts in the liver. CONCLUSION: Pyridoxamine modulates oxidative stress, advanced glycation end products, TNF-α transcripts levels, and metabolic disturbances, being a potential treatment for nonalcoholic fatty liver disease-associated microcirculatory and metabolic complications.

Cerebral microvascular dysfunction in metabolic syndrome is exacerbated by ischemia-reperfusion injury.

BACKGROUND: Metabolic syndrome (MetS) is associated with an increased risk of cerebrovascular diseases, including cerebral ischemia. Microvascular dysfunction is an important feature underlying the pathophysiology of cerebrovascular diseases. In this study, we aimed to investigate the impacts of ischemia and reperfusion (IR) injury on the cerebral microvascular function of rats with high-fat diet-induced MetS. RESULTS: We examined Wistar rats fed a high-fat diet (HFD) or normal diet (CTL) for 20 weeks underwent 30 min of bilateral carotid artery occlusion followed by 1 h of reperfusion (IR) or sham surgery. Microvascular blood flow was evaluated on the parietal cortex surface through a cranial window by laser speckle contrast imaging, functional capillary density, endothelial function and endothelial-leukocyte interactions by intravital videomicroscopy. Lipid peroxidation was assessed by TBARs analysis, the expression of oxidative enzymes and inflammatory markers in the brain tissue was analyzed by real-time PCR. The cerebral IR in MetS animals induced a functional capillary rarefaction (HFD IR 117 ± 17 vs. CTL IR 224 ± 35 capillary/mm2; p < 0.05), blunted the endothelial response to acetylcholine (HFD IR -16.93% vs. CTL IR 16.19% from baseline inner diameter p < 0.05) and increased the endothelial-leukocyte interactions in the venules in the brain. The impact of ischemia on the cerebral microvascular blood flow was worsened in MetS animals, with a marked reduction of cerebral blood flow, exposing brain tissue to a higher state of hypoxia. CONCLUSIONS: Our results demonstrate that during ischemia and reperfusion, animals with MetS are more susceptible to alterations in the cerebral microcirculation involving endothelial dysfunction and oxidative stress events.

Combined therapy with metformin and insulin attenuates systemic and hepatic alterations in a model of high-fat diet-/streptozotocin-induced diabetes.

In this study we have explored the pathogenesis of the hepatic alterations which occur in diabetes and the modulation of these complications by the combination of metformin adjunct treatment and insulin monotherapy. For this purpose, diabetic rats were treated with insulin (DM + Ins) or metformin plus insulin (DM + Met + Ins). Biochemical and cardiometabolic parameters were analysed by spectrophotometry. Intravital microscopy was used to study the hepatic microcirculation. In the liver tissue, real-time PCR was used to analyse oxidative stress enzymes, inflammatory markers and receptors for advanced glycation end products (AGE) (RAGE) gene expression. Lipid peroxidation was assessed by thiobarbituric acid reactive species (TBARs) analyses. AGE deposition and RAGE protein expression were studied by fluorescence spectrophotometry and Western blot respectively. Body weight, %HbA1c , urea, total proteins and oxidative stress parameters were found to be similarly improved by insulin or Met + Ins treatments. On the other hand, Met + Ins treatment showed a more pronounced effect on fasting blood glucose level than insulin monotherapy. Fructosamine, uric acid, creatinine, albumin and amylase levels and daily insulin dose requirements were found to be only improved by the combined Met + Ins treatment. Liver, renal and pancreatic dysfunction markers were found to be more positively affected by metformin adjunct therapy when compared to insulin treatment. Liver microcirculation damage was found to be completely protected by Met + Ins treatment, while insulin monotherapy showed no effect. Our results suggest that oxidative stress, microcirculatory damage and glycated proteins could be involved in the aetiology of liver disease due to diabetes. Additionally, metformin adjunct treatment improved systemic and liver injury in induced diabetes and showed a more pronounced effect than insulin monotherapy.

