Skeletal muscle angiogenic, regulatory, and heat shock protein responses to prolonged passive hyperthermia of the human lower limb.

Passive hyperthermia induces a range of physiological responses including augmenting skeletal muscle mRNA expression. This experiment aimed to examine gene and protein responses to prolonged passive leg hyperthermia. Seven young participants underwent 3 h of resting unilateral leg heating (HEAT) followed by a further 3 h of rest, with the contralateral leg serving as an unheated control (CONT). Muscle biopsies were taken at baseline (0 h), and at 1.5, 3, 4, and 6 h in HEAT and 0 and 6 h in CONT to assess changes in selected mRNA expression via qRT-PCR, and HSP72 and VEGFα concentration via ELISA. Muscle temperature (Tm) increased in HEAT plateauing from 1.5 to 3 h (+3.5 ± 1.5°C from 34.2 ± 1.2°C baseline value; P < 0.001), returning to baseline at 6 h. No change occurred in CONT. Endothelial nitric oxide synthase (eNOS), Forkhead box O1 (FOXO-1), Hsp72, and VEGFα mRNA increased in HEAT (P < 0.05); however, post hoc analysis identified that only Hsp72 mRNA statistically increased (at 4 h vs. baseline). When peak change during HEAT was calculated angiopoietin 2 (ANGPT-2) decreased (-0.4 ± 0.2-fold), and C-C motif chemokine ligand 2 (CCL2) (+2.9 ± 1.6-fold), FOXO-1 (+6.2 ± 4.4-fold), Hsp27 (+2.9 ± 1.7-fold), Hsp72 (+8.5 ± 3.5-fold), Hsp90α (+4.6 ± 3.7-fold), and VEGFα (+5.9 ± 3.1-fold) increased from baseline (all P < 0.05). At 6 h Tm were not different between limbs (P = 0.582; CONT = 32.5 ± 1.6°C, HEAT = 34.3 ± 1.2°C), and only ANGPT-2 (P = 0.031; -1.3 ± 1.4-fold) and VEGFα (P = 0.030; 1.1 ± 1.2-fold) differed between HEAT and CONT. No change in VEGFα or HSP72 protein concentration were observed over time; however, peak change in VEGFα did increase (P < 0.05) in HEAT (+140 ± 184 pg·mL-1) versus CONT (+7 ± 86 pg·mL-1). Passive hyperthermia transiently augmented ANGPT-2, CCL2, eNOS, FOXO-1, Hsp27, Hsp72, Hsp90α and VEGFα mRNA, and VEGFα protein.

Effect of Lonicera japonica extract on lactation performance, antioxidant status, and endocrine and immune function in heat-stressed mid-lactation dairy cows.

Here, we examined the effects of Lonicera japonica extract (LJE) on lactation performance, antioxidant status, and endocrine and immune function in heat-stressed mid-lactation dairy cows. Twenty-four healthy Chinese Holstein mid-lactation dairy cows, all with similar milk yield (30.0 ± 1.0 kg/d), parity (2.5 ± 0.3), and days in milk (105 ± 5 d) were allocated to 4 groups using a randomized complete block design: a negative control group (without LJE supplementation; CON) and groups that received LJE at 14, 28, and 56 g/d. The experiment lasted 10 wk over a hot summer, with a pre-feeding period of 2 wk. Cows were exposed to heat stress, as the average temperature-humidity index was greater than 72. The results showed that LJE had no effect on respiration rate; however, it reduced the rectal temperature of dairy cows experiencing heat stress in both a linear and quadratic manner; the lowest (39.03°C) was recorded for the LJE-28 group, lower than the CON group. Supplementation with LJE did not affect dry matter intake, milk yield, or milk composition. The majority of biochemical parameters in serum were unaffected by supplementation with different amounts of LJE; the exception was creatinine, which was reduced quadratically. Compared with the CON group, serum triiodothyronine concentrations increased significantly in the LJE-28 group. Addition of LJE to the diet increased thyroxine concentrations quadratically; values peaked at 18.62 ng/mL in the LJE-28 group. Furthermore, supplementation with increasing amounts of LJE quadratically increased the activity of glutathione peroxidase and total antioxidant capacity in serum but decreased concentration of malondialdehyde. Although we detected no differences in the concentrations of IgA, IgM, or cytokines, dairy cows in the LJE-28 group had higher IgG and IL-4 concentrations than did cows in the CON group. Supplementation with LJE increased concentrations of IgG and IL-4 in the serum quadratically but decreased that of IL-2. Finally, heat shock protein 72 concentrations in the serum tended to fall quadratically as the amount of LJE increased. In summary, LJE had no negative effects on lactation performance but helped to alleviate heat stress by improving antioxidant status and promoting endocrine and immune functions. Supplementation with LJE at 28 g/d is recommended for lactating dairy cows experiencing heat stress during hot summers.

