Nicotinamide, an acetylcholinesterase uncompetitive inhibitor, protects the blood‒brain barrier and improves cognitive function in rats fed a hypercaloric diet.

Oxidative stress and inflammation induced by abundant consumption of high-energy foods and caloric overload are implicated in the dysfunction of the blood‒brain barrier (BBB), cognitive impairment, and overactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes hydrolyse acetylcholine, affecting anti-inflammatory cholinergic signalling. Our aim was to evaluate whether nicotinamide (NAM) attenuates the impairment of the BBB and cognitive function, improving cholinergic signalling. Forty male rats were distributed into five groups: one group was fed a standard diet, and the remaining groups were fed a high-fat diet and a beverage with 40% sucrose (HFS; high-fat sucrose). In three of the HFS groups, the carbohydrate was replaced by drinking water containing different concentrations of NAM for 5 h every morning for 12 weeks. The biochemical profile, levels of stress and inflammation markers, cholinesterase activities, BBB permeability, and cognitive capacity were evaluated. The results showed that the HFS diet disturbed the metabolism of carbohydrates and lipids, causing insulin resistance. Simultaneously, AChE and BChE activities, levels of proinflammatory cytokines, oxidation of proteins and lipoperoxidation increased along with decreased antioxidant capacity in serum. In the hippocampus, increased activity of cholinesterases, protein carbonylation and lipoperoxidation were associated with decreased antioxidant capacity. Systemic and hippocampal changes were reflected in increased BBB permeability and cognitive impairment. In contrast, NAM attenuated the above changes by reducing oxidative stress and inflammation through decreasing cholinesterase activities, especially by uncompetitive inhibition. NAM may be a potential systemic and neuroprotective agent to mitigate cognitive damage due to hypercaloric diets.

Nicotinamide reduces inflammation and oxidative stress via the cholinergic system in fructose-induced metabolic syndrome in rats.

AIMS: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) have been associated with risk factors for metabolic syndrome (MetS). Our objective was to evaluate the effect of nicotinamide (NAM) on the activities, expression and protein content of cholinesterases in a MetS model. MAIN METHODS: MetS was induced in male rats administrating 40% fructose to the drinking water for 16 weeks. Additionally, from 5th week onward, the carbohydrate solution was replaced by NAM, at several concentrations for 5 h each morning for the next 12 weeks. In the 15th week, the glucose tolerance test was conducted, and blood pressure was measured. After the treatment period had concluded, the biochemical profile; oxidant stress; proinflammatory markers; and the activity, quantity and expression of cholinesterases were evaluated, and molecular docking analysis was performed. KEY FINDINGS: The MetS group showed anthropometric, hemodynamic and biochemical alterations and increased cholinesterase activity, inflammation and stress markers. In the liver, cholinesterase activity and mRNA, free fatty acid, tumor necrosis factor-alpha (TNF-α), and thiobarbituric acid-reactive substance (TBARS) levels were increased, while reduced glutathione (GSH) levels were decreased. NAM partially or totally decreased risk factors for MetS, markers of stress and inflammation, and the activity (serum and liver) and expression (liver) of cholinesterases. Molecular docking analysis showed that NAM has a greater affinity for cholinesterases than acetylcholine (ACh), suggesting NAM as an inhibitor of cholinesterases. SIGNIFICANCE: Supplementation with 40% fructose induced MetS, which increased the activity and expression of cholinesterases, oxidative stress and the inflammation. NAM attenuated these MetS-induced alterations and changes in cholinesterases.

High fructose-containing drinking water-induced steatohepatitis in rats is prevented by the nicotinamide-mediated modulation of redox homeostasis and NADPH-producing enzymes.