Levels of circulating anti-muscarinic and anti-adrenergic antibodies and their effect on cardiac arrhythmias and dysautonomia in murine models of Chagas disease.

SUMMARY Antibodies (Ab) recognizing G-protein coupled receptors, such as ß 1 and ß 2 adrenergic (anti-ß 1-AR and anti-ß 2-AR, respectively) and muscarinic cholinergic receptors (anti-M2-CR) may contribute to cardiac damage, however their role in chronic chagasic cardiomyopathy is still controversial. We describe that Trypanosoma cruzi-infected C3H/He mice show increased P and QRS wave duration, and PR and QTc intervals, while the most significant ECG alterations in C57BL/6 are prolonged P wave and PR interval. Echocardiogram analyses show right ventricle dilation in infected animals of both mouse lineages. Analyses of heart rate variability (HRV) in chronically infected C3H/He mice show no alteration of the evaluated parameters, while C57BL/6 infected mice display significantly lower values of HRV components, suggesting autonomic dysfunction. The time-course analysis of anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR Ab titres in C3H/He infected mice indicate that anti-ß 1-AR Ab are detected only in the chronic phase, while anti-ß 2-AR and anti-M2-CR are observed in the acute phase, diminish at 60 dpi and increase again in the chronic phase. Chronically infected C57BL/6 mice presented a significant increase in only anti-M2-CR Ab titres. Furthermore, anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR, exhibit significantly higher prevalence in chronically T. cruzi-infected C3H/He mice when compared with C57BL/6. These observations suggest that T. cruzi infection leads to host-specific cardiac electric alterations.

Preclinical evaluation of combined therapy with amiodarone and low-dose benznidazole in a mouse model of chronic Trypanosoma cruzi infection.

Chagasic chronic cardiomyopathy (CCC) is the primary clinical manifestation of Chagas disease (CD), caused by Trypanosoma cruzi. Current therapeutic options for CD are limited to benznidazole (Bz) and nifurtimox. Amiodarone (AMD) has emerged as most effective drug for treating the arrhythmic form of CCC. To address the effects of Bz and AMD we used a preclinical model of CCC. Female C57BL/6 mice were infected with T. cruzi and subjected to oral treatment for 30 consecutive days, either as monotherapy or in combination. AMD in monotherapy decreased the prolonged QTc interval, the incidence of atrioventricular conduction disorders and cardiac hypertrophy. However, AMD monotherapy did not impact parasitemia, parasite load, TNF concentration and production of reactive oxygen species (ROS) in cardiac tissue. Alike Bz therapy, the combination of Bz and AMD (Bz/AMD), improved cardiac electric abnormalities detected T. cruzi-infected mice such as decrease in heart rates, enlargement of PR and QTc intervals and increased incidence of atrioventricular block and sinus arrhythmia. Further, Bz/AMD therapy ameliorated the ventricular function and reduced parasite burden in the cardiac tissue and parasitemia to a degree comparable to Bz monotherapy. Importantly, Bz/AMD treatment efficiently reduced TNF concentration in the cardiac tissue and plasma and had beneficial effects on immunological abnormalities. Moreover, in the cardiac tissue Bz/AMD therapy reduced fibronectin and collagen deposition, mitochondrial damage and production of ROS, and improved sarcomeric and gap junction integrity. Our study underlines the potential of the Bz/AMD therapy, as we have shown that combination increased efficacy in the treatment of CCC.

Hair cortisol levels are associated with overweight and obesity in the ELSA-Brasil cohort.