Dietary supplementation of brown seaweed (Sargassum latifolium) alleviates the environmental heat stress-induced toxicity in male Barki sheep (Ovis aries).

Heat stress (HS) is the most potent environmental stressors for livestock in tropical and subtropical regions. HS induced splanchnic tissue hypoxia and intestinal oxidative damage, leading to endotoxemia and systemic inflammation. The present study evaluated and compared the modulatory effects of feeding Barki male sheep (Ovis aries) on a standard concentrated diet containing 2% or 4% of the brown seaweed (Sargassum latifolium) followed by roughage for 40 consecutive days on the toxicity-induced by exposure to severe environmental HS (temperature-humidity index = 28.55 ± 1.62). The present study showed that the diet containing Sargassum latifolium (especially 4%) modulated significantly (P < 0.05-0.001) almost all changes shown in the HS-exposed sheep including the increase in the thermo-respiratory responses (skin and rectal temperatures, and respiration rate) and the resulted dyslipidemia, anemia, and systemic inflammation (blood leukocytosis, the elevation in the erythrocyte sedimentation rate, and the increase in serum proinflammatory cytokines and heat shock protein-70 concentrations). In addition, Sargassum latifolium improved significantly (P < 0.05-0.001) the body-weight gain, kidney functions (especially at the high dose), and blood antioxidant defense system (total antioxidant capacity, and the activities of catalase and superoxide dismutase) in the HS-exposed sheep, as well as protected the animals from oxidative tissue damage and the risk of atherosclerosis. In conclusion, feeding sheep with the diet containing 4% of Sargassum latifolium was safe and suitable for animal nutrition, as well as efficiently alleviated the harmful effects of the environmental HS in Barki sheep through improving the animal antioxidant defense system, and regulating the thermo-respiratory and inflammatory responses.

Leucine-Enriched Essential Amino Acids Improve Recovery from Post-Exercise Muscle Damage Independent of Increases in Integrated Myofibrillar Protein Synthesis in Young Men.

BACKGROUND: Leucine-enriched essential amino acids (LEAAs) acutely enhance post-exercise myofibrillar protein synthesis (MyoPS), which has been suggested to be important for muscle repair and recovery. However, the ability of LEAAs to concurrently enhance MyoPS and muscle damage recovery in free-living humans has not been studied. METHODS: In a randomized, double-blind, placebo-controlled, parallel-group design, twenty recreationally active males consuming a controlled diet (1.2 g/kg/d of protein) were supplemented thrice daily with 4 g of LEAAs (containing 1.6 g leucine) or isocaloric placebo for four days following an acute bout of lower-body resistance exercise (RE). MyoPS at rest and integrated over 96 h of recovery was measured by D2O. Isometric and isokinetic torque, muscle soreness, Z-band streaming, muscle heat shock protein (HSP) 25 and 72, plasma creatine kinase (CK), and plasma interleukin-6 (IL-6) were measured over 96 h post-RE to assess various direct and indirect markers of muscle damage. RESULTS: Integrated MyoPS increased ~72% over 96 h after RE (p < 0.05), with no differences between groups (p = 0.98). Isometric, isokinetic, and total peak torque decreased ~21% by 48 h after RE (p < 0.05), whereas total peak torque was ~10% greater overall during recovery in LEAAs compared to placebo (p < 0.05). There were moderate to large effects for peak torque in favour of LEAAs. Muscle soreness increased during recovery with no statistical differences between groups but small to moderate effects in favour of LEAAs that correlated with changes in peak torque. Plasma CK, plasma IL-6, and muscle HSP25 increased after RE (p < 0.05) but were not significantly different between groups (p ≥ 0.13). Consistent with a trend toward attenuated Z-band streaming in LEAAs (p = 0.07), muscle HSP72 expression was lower (p < 0.05) during recovery in LEAAs compared with placebo. There were no correlations between MyoPS and any measures of muscle damage (p ≥ 0.37). CONCLUSION: Collectively, our data suggest that LEAAs moderately attenuated muscle damage without concomitant increases in integrated MyoPS in the days following an acute bout of resistance exercise in free-living recreationally active men.

