Correction: Crespo et al. Protective Effect of Protocatechuic Acid on TNBS-Induced Colitis in Mice Is Associated with Modulation of the SphK/S1P Signaling Pathway. Nutrients 2017, 9, 288.

In the original publication [...].

Effects of a resistance-training programme on endoplasmic reticulum unfolded protein response and mitochondrial functions in PBMCs from elderly subjects.

Aging has been related with a decline in the ability to handle protein folding, which leads to endoplasmic reticulum stress and alterations in unfolded protein response (UPR). Importantly, physical activity could activate the UPR and attenuate or prevent age-induced endoplasmic reticulum (ER) dysfunction. The current study evaluated the effects of a resistance exercise on UPR and mitochondrial functions in peripheral blood mononuclear cells (PBMCs) from elderly subjects. Thirty healthy women and men (age, 72.8, sx- = 2.2 years) were randomized to a training group, which performed an 8-week resistance training programme, or a control group, which followed their daily routines. The phosphorylation of PERK and IRE1, as well as ATF4, and XBP1 protein expression, significantly increased following the training, while expression of BiP, AFT6 and CHOP remain without changes. Additionally, the intervention also induced an increase in PGC-1α and Mfn1 protein levels, while no changes were found in Drp1 expression. Finally, the resistance protocol was not able to activate PINK1/Parkin and Bnip3/Nix pathways. The results obtained seem to indicate that 8-week resistance exercise activates the UPR, stimulates mitochondrial biogenesis, maintains mitochondrial dynamics and prevents mitophagy activation by unfolded proteins in PBMCs from elderly subjects.

Protective Effect of Protocatechuic Acid on TNBS-Induced Colitis in Mice Is Associated with Modulation of the SphK/S1P Signaling Pathway.

(1) Background: The present study aimed to investigate whether beneficial effects of protocatechuic acid (PCA) are associated with inhibition of the SphK/S1P axis and related signaling pathways in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) model of inflammatory bowel disease; (2) Methods: Colitis was induced in male Balb/c mice by intracolonic administration of 2 mg of TNBS. PCA (30 or 60 mg/kg body wt) was given intraperitoneally daily for five days; (3) Results: Administration of PCA prevented the macroscopic and microscopic damage to the colonic mucosa, the decrease in body weight gain and the increase in myeloperoxidase activity induced by TNBS. PCA-treated mice exhibited a lower oxidized/reduced glutathione ratio, increased expression of antioxidant enzymes and Nrf2 and reduced expression of proinflammatory cytokines. Following TNBS treatment mRNA levels, protein concentration and immunohistochemical labelling for SphK1 increased significantly. S1P production and expression of S1P receptor 1 and S1P phosphatase 2 were significantly elevated. However, there was a decreased expression of S1P lyase. Furthermore, TNBS-treated mice exhibited increased phosphorylation of AKT and ERK, and a higher expression of pSTAT3 and the NF-κB p65 subunit. PCA administration significantly prevented those changes; (4) Conclusions: Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the anti-inflammatory effect of PCA.

Impact of resistance training on the autophagy-inflammation-apoptosis crosstalk in elderly subjects.

Aging is associated with a decline in autophagy and a state of low-grade inflammation which further affects apoptosis and autophagy. Importantly, these alterations could reverse with regular physical activity. This study assessed the effects of a resistance exercise training program on autophagy, NLRP3 inflammasome, and apoptosis in peripheral blood mononuclear cells (PBMCs) from old subjects. Twenty-six healthy women and men (age, 69.6±1.5 yr) were randomized to a training (TG) or a control (CG) group. TG performed an 8-week resistance training program, while CG followed their daily routines. Protein expression of beclin-1, Atg12, Atg16 and LAMP-2 increased following the training program, while expression of p62/SQSTM1 and phosphorylation of ULK-1 at Ser757 were significantly lower. Resistance exercise also induced a decrease in NLRP3 expression and in the caspase-1/procaspase-1 ratio. Expression of Bcl-2 and Bcl-xL, as well as the Bad/BcL-2 ratio were reduced, and there was a significant decrease in the protein content of caspase-3. The results obtained seem to indicate that 8-week resistance training stimulates autophagy, prevents NLRP3 inflammasome activation, and reduces apoptosis in PBMCs from elderly subjects. These data could have a significant impact in prevention and rehabilitation programs currently employed in elderly population.

Effects of aerobic training on markers of autophagy in the elderly.