An imbalance in the redox state, increased levels of lipid precursors and overactivation of de novo lipogenesis determine the development of fibrosis during nonalcoholic steatohepatitis (NASH). We evaluated the modulation of NADPH-producing enzymes associated with the antifibrotic, antioxidant and antilipemic effects of nicotinamide (NAM) in a model of NASH induced by excess fructose consumption. Male rats were provided drinking water containing 40% fructose for 16 weeks. During the last 12 weeks of fructose administration, water containing NAM was provided to some of the rats for 5 h/day. The biochemical profiles and the ghrelin, leptin, lipoperoxidation and TNF-α levels in serum and the glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and NADP+-dependent isocitric dehydrogenase (IDP) levels, the reduced/oxidized glutathione (GSH/GSSG) and reduced/oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H/NAD(P)+) ratios, and the levels of various lipogenic and fibrotic markers in the liver were evaluated. The results showed that hepatic fibrosis induced by fructose consumption was associated with weight gain, hunger-satiety system dysregulation, hyperinsulinemia, dyslipidemia, lipoperoxidation and inflammation. Moreover, increased levels of hepatic G6PD and ME activity and expression, the NAD(P)H/NAD(P)+ ratios, and GSSG concentration and increased expression of lipogenic and fibrotic markers were detected, and these alterations were attenuated by NAM administration. Specifically, NAM diminished the activity and expression of G6PD and ME, and this effect was associated with a decrease in the NADPH/NADP+ ratios, increased GSH levels and decreased lipoperoxidation and inflammation, ameliorating fibrosis and NASH development. NAM reduces liver steatosis and fibrosis by regulating redox homeostasis through a G6PD- and ME-dependent mechanism.

Hyperglycemia promotes p53-Mdm2 interaction but reduces p53 ubiquitination in RINm5F cells.

The apoptosis of ß cells induced by hyperglycemia has been associated with p53 mobilization to mitochondria and p53 phosphorylation. Murine double minute 2 (Mdm2) induces the degradation of p53 and thereby protects cells from apoptosis. We studied the effect of glucose at high concentration on the ability of Mdm2 to ubiquitinate p53 and promote its degradation. RINm5F cells were grown in RPMI-1640 medium with 5 or 30 mM glucose for varying periods of time. After this treatment, the expression of Mdm2 was measured using real-time PCR. The phosphorylation of Mdm2 at Ser166, p53 at Ser15, and the kinases Akt and ATM were measured by Western blotting. The formation of the p53-Mdm2 complex and p53 ubiquitination was assessed by p53 immunoprecipitation and immunofluorescence. Our results showed that high glucose reduced Mdm2 mRNA expression and protein concentration and increased Mdm2 and Akt phosphorylation, albeit with slower kinetics for Akt. It also promoted p53-Mdm2 complex formation, whereas p53 ubiquitination was suppressed. Furthermore, phosphorylation of both p53 Ser15 and ATM was increased in the presence of 30 mM glucose. These data indicate that high concentration glucose decrease the mRNA expression and cytosolic concentration of Mdm2. However, although the increase in glucose promoted the phosphorylation of Mdm2, it also decreased p53 ubiquitination, thus avoiding p53 degradation. In hyperglycemic conditions, such as diabetes mellitus, the reduction of pancreatic ß cells mass is favored by stabilization of p53 in association with low p53 ubiquitination and reduced expression of Mdm2.

Effect of an aqueous extract of Cucurbita ficifolia Bouché on the glutathione redox cycle in mice with STZ-induced diabetes.

ETHNOPHARMACOLOGICAL IMPORTANCE: Cucurbita ficifolia is used in Mexican traditional medicine as an anti-diabetic and anti-inflammatory agent and its actions can be mediated by antioxidant mechanisms. Disturbance in the homeostasis of glutathione has been implicated in the etiology and progression of diabetes mellitus and its complications. MATERIAL AND METHODS: It was evaluated, the effect of an aqueous extract of Cucurbita ficifolia on glycemia, plasma lipid peroxidation; as well as levels of reduced (GSH) and oxidized (GSSG) glutathione and activities of enzymes involved in glutathione redox cycle: glutathione peroxidase (GPx) and glutathione reductase (GR) in liver, pancreas, kidney and heart homogenates of streptozotocin-induced diabetic mice. RESULTS: Increased blood glucose and lipid peroxidation, together with decreased of GSH concentration, GSH/GSSG ratio and its redox potential (E(h)), and enhanced activity of GPx and GR in liver, pancreas and kidney were the salient features observed in diabetic mice. Administration of the aqueous extract of Cucurbita ficifolia to diabetic mice for 30 days, used at a dose of 200 mg/kg, resulted in a significant reduction in glycemia, polydipsia, hyperphagia and plasma lipid peroxidation. Moreover, GSH was increased in liver, pancreas and kidney, and GSSG was reduced in liver, pancreas and heart, therefore GSH/GSSG ratio and its E(h) were restored. Also, the activities involved in the glutathione cycle were decreased, reaching similar values to controls. CONCLUSIONS: An aqueous extract of Cucurbita ficifolia with hypoglycemic action, improve GSH redox state, increasing glutathione pool, GSH, GSH/GSSG ratio and its E(h), mechanism that can explain, at least in part, its antioxidant properties, supporting its use as an alternative treatment for the control of diabetes mellitus, and prevent the induction of complications by oxidative stress.