Elevating body termperature to reduce low-grade inflammation: a welcome strategy for those unable to exercise?

Chronic low-grade inflammation is increasingly recognized in the aetiology of a range of chronic diseases, including type 2 diabetes mellitus and cardiovascular disease, and may therefore serve as a promising target in their prevention or treatment. An acute inflammatory response can be induced by exercise; this is characterised by the acute increase in proinflammatory markers that subsequently stimulate the production of anti-inflammatory proteins. This may help explain the reduction in basal concentrations of pro-inflammatory markers following chronic exercise training. For sedentary populations, such as people with a disability, wheelchair users, or the elderly, the prevalence of chronic low-grade inflammation- related disease is further increased above that of individuals with a greater capacity to be physically active. Performing regular exercise with its proposed anti-inflammatory potential may not be feasible for these individuals due to a low physical capacity or other barriers to exercise. Therefore, alternatives to exercise that induce a transient acute inflammatory response may benefit their health. Manipulating body temperature may be such an alternative. Indeed, exercising in the heat results in a larger acute increase in inflammatory markers such as interleukin-6 and heat shock protein 72 when compared with exercising in thermoneutral conditions. Moreover, similar to exercise, passive elevation of body temperature can induce acute increases and chronic reductions in inflammatory markers and positively affect markers of glycaemic control. Here we discuss the potential benefits and mechanisms of active (i.e., exercise) and passive heating methods (e.g., hot water immersion, sauna therapy) to reduce chronic low-grade inflammation and improve metabolic health, with a focus on people who are restricted from being physically active.

Effects of severe whole-body hyperthermia on ovarian hormone and extracellular Hsp72 responses in young adult women.

Background: Although acute thermal stress appears to be one of the most effective stressors that increase the intra- and extracellular concentrations of heat shock protein 72 (Hsp72), 17ß-estradiol has been shown to inhibit heat-induced Hsp72 expression. Materials and Methods: To determine whether severe whole-body hyperthermia (increase in rectal temperature up to 39.5 °C) induced by lower-body heating is a sufficient stimulus to modulate hormonal (17ß-estradiol, progesterone, prolactin, epinephrine, and norepinephrine) and extracellular Hsp72 responses, we investigated young adult women (21 ± 1 yr). Results and Conclusions: In the present study, we show that a severe whole-body hyperthermia (increase in rectal temperature of approximately 2.6 °C and heart rate of approximately 80 bpm from baseline) was sufficient to increase 17ß-estradiol, progesterone, and prolactin and catecholamine norepinephrine concentration. Moreover, we show that the concentration of extracellular Hsp72 and catecholamine epinephrine were not affected by severe whole-body hyperthermia in young adult women. From the functional point of view, expression of ovarian hormones induced by passive heat stress may have therapeutic potential for young adult women in, for example, estrogen treatment and overall women's health.

Betaine protects against heat exposure-induced oxidative stress and apoptosis in bovine mammary epithelial cells via regulation of ROS production.