Autophagy is a molecular process essential for the maintenance of cellular homeostasis, which appears to (i) decline with age and (ii) respond to physical exercise. In addition, recent evidence suggests a crosstalk between autophagy and toll-like receptor (TLR)-associated inflammatory responses. This study assessed the effects of aerobic exercise training on autophagy and TLR signaling in older subjects. Twenty-nine healthy women and men (age, 69.7 ± 1.0 year) were randomized to a training (TG) or a control (CG) group. TG performed an 8-week aerobic training program, while CG followed their daily routines. Peripheral blood mononuclear cells were isolated from blood samples obtained before and after the intervention, and protein levels of protein 1 light chain 3 (LC3), sequestosome 1 (p62/SQSTM1), beclin-1, phosphorylated unc-51-like kinase (ULK-1), ubiquitin-like autophagy-related (Atg)12, Atg16, and lysosome-associated membrane protein (LAMP)-2 were measured. TLR2 and TLR4 signaling pathways were also analyzed. Peak oxygen uptake increased in TG after the intervention. Protein expression of beclin-1, Atg12, Atg16, and the LC3II/I ratio increased following the training program (p < 0.05), while expression of p62/SQSTM1 and phosphorylation of ULK-1 at Ser(757) were lower (p < 0.05). Protein content of TLR2, TLR4, myeloid differentiation primary response gen 88 (MyD88), and TIR domain-containing adaptor-inducing interferon (TRIF) were not significantly modified by exercise. The current data indicate that aerobic exercise training induces alterations in multiple markers of autophagy, which seem to be unrelated to changes in TLR2 and TLR4 signaling pathways. These results expand knowledge on exercise-induced autophagy adaptations in humans and suggest that the exercise type employed may be a key factor explaining the potential relationship between autophagy and TLR pathways.

Whole-body vibration improves the anti-inflammatory status in elderly subjects through toll-like receptor 2 and 4 signaling pathways.

Regular physical exercise has anti-inflammatory effects in elderly subjects. Yet, the inflammatory responses after whole body vibration (WBV) training, a popular exercise paradigm for the elderly, remain to be elucidated. This study assessed the effects of WBV training on the inflammatory response associated with toll-like receptors (TLRs) signaling pathways. Twenty-eight subjects were randomized to a training group (TG) or a control group (CG). TG followed an 8-week WBV training program. Blood samples were obtained before and after the training period in both groups. Peripheral blood mononuclear cells were isolated, and mRNA and protein levels of makers involved in the TLR2/TLR4 myeloid differentiation primary response gen 88 (MyD88) and TIR domain-containing adaptor inducing interferon (TRIF)-dependent pathways were analyzed. Plasma TNFα and C-reactive protein levels were also assessed. The WBV program reduced protein expression of TLR2, TLR4, MyD88, p65, TRIF and heat shock protein (HSP) 60, while HSP70 content increased. IL-10 mRNA level and protein concentration were upregulated, and TNFα protein content decreased, after WBV training. Plasma concentration of C-reactive protein and TNFα decreased in the TG. The current data suggest WBV may improve the anti-inflammatory status of elderly subjects through an attenuation of MyD88- and TRIF-dependent TLRs signaling pathways.

Hypoxia-inducible factor-1 modulates the expression of vascular endothelial growth factor and endothelial nitric oxide synthase induced by eccentric exercise.

The present study investigated the effects of acute and chronic eccentric exercise on the hypoxia-inducible factor (HIF)-1α activation response and the concomitant modulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression in rat skeletal muscle. Twenty-four male Wistar rats were randomly assigned to three experimental groups: rested control group, acutely exercised group after an intermittent downhill protocol for 90 min, and acutely exercise group with a previous eccentric training of 8 wk. HIF-1α activation, VEGF and eNOS gene expression, protein content, and promoter activation were assessed in vastus lateralis muscle biopsies. Acute eccentric exercise induced a marked activation of HIF-1α and resulted in increased VEGF and eNOS mRNA level and protein concentration. The binding of HIF-1α to the VEGF and eNOS promoters, measured by a chromatin immunoprecipitation assay, was undetectable in rested rats, whereas it was evident in acutely exercised animals. Acute exercise also increased myeloperoxidase, toll-like receptor-4, tumor necrosis factor-α, and interleukin-1ß protein content, suggesting a contribution of proinflammatory stimuli to HIF-1α activation and VEGF overexpression. All of these effects were partially abolished by training. Moreover, training resulted in an increased capillary density. In summary, our findings indicate that eccentric exercise prompts an HIF-1α response in untrained skeletal muscle that contributes to the upregulation of VEGF and eNOS gene expression and is attenuated after an eccentric training program.