Regulation of proteinases during mouse peri-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9.

Trophoblast cells express urokinase-type plasminogen activator (PLAU) and may depend on its activity for endometrial invasion and tissue remodeling during peri-implantation development. However, the developmental regulation, tissue distribution, and function of PLAU are not completely understood. In this study, the expression of PLAU and its regulation by extracellular matrix proteins was examined by RT-PCR, immunocytochemistry, and plasminogen-casein zymography in cultured mouse embryos. There was a progressive increase in Plau mRNA expression in blastocysts cultured on gestation days 4-8. Tissue-type plasminogen activator (55 kDa) and PLAU (a triplet of 40, 37, and 31 kDa) were present in conditioned medium and embryo lysates, and were adsorbed to the culture plate surface. The temporal expression pattern of PLAU, according to semi-quantitative gel zymography, was similar in non-adhering embryos and embryos cultured on fibronectin, laminin, or type IV collagen, although type IV collagen and laminin upregulated Plau mRNA expression. Immunofluorescence revealed PLAU on the surface of the mural trophectoderm and in non-spreading giant trophoblast cells. Exogenous human plasminogen was transformed to plasmin by cultured embryos and activated endogenous matrix metalloproteinase 9 (MMP9). Indeed, the developmental expression profile of MMP9 was similar to that of PLAU. Our data suggest that the intrinsic developmental program predominantly regulates PLAU expression during implantation, and that PLAU could be responsible for activation of MMP9, leading to localized matrix proteolysis as trophoblast invasion commences.

Matrix metalloproteinases 2 and 9 in central nervous system and their modification after vanadium inhalation.

Vanadium (V) derivatives are well-known environmental pollutants and its toxicity has been related with oxidative stress. Toxicity after vanadium inhalation on the substantia nigra, corpus striatum, hippocampus and ependymal epithelium was reported previously. The purpose of this study was to analyse the role of matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) in the changes observed in brain tissue after chronic V inhalation. Mice were exposed to vaporized, vanadium pentoxide 0.02 m in deionized water for 1 h twice a week, and killed at 1 h, 1, 2 and 4 weeks after exposure. The brain was removed and the olfactory bulb, prefrontal cortex, striatum and hippocampus were dissected and the MMP content was obtained by zymography. The results showed that MMP-9 increased in all the structures at the end of the exposure, although in the hippocampus this increment was evident after 1 week of exposure. When MMP-2 was analysed in the olfactory bulb and prefrontal cortex it remained unchanged throughout the whole exposure, while in the hippocampus it increased at week 4, while in the striatum MMP-2 increased from the second week only, through the whole experiment. These results demonstrate that V increased MMPs in different structures of the CNS and this change might be associated with the previously reported modifications, such as dendritic spine loss and neuronal cell death. The modifications in MMPs could be related with blood-brain barrier (BBB) disruption which was reported previously. Oxidative stress might also be involved in the activation of these gelatinases as part of the different mechanisms which take place in V toxicity in the CNS.

[Nitric oxide as main effector in the interleukin-1 system in ovulation]. / El óxido nítrico como principal efector del sistema de la interleucina-1 en la ovulación.

Ovulation is a complex process involving not only gonadotropins and steroid hormones, but also many local mediators common to inflammatory reactions, such as cytokines. Of particular interest is the ovarian interleukin-1 (IL-1) system, which may be an intermediary of gonadotropins in the ovulatory process. The preovulatory follicles have a complete and highly compartmentalized intraovarian IL-1 system including ligands, receptor, and receptor antagonist. IL-1 has been considered as the inductor of several ovulation-associated events such as prostaglandin and progesterone biosynthesis, plasminogen activator production, glycosaminoglycan generation, and enhancement of vascular permeability. The principal effector of the IL-1 system is nitric oxide. This paper analyzes the sites of synthesis and action of the IL-1 system in preovulatory follicle and its vascular dynamics as well as IL-1's mechanism of action in triggering follicular rupture.