Heat stress is one of the wide varieties of factors which cause oxidative stress in vivo; elevated temperature can lead to oxidative stress of dairy cows that affects milk production. The aim of this study was to determine the capacity of the betaine to act as an antioxidant against oxidative stress induced by heat exposure and apoptosis in mammary epithelial cells (mammary alveolar cells, MAC-T). The MAC-T were divided into four treatment groups: control (37 °C), heat stress (HS, 42 °C), betaine (37 °C), and HS + betaine. MAC-T under heat stress (HS) showed increased ROS accumulation, malondialdehyde (MDA) content, superoxide dismutase (SOD) concentration, and catalase (CAT) activity. During heat stress, betaine decreased the mRNA expression level of HSP70 and HSP27 in MAC-T. Bax/Bcl-2 ratio and caspase-3, the markers of apoptosis, were also elevated in MAC-T under heat stress. The markers of oxidative stress Nrf-2/HO-1 genes were also elevated in MAC-T under heat stress. Pretreatment of betaine reversed the heat-induced depletion in total antioxidant status, ROS accumulation, and SOD and CAT contents in MAC-T. Bax/Bcl-2 ratio and Nrf-2/HO-1 expression of heat-exposed MAC-T were also reduced with betaine supplementation. In conclusion, betaine alleviated oxidative stress and apoptosis of MAC-T by inhibiting ROS accumulation.

Auto-fluorescent polymer nanotheranostics for self-monitoring of cancer therapy via triple-collaborative strategy.

Aberrant regulation of angiogenesis supply sufficient oxygen and nutrients to exacerbate tumor progression and metastasis. Taking this hallmark of cancer into account, reported here is a self-monitoring and triple-collaborative therapy system by auto-fluorescent polymer nanotheranostics which could be concurrently against angiogenesis and tumor cell growth by combining the benefits of anti-angiogenesis, RNA interfere and photothermal therapy (PTT). Auto-fluorescent amphiphilic polymer polyethyleneimine-polylactide (PEI-PLA) with positive charge can simultaneously load hydrophobic antiangiogenesis agent combretastatin A4 (CA4), NIR dye IR825 and absorb negatively charged heat shock protein 70 (HSP70) inhibitor (siRNA against HSP70) to construct self-monitoring nanotheranostics (NPICS). NPICS can effectively restrain the expression of HSP70 to reduce their endurance to the IR825-mediated PTT, leading to an enhanced photocytotoxicity. In a xenograft mouse tumor model, NPICS show an effect of inhibition of tumor angiogenesis and also display a highly synergistic anticancer efficacy with NIR laser irradiation. Significantly, based on its inherent auto-fluorescence, PEI-PLA not only serves as the drug carrier, but also as the self-monitor to real-time track NPICS biodistribution and tumor accumulation via fluorescence imaging. Moreover, IR825 endows NPICS could also be used as photoacoustic (PA) agents for in vivo PA imaging. This nanoplatform shows enormous potentials in cancer theranostics.

Acute and chronic effects of hot water immersion on inflammation and metabolism in sedentary, overweight adults.

Regular exercise-induced acute inflammatory responses are suggested to improve the inflammatory profile and insulin sensitivity. As body temperature elevations partly mediate this response, passive heating might be a viable tool to improve the inflammatory profile. This study investigated the acute and chronic effects of hot water immersion on inflammatory and metabolic markers. Ten sedentary, overweight men [body mass index (BMI): 31.0 ± 4.2 kg/m2, mean ± SD] were immersed in water set at 39°C for 1 h (HWI) or rested for 1 h at ambient temperature (AMB). Venous blood was obtained before the session, immediately postsession, and 2 h postsession for assessment of monocyte intracellular heat shock protein-72 (iHsp72) and plasma concentrations of extracellular Hsp72 (eHsp72), interleukin-6 (IL-6), fasting glucose, insulin, and nitrite. Thereafter, participants underwent a 2-wk intervention period, consisting of 10 hot water immersion sessions (INT). Eight BMI-matched participants (BMI: 30.0 ± 2.5 kg/m2) were included as control (CON). Plasma IL-6 and nitrite concentrations were higher immediately following HWI compared with AMB (IL-6 P < 0.001, HWI: 1.37 ± 0.94 to 2.51 ± 1.49 pg/ml; nitrite P = 0.04, HWI: 271 ± 52 to 391 ± 72 nM), whereas iHsp72 expression was unchanged ( P = 0.57). In contrast to resting iHsp72 expression ( P = 0.59), fasting glucose ( P = 0.04; INT: 4.44 ± 0.93 to 3.98 ± 0.98 mmol/l), insulin ( P = 0.04; INT: 68.1 ± 44.6 to 55.0 ± 29.9 pmol/l), and eHsp72 ( P = 0.03; INT: 17 ± 41% reduction) concentrations were lowered after INT compared with CON. HWI induced an acute inflammatory response and increased nitric oxide bioavailability. The reductions in fasting glucose and insulin concentrations following the chronic intervention suggest that hot water immersion may serve as a tool to improve glucose metabolism. NEW & NOTEWORTHY A single hot water immersion (HWI) session induces an acute increase in plasma interleukin-6 and nitrite concentrations but does not acutely elevate heat shock protein-72 expression in monocytes [intracellular Hsp72 (iHsp72)]. A chronic HWI intervention reduces fasting glucose and insulin concentrations in the absence of changes in resting iHsp72. Therefore, HWI shows potential as a strategy to combat chronic low-grade inflammation and improve glucose metabolism in individuals without the physical capacity to do so using exercise.