Role of Toll-like receptor 2 and 4 signaling pathways on the inflammatory response to resistance training in elderly subjects.

This study assessed the effects of a resistance exercise training program on the inflammatory response associated with Toll-like receptor (TLR) 2 and TLR4 signaling pathways in senior participants. Twenty-six healthy subjects (age, 69.5 ± 1.3) were randomized to a training (TG; n = 16) or a control (CG; n = 10) group. TG performed an 8-week resistance training program, while CG followed their daily routines. Peripheral blood mononuclear cells were isolated from blood samples obtained before and after the intervention, and levels of proteins involved in the TLR2, TLR4, and myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways were analyzed. The inflammatory status was evaluated through messenger RNA (mRNA) and protein content of interleukin (IL)-10 and tumor necrosis factor alpha (TNF-α) and plasma levels of C-reactive protein (CRP). After the 8-week resistance training, TLR2 and TLR4 protein expression was reduced in TG. MyD88, p65, phospho-p38, TIR domain-containing adaptor inducing interferon (TRIF), IKKi/IKKε, phospho-interferon regulatory factor (IRF) 3, and phosho-IRF7 were also downregulated in TG after the intervention. The training program induced an increase of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Hsp70 and a reduction of Hsp60. While TNF-α mRNA and protein values remained unchanged in both TG and CG, IL-10 mRNA and protein content were upregulated in TG after the intervention. CRP values decreased in TG only. The increase in Hsp70 negatively correlated with TLR2 and TLR4 downregulation. These data suggest that resistance exercise may represent an effective tool to ameliorate the pro-inflammatory status of old participants through an attenuation of MyD88-dependent and MyD88-independent TLR2 and TLR4 signaling pathways.

Eccentric exercise induces nitric oxide synthase expression through nuclear factor-kappaB modulation in rat skeletal muscle.

This study aimed to investigate the effect of eccentric exercise on the expression of the different nitric oxide synthase (NOS) isoforms in rat deep vastus lateralis muscle. Twenty-four rats were allocated to four experimental groups: rested control group, acutely exercised group after an intermittent downhill protocol for 90 min, acutely exercised group treated with pyrrolidine dithiocarbamate (100 mg/kg ip) for 24 and 1 h before the acute exercise bout, and acutely exercised group with a previous submaximal eccentric training of 8 wk. Acutely exercised rats showed increased levels of protein tyrosine nitration, NF-kappaB binding, and phospho-I kappaB alpha content. A significant increase was observed in mRNA level and protein content of neuronal NOS, inducible NOS, and endothelial NOS. The binding of NF-kappaB to the NOS isoform promoters, measured by a chromatin immunoprecipitation assay, was undetectable in rested rats, whereas it was evident in acutely exercised animals. All of these effects were partially abolished by pyrrolidine dithiocarbamate treatment and by training. In summary, our findings provide a direct link between the NF-kappaB signaling cascade and NOS expression in skeletal muscle following eccentric exercise and suggest a modulation of the expression of the three NOS isoforms by this transcription factor.

Cytokine and hormone responses to resistance training.

This study examined the effects of heavy resistance training on physiological acute exercise-induced fatigue (5 x 10 RM leg press) changes after two loading protocols with the same relative intensity (%) (5 x 10 RM(Rel)) and the same absolute load (kg) (5 x 10 RM(Abs)) as in pretraining in men (n = 12). Exercise-induced neuromuscular (maximal strength and muscle power output), acute cytokine and hormonal adaptations (i.e., total and free testosterone, cortisol, growth hormone (GH), insulin-like growth factor-1 (IGF-1), IGF binding protein-3 (IGFBP-3), interleukin-1 receptor antagonist (IL-1ra), IL-1beta, IL-6, and IL-10 and metabolic responses (i.e., blood lactate) were measured before and after exercise. The resistance training induced similar acute responses in serum cortisol concentration but increased responses in anabolic hormones of FT and GH, as well as inflammation-responsive cytokine IL-6 and the anti-inflammatory cytokine IL-10, when the same relative load was used. This response was balanced by a higher release of pro-inflammatory cytokines IL-1beta and cytokine inhibitors (IL-1ra) when both the same relative and absolute load was used after training. This enhanced hormonal and cytokine response to strength exercise at a given relative exercise intensity after strength training occurred with greater accumulated fatigue and metabolic demand (i.e., blood lactate accumulation). The magnitude of metabolic demand or the fatigue experienced during the resistance exercise session influences the hormonal and cytokine response patterns. Similar relative intensities may elicit not only higher exercise-induced fatigue but also an increased acute hormonal and cytokine response during the initial phase of a resistance training period.