Presence of uterine peroxidase activity in the rat early pregnancy.

Peroxidase has been associated with estrogen action in the uterus. This enzyme plays an important role in the control of hydrogen peroxide levels and in catechol estrogen production. Since the uterus, during early pregnancy, is subjected to estrogen and progesterone regulation, we analyzed the changes of peroxidase activity in relation to receptivity and uterine early response to the embryo. Soluble and microsomal peroxidase activity were determined in the rat uterus during the estrus phase and early pregnancy (days 3 through 6). Soluble peroxidase activity increased significantly (p < 0.01) from day 3 (1.50 +/- 0.24) to day 4 (3.5 +/- 0.3) and 5 (5 +/- 0.5 U/mg protein, mean +/- S.D., n = 6) of pregnancy. During day 6, a significant decrease was noted in both the implantation site and the nonimplantation uterine tissue. Microsomal calcium-extractable peroxidase showed a similar pattern, with lower specific activity than, the soluble peroxidase. During estrus, the uterine tissue showed the highest activity of calcium-extracted peroxidase (8.7 +/- 1.35 U/mg protein), statistically greater when compared with days 3, 4, 5 and 6 of pregnancy. In conclusion, high peroxidase activity was associated with uterine receptivity. The decrease of activity on day 6 might be due to a progesterone-estrogen interaction, and consequently, hydrogen peroxide can be utilized for hydroxile production by means of the Fenton reaction. Lipoperoxidation may be necessary for changes in membrane fluidity for embryo attachment to endometrial epithelium.

[Cytokines and growth factors as autocrine and paracrine modulators in the peri-implantation period]. / Las citocinas y los factores de crecimiento como reguladores autocrinos y paracrinos durante el periodo peri-implantacional.

The embryo implantation is a complex event that involve a interactions sequence among conceptus and uterine endometrium. Several cytokines and growth factors participate as autocrine/paracrine modulators in such interrelations. In this paper the role and expression and functions of cytokines and growth factors in early pregnancy are analyzed. Post-coito expression of cytokines in uterine cells and leukocytes promoting a uterine inflammatory response to semen. The growth factors are expressed in early pregnancy for several uterine tissues. The ovarian steroid hormones modulate the synthesis and secretion of this molecules in uterus. Autocrine and paracrine regulation are require for embryo implantation. At least interleukin-1, leukemia inhibitor factor and epidermal growth factor receptor are indispensable to mice embryo implantation.

Uterine glutathione reductase activity: modulation by estrogens and progesterone.

The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.

[New endometrial modulators in early pregnancy]. / Los nuevos moduladores endometriales en el embarazo temprano.

Cytokines synthesized by the uterus or placenta include those thought to be produced exclusively by, or though to act on, cells of the lymphohematopoietic system. Although many of these cytokines are protein mediators of the immune system effector phase, in the female reproductive tract their principal target cells and sites of synthesis are non-lymphohematopoietic cells. During pregnancy, uterine epithelial cells, decidual cells and trophoblast appear to be major sources of the classic lymphohematopoietic cytokines. This suggests two not necessarily exclusive alternatives: that these cells are extensions of, or are involved in, regulating the immune system, or that these factors regulate growth and differentiation of uterine and embryonic tissues. This paper analyzes the sites of synthesis, targets and possible functions of the cytokines during early pregnancy.

[Molecular systems in cellular interactions. II: Embryonal implantation in mammals]. / Sistemas moleculares en las interacciones celulares. II: Implantación embrionaria en mamíferos.

In the present paper we consider the molecular mechanisms for cell recognition and cell adhesion on embryo implantation in mammals. In mammalian embryo implantation, the cellular interactions are complex, because several kinds of cells are involved: embryo trophoblast cells interact with several uterine cells and their respective extracellular matrices, participating lactosaminoglycans, integrins, cadherins and galactosyl transferases.