Dietary curcumin supplementation does not alter peripheral blood mononuclear cell responses to exertional heat stress.

INTRODUCTION: Curcumin reduces gut barrier damage and plasma cytokine responses to exertional heat stress. However, the role of peripheral blood mononuclear cell (PBMC) in this response remains unclear. PURPOSE: This work investigated the effect of 3 days of 500 mg/day dietary curcumin supplementation on PBMC responses to exertional heat stress in non-heat acclimated humans. METHODS: Eight participants ran (65% VO2max) for 60 min in an environmental chamber (37 °C/25% RH) two times (curcumin/placebo). Blood samples were collected pre, post, 1 h post, and 4 h post-exercise. PBMC were isolated from blood samples and the protein content of markers along the TLR4 signaling pathway (TLR4, MyD88, pNF-κB, NF-κB), indicators of cellular energy status (SIRT1 and p-AMPK), and mediators of cellular heat shock response (pHSF-1 and HSP70) were examined with Western blot. Data were analyzed with two-way (condition × time) RM-ANOVAs with Newman-Keuls post hocs. RESULTS: As compared to placebo, curcumin did not alter protein expression in PBMC (p > 0.05). However, in both study conditions at 1 h post-reductions were noted in TLR 4 (- 21.5%; p = 0.03), HSP70 (- 11.0%; p = 0.04), pAMPK (- 48.5%; p < 0.01), and SIRT1 (- 47.8%; p < 0.01). Remarkably, the ratio of pNF-κB to NF-κB was elevated in both conditions at this same timepoint (+ 75.4%; p = 0.02). CONCLUSIONS: Inflammatory protein expression in PBMC did not differ between curcumin and placebo conditions. Downregulation of pAMPK/SIRT1 and release of HSP70 to the bloodstream may compensate for reduced TLR4, allowing PBMC to maintain inflammatory capacity and preventing an "open window" during the hours following hyperthermic exercise.

Enzyme-treated Asparagus Extract Down-regulates Heat Shock Protein 27 of Pancreatic Cancer Cells.

BACKGROUND/AIM: From the standpoint of cancer therapy, it is valuable to enhance the anticancer effects of chemotherapy. Our previous reports revealed that up-regulation of heat-shock protein 27 (HSP27) has been linked to gemcitabine resistance of pancreatic cancer cells. Enzyme-treated asparagus extract (ETAS) is an extract that is produced from asparagus. The purpose of this study was to investigate the effect of ETAS on the expression of HSP27 and other HSPs in the gemcitabine-resistant pancreatic cancer cell line KLM1-R. MATERIALS AND METHODS: KLM1-R cells were treated with ETAS, and expression levels of HSPs, including HSP27, were investigated by western blotting. RESULTS: ETAS down-regulated HSP27 and pHSP27 (serine 78) in KLM1-R cells, but, HSP70 and GRP78 levels were not altered. CONCLUSION: This study suggests the potential therapeutic benefit of ETAS in enhancing anticancer effects by its combination with gemcitabine for patients with pancreatic cancer.

Heat shock protein 72 regulates hepatic lipid accumulation.