Eccentric training impairs NF-kappaB activation and over-expression of inflammation-related genes induced by acute eccentric exercise in the elderly.

The present study was aimed to investigate in elderly humans changes in NF-kappaB activation and in the expression of the inflammation-related genes inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) induced in peripheral blood mononuclear cells (PBMC) by acute eccentric exercise and by submaximal eccentric training. Eleven subjects, aged 66-75 years, carried out 2 bouts of eccentric exercise separated by 8 weeks of training. Following the first bout, NF-kappaB activation, and protein level of p50/p65 subunits, phospho-IkappaBalpha and phospho-IKKalpha increased, while IkappaBalpha protein level was significantly reduced. This was accompanied by a significant increase in iNOS, COX-2 and IL-6 mRNA protein level and protein content. Changes were significantly attenuated following the second exercise bout. In conclusion, acute eccentric exercise increases NF-kappaB activation and the expression of several inflammation-related genes in PBMC from elderly individuals. Regular eccentric training might be an effective method of preventing undesirable inflammatory responses induced by eccentric exercise.

Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells.

This study was aimed to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by proinflammatory stimuli in parenchymal liver cells. Chang Liver cells were incubated with a cytokine mixture (CM) supplemented with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin (5-50 microM). Concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were measured. Oxidised glutathione concentration and the oxidised/reduced glutathione ratio were increased by the CM. These effects were significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. Effects of apigenin reached a lesser extent and were not significant at 25 microM. Treatment with quercetin and kaempferol prevented the production of peroxides, superoxide anion and nitric oxide induced by CM. Taxifolin 50 microM and apigenin 25-50 microM caused a significant increase in peroxides and nitric oxide generation. Protein concentration of the different antioxidant enzymes was generally reduced by kaempferol and quercetin in comparison to CM, although quercetin 25 and 50 microM increased Mn SOD protein concentration. GPx protein level was significantly increased by apigenin 25 and 50 microM. Changes in mRNA tended to be parallel to those in protein concentration. Our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells.

Effects of eccentric exercise on NF-kappaB activation in blood mononuclear cells.

PURPOSE: Changes in nuclear factor kappaB (NF-kappaB) activation induced in peripheral blood mononuclear cells (PBMC) by acute eccentric exercise and by submaximal eccentric training were investigated. METHODS: Eleven subjects carried out two bouts of eccentric exercise separated by 6 wk of training. RESULTS: Soreness, vertical jump height, and plasma creatine kinase were significantly modified after the first bout. NF-kappaB activation, p50 and p65, phospho-IkappaBalpha and phospho-IKK protein level, and Mn-SOD expression increased in PBMC, whereas IkappaBalpha protein level was significantly reduced. Changes were significantly attenuated after the second exercise bout. An additional group of nine subjects carried out the two bouts of exercise without training. Effects on NF-kappaB activation were similar after the second bout compared with the first, despite a reduction in markers of muscle injury (repeated bout effect). CONCLUSION: Training significantly attenuates the NF-kappaB-dependent pathway changes induced in PBMC by eccentric exercise, with no contribution from the repeated bout effect.

Exercise, oxidative stress and risk of cardiovascular disease in the elderly. Protective role of antioxidant functional foods.

Free radicals and oxidative stress are involved in the pathogenic mechanisms of cardiovascular disease (CVD), diabetes and cancer. Exercise is a useful strategy for preventing CVD but in elderly persons it can enhance oxidative stress, which is why some studies recommend antioxidant supplementation for exercising elderly subjects. This intervention study was performed on 320 elderly subjects following a Geriatric Revitalization Program (GEREPRO) to maintain physical health and reduce CVD risk. GEREPRO was based on regular exercise concurrent with a nutritional antioxidant treatment based on daily intake of a functional antioxidant food, Biofrutas. Sustained exercise (10 months, 3 sessions/week) significantly increased cardiorespiratory fitness and plasma HDL-cholesterol; it reduced some predictors of cardiovascular risk (arterial pressure, LDL-cholesterol, total cholesterol/LDL-C, LDL-C/HDL-C), but significantly enhanced some biomarkers of oxidative stress. Concurrent antioxidant supplementation did not produce any ergogenic effects but, meaningfully, enhanced some positive effects of exercise on physical health and the CDV risk index, and it totally prevented the exercise-induced oxidative stress. Our results show that regular and moderate exercise improves cardiorespiratory function and reduces CVD risk in elderly people, while concurrent antioxidant supplementation modulates oxidative insult during exercise in the elderly and enhances the beneficial effects of exercise.