Introduction: Hair cortisol level has recently been identified as a promising marker for detecting long-term cortisol levels and a marker of hypothalamic-pituitary-adrenal cortex (HPA) axis activity. However, research on the association between obesity and an altered cortisol metabolism remains controversial. Objective: This study aimed to investigate the relationship between hair cortisol levels and overweight and obesity in participants from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). Methods: This was a cross-sectional study involving 2,499 participants from the second follow-up (visit 3, 2017-2019) attending research centers in Rio de Janeiro and Rio Grande do Sul states. Hair samples were collected, and cortisol levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits. Cortisol levels were classified as low (< 40 pg/mg), medium (40-128 pg/mg), or high (> 128 pg/mg). The participants were classified as eutrophic, overweight, or obese according to their weight (kg) and height (m2). Odds ratios (ORs) with 95% confidence intervals (95%CI) were estimated. Results: Of the 2499 individuals, 30% had eutrophic weight, 40% were overweight, and 30% were obese. Notably, cortisol levels gradually increased with increasing body weight. Among participants with high hair cortisol levels, 41.2% were classified as overweight and 34.2% as obese. Multinomial logistic regression analysis indicated that participants with high cortisol levels were 43% (OR =1.43; 95%CI: 1.02-2.03) more likely to be overweight and 72% (OR =1.72; 95%CI:1.20-2.47) more likely to be obese than participants with low hair cortisol levels. After adjustment for all covariates, high cortisol levels remained associated with obesity (OR = 1.54; 95%CI:1.02-2.31) and overweight (OR =1.33; 95%CI:0.91-1.94). Conclusion: In the ELSA-Brazil cohort, hair stress were positively associated with overweight and obesity. These results underscore the importance of considering stress and cortisol as potential factors in obesity prevention and intervention efforts, and highlight a novel aspect of the complex relationship between stress and obesity in the Brazilian population.

Cholesterol Exacerbates the Pathophysiology of Non-Alcoholic Steatohepatitis by Upregulating Hypoxia-Inducible Factor 1 and Modulating Microcirculatory Dysfunction.

Cholesterol is a pivotal lipotoxic molecule that contributes to the progression of Non-Alcoholic Steatohepatitis NASH). Additionally, microcirculatory changes are critical components of Non-Alcoholic Fatty Liver Disease (NAFLD) pathogenesis. This study aimed to investigate the role of cholesterol as an insult that modulates microcirculatory damage in NAFLD and the underlying mechanisms. The experimental model was established in male C57BL/6 mice fed a high-fat high-carbohydrate (HFHC) diet for 39 weeks. Between weeks 31-39, 2% cholesterol was added to the HFHC diet in a subgroup of mice. Leukocyte recruitment and hepatic stellate cells (HSC) activation in microcirculation were assessed using intravital microscopy. The hepatic microvascular blood flow (HMBF) was measured using laser speckle flowmetry. High cholesterol levels exacerbated hepatomegaly, hepatic steatosis, inflammation, fibrosis, and leukocyte recruitment compared to the HFHC group. In addition, cholesterol decreased the HMBF-cholesterol-induced activation of HSC and increased HIF1A expression in the liver. Furthermore, cholesterol promoted a pro-inflammatory cytokine profile with a Th1-type immune response (IFN-γ/IL-4). These findings suggest cholesterol exacerbates NAFLD progression through microcirculatory dysfunction and HIF1A upregulation through hypoxia and inflammation. This study highlights the importance of cholesterol-induced lipotoxicity, which causes microcirculatory dysfunction associated with NAFLD pathology, thus reinforcing the potential of lipotoxicity and microcirculation as therapeutic targets for NAFLD.

Cirrhotic Liver Sustains In Situ Regeneration of Acellular Liver Scaffolds after Transplantation into G-CSF-Treated Animals.