Induction of the chaperone heat shock protein 72 (HSP72) through heat treatment (HT), exercise, or overexpression improves glucose tolerance and mitochondrial function in skeletal muscle. Less is known about HSP72 function in the liver where lipid accumulation can result in insulin resistance and nonalcoholic fatty liver disease (NAFLD). The purpose of this study was 1) to determine whether weekly in vivo HT induces hepatic HSP72 and improves glucose tolerance in rats fed a high-fat diet (HFD) and 2) to determine the ability of HSP72 to protect against lipid accumulation and mitochondrial dysfunction in primary hepatocytes. Male Wistar rats were fed an HFD for 15 wk and were given weekly HT (41°C, 20 min) or sham treatments (37°C, 20 min) for the final 7 wk. Glucose tolerance and insulin sensitivity were assessed, along with HSP72 induction and triglyceride storage, in the skeletal muscle and liver. The effect of an acute loss of HSP72 in primary hepatocytes was examined via siRNA. Weekly in vivo HT improved glucose tolerance, elevated muscle and hepatic HSP72 protein content, and reduced muscle triglyceride storage. In primary hepatocytes, mitochondrial morphology was changed, and fatty acid oxidation was reduced in small interfering HSP72 (siHSP72)-treated hepatocytes. Lipid accumulation following palmitate treatment was increased in siHSP72-treated hepatocytes. These data suggest that HT may improve systemic metabolism via induction of hepatic HSP72. Additionally, acute loss of HSP72 in primary hepatocytes impacts mitochondrial health as well as fat oxidation and storage. These findings suggest therapies targeting HSP72 in the liver may prevent NAFLD.

The earthworm Dendrobaena veneta (Annelida): A new experimental-organism for photobiomodulation and wound healing.

Photobiomodulation (PBM) is a manipulation of cellular behavior using non-ablative low intensity light sources. This manipulation triggers a cascade of metabolic effects and physiological changes resulting in improved tissue repair, of benefit in the treatment of tissue injury, degenerative or autoimmune diseases. PBM has witnessed an exponential increase in both clinical instrument technology and applications. It is therefore of benefit to find reliable experimental models to test the burgeoning laser technology for medical applications. In our work, we proposed the earthworm Dendrobaena veneta for the study of non-ablative laser-light effects on wound healing. In our preliminary work, D. veneta has been shown to be positively affected by PBM. New tests using D. veneta were set up to evaluate the effectiveness of a chosen 808 nm-64 J/cm2-1W-CW laser therapy using the AB2799 hand-piece with flat-top bean profile, on the wound healing process of the earthworm. Effective outcome was assimilated through examining the macroscopic, histological, and molecular changes on the irradiated posterior-segment of excised-earthworms with respect to controls. Three successive treatments, one every 24 hours, were concluded as sufficient to promote the wound healing, by effects on muscular and blood vessel contraction, decrement of bacteria load, reduction of inflammatory processes and tissue degeneration. D. veneta was demonstrated to be a reliable experimental organism that meets well the 3Rs principles and the National Science Foundation statement. Through their genetic and evolutionary peculiarity, comparable to those of scientifically accredited models, D. veneta allows the effect of laser therapies by multidisciplinary methods, at various degree of complexity and costs to be investigated.

Disruption of De Novo Serine Synthesis in Müller Cells Induced Mitochondrial Dysfunction and Aggravated Oxidative Damage.

De novo serine synthesis plays important roles in normal mitochondrial function and cellular anti-oxidative capacity. It is reported to be mainly activated in glial cells of the central nervous system, but its role in retinal Müller glia remains unclear. In this study, we inhibited de novo serine synthesis using CBR-5884, a specific inhibitor of phosphoglycerate dehydrogenase (PHGDH, a rate limiting enzyme in de novo serine metabolism) in MIO-M1 cells (immortalized human Müller cells) and huPMCs (human primary Müller cells) under mild oxidative stress. Alamar blue and LDH (lactate dehydrogenase) assays showed significantly reduced metabolic activities and increased cellular damage of Müller cells, when exposed to CBR-5884 accompanied by mild oxidative stress; however, CBR-5884 alone had little effect. The increased cellular damage was partially reversed by supplementation with exogenous serine/glycine. HSP72 (an oxidative stress marker) and reactive oxygen species (ROS) levels were significantly increased; glutathione and NADPH/NADP+ levels were pronouncedly reduced under PHGDH inhibition accompanied by oxidative stress. JC-1 staining and Seahorse respiration experiments showed that inhibition of de novo serine synthesis in Müller cells can also increase mitochondrial stress and decrease mitochondrial ATP production. qPCR and Western blot demonstrated an increased expression of HSP60 (a key mitochondrial stress-related gene), and this was further validated in human retinal explants. Our study suggests that de novo serine synthesis is important for Müller cell survival, particularly when they are exposed to mild oxidative stress, possibly by maintaining mitochondrial function and generating glutathione and NADPH to counteract ROS.

Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling.

BACKGROUND & AIMS: Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals. METHODS: Double transgenic mice overexpressing Hsp72 (gene Hspa1a) under the control of a tissue-specific tetracycline-inducible system (Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory-Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid. RESULTS: Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1ß or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo. Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation. CONCLUSIONS: Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. LAY SUMMARY: HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress.

Protective effects of red grape (Vitis vinifera) juice through restoration of antioxidant defense, endocrine swing and Hsf1, Hsp72 levels in heat stress induced testicular dysregulation of Wister rat.

Ability of red grape juice (RGJ), a known antioxidant, on testis of adult Wister rat to protect from oxidative stress induced damages by heat stress has been investigated in this study. Heat stress was induced maintaining body and testicular temperature at 43°C for 30min/day for 15 days using a hyperthermia induction chamber. Four groups of rats (n=6 per group) comprising of Group-I (control) -kept at 32°C, Group-II -exposed to heat stress alone, Group-III received RGJ (0.8ml/rat/day) alone and Group-IV -exposed to heat stress and received RGJ at same dose. Analysis of blood and testicular tissue exhibited significant reduction in serum testosterone, testicular superoxide dismutase, testicular catalase and testicular glutathione (all p < 0.001); whereas, significant rise in the level of serum corticosteroid, testicular lipid peroxidase and the apoptotic enzyme caspase-3 of testis (all p < 0.001) were observed along with substantial increase in testicular Hsp72 and Hsf-1, and decrease in 17ß-HSD3 were noted in heat stressed rats compared to controls. In Group-IV rats, RGJ administration could restore these parameters to normal levels. The signs of retention were clear in Group-IV rats and found to be significantly different as compared to that of the Group-II rats. In testicular histology of rats exposed to heat stress alone revealed remarkable germ cell degeneration and tubular deformations which were prevented by RGJ treatment (Group-IV). The reduced number of sperm level in Group-II also restored in RGJ treatment (Group-IV). The above results indicate that consumption of RGJ may substantially protect testis from heat stress induce dysfunctions.

Effect of Supplemental Trace Minerals on Hsp-70 mRNA Expression in Commercial Broiler Chicken.

The effects of supplementing the organic forms of selenium (Se), chromium (Cr), and zinc (Zn) on Hsp-70 mRNA expression and body weight in broiler chickens were evaluated. 200 chicks were equally distributed into stainless steel battery brooders at the rate of 5 birds per pen and reared under heat stress condition up to 42nd day. The chicks were fed with three experimental diets supplemented with organic forms of Se (0.30 mg/kg), Cr (2 mg/kg), and Zn (40 mg/kg) during the starter and finisher phases and a control diet without any supplementation. On the 21st and 42nd day, 20 birds from each period were sacrificed and samples were collected for analysis. Organic Se, Cr, and Zn supplementation significantly (P < 0.05) reduced the expression of Hsp-70 mRNA levels. The Hsp-70 mRNA expression levels were significantly (P < 0.05) different between the tissues studied with spleen having the lowest expression level. Hsp-70 mRNA expression level was not affected by age of the birds. The study concluded that organic trace mineral (oTM) supplementation resulted in low Hsp-70 mRNA expression, indicating reduced heat stress in broilers.

Thermal Preconditioning May Prevent Tendon Adhesion by Up-Regulating HSP72 in Rats.