Changes in oxidative stress markers and NF-kappaB activation induced by sprint exercise.

This study was aimed to investigate changes in blood markers of oxidative damage induced by short-term supramaximal anaerobic exercise and to determine whether oxidative stress was associated to activation of the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB). Both a single Wingate test (WAnT) test and series of four WAnTs separated by 60 min rest intervals were carried out by eight professional cyclists. Leukocyte 8-OH-2-deoxyguanosine levels were significantly elevated 24 h after both exercise protocols. A significant decrease in blood reduced glutathione (GSH) concentration was observed immediately after and at 15, 60 and 120 min of the single WAnT, followed by a return to basal value after 24 h. This decrease was parallel to a significant increase of the oxidised/reduced glutathione (GSSG/GSH) ratio, to an activation of NF-kappaB and to a significant decrease in the protein level of its inhibitor IkappaB. GSH concentration and the GSSG/GSH ratio changed significantly for the first three of the WAnTs series and normalised thereafter. A significant activation of NF-kappaB and a decrease in the IkappaB protein level were also detected. We conclude that short-term supramaximal anaerobic exercise induces oxidative stress, as evidenced by non cumulative damage to macromolecules and changes in the glutathione status. Our data also indicate that high intensity anaerobic work gives rise to an activation of the transcription factor NF-kappaB accompanied by a degradation of IkappaB.

Effects of epomediol on ethinyloestradiol-induced changes in glutathione homeostasis in the rat.

Epomediol is a synthetic terpenoid compound that has been reported to reduce ethinyloestradiol-induced cholestasis. The choleretic action of epomediol is related to an increase in both the bile acid-dependent and independent fractions of bile flow, but the role of glutathione metabolism and transport is still unknown. This study was aimed to evaluate if changes in glutathione homeostasis could contribute to the beneficial effects of epomediol in rats with ethinyloestradiol-induced cholestasis. When compared to control animals, ethinyloestradiol treatment resulted in a significant decrease in the liver concentration of reduced (GSH) and oxidized glutathione. Both GSH and oxidized glutathione concentrations returned to normal in animals receiving ethinyloestradiol plus epomediol. Ethinyloestradiol administration induced a significant decrease in plasma and renal GSH and the tripeptide was almost absent from bile. Combined treatment with epomediol plus ethinyloestradiol normalised renal GSH and both biliary and liver cysteine were significantly increased. Liver and kidney gamma-glutamyltranspeptidase activities were higher in rats receiving ethinyloestradiol and still remained elevated in animals with the combined treatment. Liver gamma-glutamylcysteine synthetase activity rose significantly by administration of ethinyloestradiol plus epomediol but the corresponding mRNA levels were not modified. Changes in glutathione homeostasis and higher biliary levels of GSH amino acid constituents could contribute to the beneficial effects of epomediol in rats with ethinyloestradiol-induced cholestasis.

Urinary levels of 8-hydroxydeoxyguanosine as a marker of oxidative damage in road cycling.

We have determined the urinary 8-hydroxydeoxyguanosine (8-OHdG) levels of eight professional cyclists during a 4-day and a 3-week stage races. Monitoring of heart rates was used to establish zones corresponding to different intensities of exercise. The urinary 8-OHdG excretion, expressed by body weight, increased significantly in the first day or the first week of each race, respectively, and did not show further increases thereafter. Maximum 8-OHdG levels were reached in parallel to longer times spent at high intensities of exercise. Urinary excretion of creatinine increased with exercise, and changes in 8-OHdG levels were not detected when corrected by creatinine excretion. Serum glutathione concentrations did not change significantly at any point during exercise. We conclude that road cycling courses with an oxidative damage to DNA, which is sustained as long as the exercise is repeated. Both adaptation of antioxidant defenses and a decreased capacity to maintain a high intensity of effort may contribute to explain the absence of progressive increases in 8-OHdG excretion. The results of this study also confirm that the correction procedure using the amount of creatinine excreted should not be used when studying effects of exercise on urinary 8-OHdG.