Acellular liver scaffolds (ALS) produced by decellularization have been successfully explored for distinct regenerative purposes. To date, it is unknown whether transplanted ALSs are affected by cirrhotic livers, either becoming cirrhotic themselves or instead remaining as a robust template for healthy cell growth after transplantation into cirrhotic rats. Moreover, little is known about the clinical course of recipient cirrhotic livers after ALS transplantation. To address these questions, we transplanted ALSs into cirrhotic rats previously treated with the granulocyte colony-stimulating factor. Here, we report successful cellular engraftment within the transplanted ALSs at 7, 15, and 30 days after transplantation. Recellularization was orchestrated by liver tissue cell activation, resident hepatocytes and bile duct proliferation, and an immune response mediated by the granulocyte components. Furthermore, we showed that transplanted ALSs ensured a pro-regenerative and anti-inflammatory microenvironment, attracted vessels from the host cirrhotic tissue, and promoted progenitor cell recruitment. ALS transplantation induced cirrhotic liver regeneration and extracellular matrix remodeling. Moreover, the transplanted ALS sustained blood circulation and attenuated alterations in the ultrasonographic and biochemical parameters in cirrhotic rats. Taken together, our results confirm that transplanted ALSs are not affected by cirrhotic livers and remain a robust template for healthy cell growth and stimulated cirrhotic liver regeneration.

Simvastatin Improves Microcirculatory Function in Nonalcoholic Fatty Liver Disease and Downregulates Oxidative and ALE-RAGE Stress.

Increased reactive oxidative stress, lipid peroxidation, inflammation, and fibrosis, which contribute to tissue damage and development and progression of nonalcoholic liver disease (NAFLD), play important roles in microcirculatory disorders. We investigated the effect of the modulatory properties of simvastatin (SV) on the liver and adipose tissue microcirculation as well as metabolic and oxidative stress parameters, including the advanced lipoxidation end product-receptors of advanced glycation end products (ALE-RAGE) pathway. SV was administered to an NAFLD model constructed using a high-fat-high-carbohydrate diet (HFHC). HFHC caused metabolic changes indicative of nonalcoholic steatohepatitis; treatment with SV protected the mice from developing NAFLD. SV prevented microcirculatory dysfunction in HFHC-fed mice, as evidenced by decreased leukocyte recruitment to hepatic and fat microcirculation, decreased hepatic stellate cell activation, and improved hepatic capillary network architecture and density. SV restored basal microvascular blood flow in the liver and adipose tissue and restored the endothelium-dependent vasodilatory response of adipose tissue to acetylcholine. SV treatment restored antioxidant enzyme activity and decreased lipid peroxidation, ALE-RAGE pathway activation, steatosis, fibrosis, and inflammatory parameters. Thus, SV may improve microcirculatory function in NAFLD by downregulating oxidative and ALE-RAGE stress and improving steatosis, fibrosis, and inflammatory parameters.

Experimental Combination Therapy with Amiodarone and Low-Dose Benznidazole in a Mouse Model of Trypanosoma cruzi Acute Infection.

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately 6 to 7 million people in Latin America, with cardiomyopathy being the clinical manifestation most commonly associated with patient death during the acute phase. The etiological treatment of CD is restricted to benznidazole (Bz) and nifurtimox (Nif), which involve long periods of administration, frequent side effects, and low efficacy in the chronic phase. Thus, combined therapies emerge as an important tool in the treatment of CD, allowing the reduction of Bz dose and treatment duration. In this sense, amiodarone (AMD), the most efficient antiarrhythmic drug currently available and prescribed to CD patients, is a potential candidate for combined treatment due to its known trypanocidal activity. However, the efficacy of AMD during the acute phase of CD and its interaction with Bz or Nif are still unknown. In the present study, using a well-established murine model of the acute phase of CD, we observed that the Bz/AMD combination was more effective in reducing the peak parasitemia than both monotherapy treatments. Additionally, the Bz/AMD combination reduced (i) interleukin-6 (IL-6) levels in cardiac tissue, (ii) P-wave duration, and (iii) frequency of arrhythmia in infected animals and (iv) restored gap junction integrity in cardiac tissue. Therefore, our study validates AMD as a promising candidate for combined therapy with Bz, reinforcing the strategy of combined therapy for CD. IMPORTANCE Chagas disease affects approximately 6 to 7 million people worldwide, with cardiomyopathy being the clinical manifestation that most commonly leads to patient death. The etiological treatment of Chagas disease is limited to drugs (benznidazole and nifurtimox) with relatively high toxicity and therapeutic failures. In this sense, amiodarone, the most effective currently available antiarrhythmic drug prescribed to patients with Chagas disease, is a potential candidate for combined treatment due to its known trypanocidal effect. In the present study, we show that combined treatment with benznidazole and amiodarone improves the trypanocidal effect and reduces cardiac damage in acutely T. cruzi-infected mice.

