Tricarboxylic acid cycle dehydrogenases inhibition by naringenin: experimental and molecular modelling evidence.

The study of polyphenols' effects on health has been gaining attention lately. In addition to reacting with important enzymes, altering the cell metabolism, these substances can present either positive or negative metabolic alterations depending on their consumption levels. Naringenin, a citrus flavonoid, already presents diverse metabolic effects. The objective of this work was to evaluate the effect of maternal naringenin supplementation during pregnancy on the tricarboxylic acid cycle activity in offspring's cerebellum. Adult female Wistar rats were divided into two groups: (1) vehicle (1 ml/kg by oral administration (p.o.)) or (2) naringenin (50 mg/kg p.o.). The offspring were euthanised at 7th day of life, and the cerebellum was dissected to analyse citrate synthase, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (α-KGDH) and malate dehydrogenase (MDH) activities. Molecular docking used SwissDock web server and FORECASTER Suite, and the proposed binding pose image was created on UCSF Chimera. Data were analysed by Student's t test. Naringenin supplementation during pregnancy significantly inhibited IDH, α-KGDH and MDH activities in offspring's cerebellum. A similar reduction was observed in vitro, using purified α-KGDH and MDH, subjected to pre-incubation with naringenin. Docking simulations demonstrated that naringenin possibly interacts with dehydrogenases in the substrate and cofactor binding sites, inhibiting their function. Naringenin administration during pregnancy may affect cerebellar development and must be evaluated with caution by pregnant women and their physicians.

Guarana supplementation attenuated obesity, insulin resistance, and adipokines dysregulation induced by a standardized human Western diet via brown adipose tissue activation.

Obesity is a metabolic disorder associated with adverse health consequences that has increased worldwide at an epidemic rate. This has encouraged many people to utilize nonprescription herbal supplements for weight loss without knowledge of their safety or efficacy. However, mounting evidence has shown that some herbal supplements used for weight loss are associated with adverse effects. Guarana seed powder is a popular nonprescription dietary herb supplement marketed for weight loss, but no study has demonstrated its efficacy or safety when administered alone. Wistar rats were fed four different diets (low-fat diet and Western diet with or without guarana supplementation) for 18 weeks. Metabolic parameters, gut microbiota changes, and toxicity were then characterized. Guarana seed powder supplementation prevented weight gain, insulin resistance, and adipokine dysregulation induced by Western diet compared with the control diet. Guarana induced brown adipose tissue expansion, mitochondrial biogenesis, uncoupling protein-1 overexpression, AMPK activation, and minor changes in gut microbiota. Molecular docking suggested a direct activation of AMPK by four guarana compounds tested here. We propose that brown adipose tissue activation is one of the action mechanisms involved in guarana supplementation-induced weight loss and that direct AMPK activation may underlie this mechanism. In summary, guarana is an attractive potential therapeutic agent to treat obesity.

Putative model for heat shock protein 70 complexation with receptor of advanced glycation end products through fluorescence proximity assays and normal mode analyses.

Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.

The oxidation of HSP70 is associated with functional impairment and lack of stimulatory capacity.

Expression of intracellular HSP70 is associated with cytoprotective effects against a wide range of stressful stimuli, such as inflammation, oxidative stress, hypoxia, endotoxins, infections, and fever. This cytoprotective effect is mainly attributed to their ability to stabilize protein structures through chaperone-like reversible interactions. HSP70 was recently detected in the extracellular medium, and its presence in serum is commonly associated with pathological situations, where it exerts modulatory effects on cells of the immune system. Previously, we have described the relationship between serum HSP70 levels, oxidant status, and clinical outcome of septic patients; the group of patients with higher prooxidant status and higher serum HSP70 had also higher mortality. To investigate the possible association between oxidized HSP70 and cytoprotection or cell death, we incubated RAW 264.7 macrophages with oxidized HSP70 and evaluated nitrite production, cell proliferation, cell viability, TNF-α release, and phagocytic activity. We also evaluated structural modifications caused by oxidation in purified HSP70. Oxidation of HSP70 altered its protein structure; besides, the modulatory effect of oxidized HSP70 on RAW264.7 cells was different from that of native HSP70. Macrophages treated with oxidized HSP70 presented lower proliferation and viability, lower phagocytic activity, and lower TNF-α release. These results indicate that oxidation of extracellular HSP70 modified its signaling properties, causing alterations on its modulatory effects on macrophage function and viability.

Serum heat shock protein 70 levels, oxidant status, and mortality in sepsis.

Animal studies as well as prospective randomized clinical trials associated sepsis with redox imbalance and oxidative stress, but other studies failed to establish a correlation between antioxidant-based therapies and improvement of sepsis condition. This is also true for studies on the role of the chaperone heat shock protein 70 (HSP70), which is increased in serum during sepsis. Heat shock protein 70 is affected at several levels by oxidative stress, but this relationship has never been studied in sepsis. Here, we evaluated the relationship between serum HSP70 immunocontent and oxidant status in sepsis. Patients with severe sepsis were followed up for 28 days after diagnosis, or until death. Up to a maximum of 12 h after sepsis diagnosis, serum was collected for determination of HSP70 immunocontent by Western blot and evaluation of oxidative parameters (TRAP [total radical-trapping antioxidant parameter], TBARSs [thiobarbituric acid-reactive substances], and carbonyl levels). Serum of sepsis patients presented enhanced HSP70 levels. Analysis of oxidative parameters revealed that septic patients with pronounced oxidative damage in serum had also increased HSP70 serum levels. Sepsis patients in whom serum oxidative stress markers were not different from control presented normal serum HSP70. Analysis of septic patients according to survival outcome also indicated that patients with increased HSP70 serum levels presented increased mortality. We concluded that serum HSP70 levels are modulated according to the patient oxidant status, and increased serum HSP70 is associated to mortality in sepsis.