BACKGROUND/AIMS: The study aims to determine the effects of thermal preconditioning on tendon adhesion by regulating the expression of heat shock protein 72 (HSP72) in rat models. METHODS: Sixty male Wistar rats were collected and randomly assigned into the thermal preconditioning and control groups. During the 4th and 8th weeks following surgery, 15 rats were sacrificed in each period respectively, and their tendon adhesion was observed and evaluated. Biomechanical testing was performed to measure the tensile strength and gliding distance of tendons. Hematoxylin-eosin (HE) was used to observe the morphological structure of the tendons. Immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the HSP72, fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR-1), ß-catenin, epithelial cell adhesion molecule (EPCAM), Tenomodulin and scleraxis protein expressions. Pearson correlation analysis was applied to analyze the correlation between HSP72 expression and tendon adhesion. RESULTS: At the 4th week after surgery, we found no differences in the tendon adhesion scores or mRNA and protein expressions of HSP72 between the thermal preconditioning and control groups. However, after the 8th week after surgery, the thermal preconditioning group had a lower tendon adhesion score and higher mRNA and protein expressions of HSP72 than the control group. During the same period, we found longer gliding distance and higher expression levels of FGF-2, FGFR-1, ß-catenin, Tenomodulin and scleraxis, but lower EPCAM expression in the thermal preconditioning group. Pearson correlation analysis indicated that HSP72 mRNA and protein expression levels were negatively correlated with tendon adhesion. CONCLUSIONS: These findings provide evidence that thermal preconditioning may alleviate tendon adhesions via upregulation of HSP72 expression.

Chronic probiotic supplementation with or without glutamine does not influence the eHsp72 response to a multi-day ultra-endurance exercise event.

Probiotic and glutamine supplementation increases tissue Hsp72, but their influence on extracellular Hsp72 (eHsp72) has not been investigated. The aim of this study was to investigate the effect of chronic probiotic supplementation, with or without glutamine, on eHsp72 concentration before and after an ultramarathon. Thirty-two participants were split into 3 independent groups, where they ingested probiotic capsules (PRO; n = 11), probiotic + glutamine powder (PGLn; n = 10), or no supplementation (CON; n = 11), over a 12-week period prior to commencement of the Marathon des Sables (MDS). eHsp72 concentration in the plasma was measured at baseline, 7 days pre-race, 6-8 h post-race, and 7 days post-race. The MDS increased eHsp72 concentrations by 124% (F[1,3] = 22.716, p < 0.001), but there was no difference in the response between groups. Additionally, PRO or PGLn supplementation did not modify pre- or post-MDS eHsp72 concentrations compared with CON (p > 0.05). In conclusion, the MDS caused a substantial increase in eHsp72 concentration, indicating high levels of systemic stress. However, chronic PRO or PGLn supplementation did not affect eHsp72 compared with control pre- or post-MDS. Given the role of eHsp72 in immune activation, the commercially available supplements used in this study are unlikely to influence this cascade.

Anti-inflammatory effect as a mechanism of effectiveness underlying the clinical benefits of pelotherapy in osteoarthritis patients: regulation of the altered inflammatory and stress feedback response.

The purpose of the present investigation was to evaluate whether an anti-inflammatory effect together with an improvement of the regulation of the interaction between the inflammatory and stress responses underlies the clinical benefits of pelotherapy in osteoarthritis (OA) patients. This study evaluated the effects of a 10-day cycle of pelotherapy at the spa centre 'El Raposo' (Spain) in a group of 21 OA patients diagnosed with primary knee OA. Clinical assessments included pain intensity using a visual analog scale; pain, stiffness and physical function using the Western Ontario and McMaster Universities Arthritis Index; and health-related quality of life using the EuroQol-5D questionnaire. Serum inflammatory cytokine levels (IL-1ß, TNF-α, IL-8, IL-6, IL-10 and TGF-ß) were evaluated using the Bio-Plex® Luminex® system. Circulating neuroendocrine-stress biomarkers, such as cortisol and extracellular 72 kDa heat shock protein (eHsp72), were measured by ELISA. After the cycle of mud therapy, OA patients improved the knee flexion angle and OA-related pain, stiffness and physical function, and they reported a better health-related quality of life. Serum concentrations of IL-1ß, TNF-α, IL-8, IL-6 and TGF-ß, as well as eHsp72, were markedly decreased. Besides, systemic levels of cortisol increased significantly. These results confirm that the clinical benefits of mud therapy may well be mediated, at least in part, by its systemic anti-inflammatory effects and neuroendocrine-immune regulation in OA patients. Thus, mud therapy could be an effective alternative treatment in the management of OA.