Aerobic Exercise Training Improves Microvascular Function and Oxidative Stress Parameters in Diet-Induced Type 2 Diabetic Mice.

Purpose: Type 2 diabetic (T2D) patients have liver and adipose tissue microcirculation disturbances associated with metabolic dysfunction and disease progression. However, the potential role of aerobic training on hepatic and white adipose tissue (WAT) microcirculation and the underlying mechanisms have not been elucidated to date. Therefore, we investigated the role of aerobic training on liver and WAT microcirculation and AGE-RAGE modulation in T2D mice. Methods: The control group (CTL) was fed standard chow, and T2D was induced by feeding male C57BL/6 a high-fat, high-carbohydrate diet for 24 weeks. In the following 12 weeks, mice underwent aerobic training (CTL EX and T2D EX groups), or were kept sedentary (CTL and T2D groups). We assessed metabolic parameters, biochemical markers, oxidative damage, the AGE-RAGE axis, hepatic steatosis, hepatic stellate cells activation (HSC) and liver and WAT microcirculation. Results: Hepatic microcirculation was improved in T2D EX mice which were associated with improvements in body, liver and fat mass, blood pressure, hepatic steatosis and fibrosis, and decreased HSC and AGE-RAGE activation. In contrast, improvement in WAT microcirculation, that is, decreased leukocyte recruitment and increased perfusion, was associated with increased catalase antioxidant activity. Conclusion: Physical training improves hepatic and adipose tissue microcirculatory dysfunction associated with T2D, likely due to downregulation of AGE-RAGE axis, decreased HSC activation and increased antioxidant activity.

Benznidazole and amiodarone combined treatment attenuates cytoskeletal damage in Trypanosoma cruzi-infected cardiac cells.

Chagas disease (CD), a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, is an important public health problem mainly in Latin America, leading to approximately 12,000 annual deaths. Current etiological treatment for CD is limited to two nitro compounds, benznidazole (Bz) and nifurtimox (Nif), both presenting relevant limitations. Different approaches have been employed to establish more effective and safer schemes to treat T. cruzi infection, mostly based on drug repurposing and combination therapies. Amiodarone (AMD), an antiarrhythmic medicament of choice for patients with the chronic cardiac form of CD, is also recognized as a trypanocidal agent. Therefore, our aim is to investigate the combined treatment Bz + AMD on trypomastigote viability, control of T. cruzi intracellular form proliferation, and recovery of the infection-induced cytoskeleton alterations in cardiac cells. The combination of Bz + AMD did not improve the direct trypanocidal effect of AMD on the infective blood trypomastigote and replicative intracellular forms of the parasite. Otherwise, the treatment of T. cruzi-infected cardiac cells with Bz plus AMD attenuated the infection-triggered cytoskeleton damage of host cells and the cytotoxic effects of AMD. Thus, the combined treatment Bz + AMD may favor parasite control and hamper tissue damage.

The efficacy of novel arylimidamides against Trypanosoma cruzi in vitro.

The present study aimed to determine the in vitro biological efficacy and selectivity of 7 novel AIAs upon bloodstream trypomastigotes and intracellular amastigotes of Trypanosoma cruzi. The biological activity of these aromatic compounds was assayed for 48 and 24 h against intracellular parasites and bloodstream forms of T. cruzi (Y strain), respectively. Additional assays were also performed to determine their potential use in blood banks by treating the bloodstream parasites with the compounds diluted in mouse blood for 24 h at 4°C. Toxicity against mammalian cells was evaluated using primary cultures of cardiac cells incubated for 24 and 48 h with the AIAs and then cellular death rates were determined by MTT colorimetric assays. Our data demonstrated the outstanding trypanocidal effect of AIAs against T. cruzi, especially DB1853, DB1862, DB1867 and DB1868, giving IC50 values ranging between 16 and 70 nanomolar against both parasite forms. All AIAs presented superior efficacy to benznidazole and some, such as DB1868, also demonstrated promising activity as a candidate agent for blood prophylaxis. The excellent anti-trypanosomal efficacy of these novel AIAs against T. cruzi stimulates further in vivo studies and justifies the screening of new analogues with the goal of establishing a useful alternative therapy for Chagas disease.

Role of Maternal Microbiota and Nutrition in Early-Life Neurodevelopmental Disorders.

The rise in the prevalence of obesity and other related metabolic diseases has been paralleled by an increase in the frequency of neurodevelopmental problems, which has raised the likelihood of a link between these two phenomena. In this scenario, maternal microbiota is a possible linking mechanistic pathway. According to the "Developmental Origins of Health and Disease" paradigm, environmental exposures (in utero and early life) can permanently alter the body's structure, physiology, and metabolism, increasing illness risk and/or speeding up disease progression in offspring, adults, and even generations. Nutritional exposure during early developmental stages may induce susceptibility to the later development of human diseases via interactions in the microbiome, including alterations in brain function and behavior of offspring, as explained by the gut-brain axis theory. This review provides an overview of the implications of maternal nutrition on neurodevelopmental disorders and the establishment and maturation of gut microbiota in the offspring.

Advanced glycation end product: A potential biomarker for risk stratification of non-alcoholic fatty liver disease in ELSA-Brasil study.

BACKGROUND: Liver diseases are associated with the excess formation of advanced glycation end products (AGEs), which induce tissue inflammation and oxidative damage. However, the trend of oxidative marker levels according to the steatosis grade in non-alcoholic fatty liver disease (NAFLD) is unclear. AIM: To compare serum AGE levels between participants with NAFLD accordingly to steatosis severity in the baseline ELSA-Brasil population. METHODS: In 305 individuals at baseline ELSA-Brasil, NAFLD-associated steatosis was classified by ultrasound hepatic attenuation. The participants were grouped according to the severity of steatosis: mild and moderate/severe pooled. The measurement of serum fluorescent AGE concentrations was based on spectrofluorimetric detection. Serum AGE content and clinical and laboratory characteristics of the participants were compared between groups. The correlation between serum AGE levels and the grade of steatosis was analyzed. Logistic regression analysis was used to investigate the relationship between serum AGE levels and steatosis severity. A P value < 0.05 was considered statistically significant. RESULTS: According to the steatosis severity spectrum in NAFLD, from mild to moderate/severe, individuals with the most severe steatosis grade had a higher incidence of metabolic syndrome (63% vs 34%, P ≤ 0.001), diabetes mellitus (37% vs 14%, P ≤ 0.001), and high cholesterol levels (51% vs 33%, P < 0.001). Moreover, individuals with increasing severity of steatosis presented increasing waist circumference, body mass index, systolic and diastolic blood pressure, fasting blood glucose, glycated hemoglobin, insulin, triglycerides, alanine aminotransferase, gamma-glutamyl transferase, C-reactive protein, and uric acid levels and lower high-density lipoprotein. Higher serum AGE content was present in the moderate/severe group of individuals than in the mild group (P = 0.008). In addition, the serum AGE levels were correlated with the steatosis grade in the overall sample (rho = 0.146, P = 0.010). Logistic regression analysis, after adjusting for confounding variables, showed that subjects with higher serum AGE content had a 4.6-fold increased chance of having moderate or severe steatosis when compared to low levels of serum AGEs. According to the results of the receiver operator characteristic curves analyses (areas under the curve, AUC = 0.83), AGEs could be a good marker of steatosis severity in patients with NAFLD and might be a potential biomarker in predicting NAFLD progression, strengthening the involvement of AGE in NAFLD pathogenesis. CONCLUSION: NAFLD-associated steatosis was associated with serum AGE levels; therefore, plasmatic fluorescent AGE quantification by spectroscopy could be a promising alternative method to monitor progression from mild to severe NAFLD accordingly to steatosis grade.

The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi.

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease.

Effects of short-term high-intensity interval and continuous exercise training on body composition and cardiac function in obese sarcopenic rats.

AIM: We investigated the effects of high-intensity interval and continuous short-term exercise on body composition and cardiac function after myocardial ischemia-reperfusion injury (IRI) in obese rats. METHODS: Rats fed with a standard chow diet (SC) or high-fat diet (HFD) for 20 weeks underwent systolic blood pressure (SBP), glycemia and dual-energy X-ray absorptiometry analyses. Then, animals fed with HFD were subdivided into three groups: sedentary (HFD-SED); moderate-intensity continuous training (HFD-MICT); and high-intensity interval training (HFD-HIIT). Exercised groups underwent four isocaloric aerobic exercise sessions, in which HFD-MICT maintained the intensity continuously and HFD-HIIT alternated it. After exercise sessions, all groups underwent global IRI and myocardial infarct size (IS) was determined histologically. Fat and muscle mass were weighted, and protein levels involved in muscle metabolism were assessed in skeletal muscle. RESULTS: HFD-fed versus SC-fed rats reduced lean body mass by 31% (P < 0.001), while SBP, glycemia and body fat percentage were increased by 10% (P = 0.04), 30% (P = 0.006) and 54% (P < 0.001); respectively. HFD-induced muscle atrophy was restored in exercised groups, as only HFD-SED presented lower gastrocnemius (32%; P = 0.001) and quadriceps mass (62%; P < 0.001) than SC. PGC1-α expression was 2.7-fold higher in HFD-HIIT versus HFD-SED (P = 0.04), whereas HFD-HIIT and HFD-MICT exhibited 1.7-fold increase in p-mTORSer2481 levels compared to HFD-SED (P = 0.04). Although no difference was detected among groups for IS (P = 0.30), only HFD-HIIT preserved left-ventricle developed pressure after IRI (+0.7 mmHg; P = 0.9). SIGNIFICANCE: Short-term exercise, continuous or HIIT, restored HFD-induced muscle atrophy and increased mTOR expression, but only HIIT maintained myocardial contractility following IRI in obese animals.

In vitro analyses of the effect of aromatic diamidines upon Trypanosoma cruzi.

OBJECTIVES: Aromatic diamidines (ADs) have been recognized as promising antiparasitic agents. Therefore, in the present work, the in vitro trypanocidal effect of 11 ADs upon the relevant clinical forms of Trypanosoma cruzi was evaluated, as well as determining their toxicity to mammalian cells and their subcellular localization. METHODS: The trypanocidal effect upon trypomastigotes and amastigotes was evaluated by light microscopy through the determination of the IC(50) values. The cytotoxicity was determined by the MTT colorimetric assay against mouse cardiomyocytes. For the subcellular localization, transmission electron microscopy and fluorescence approaches were used. The fluorescence intensity within the kinetoplast DNA (kDNA) and nuclear DNA (nDNA) of treated parasites was determined using the Image J program. RESULTS: Compounds 2, 5 and 7 showed the lowest IC(50) values (micromolar range) against intracellular amastigotes and trypomastigotes. In the presence of blood, all the tested ADs exhibited a reduction of their activity. The compounds did not exhibit toxicity to cardiac cells and the highest selectivity index (SI) was achieved by compound 5 with an SI of >137 for trypomastigotes and compound 7 with an SI of >107 for intracellular parasites. The subcellular effects upon bloodstream forms treated with compounds 5 and 7 were mainly on kDNA, leading to its disorganization. The higher accumulation in the kDNA observed for all tested ADs was not directly related to their efficacy. CONCLUSIONS: Our results show the high activity of this new series of ADs against both trypomastigote and amastigote forms, with excellent SIs, especially compound 7, which merits further in vivo evaluation.

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies.

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.