Nanoparticles Exacerbate Both Ubiquitin and Heat Shock Protein Expressions in Spinal Cord Injury: Neuroprotective Effects of the Proteasome Inhibitor Carfilzomib and the Antioxidant Compound H-290/51.

Increased levels of ubiquitin and heat shock protein (HSP) 72 kD are often seen in spinal cord injury (SCI). However, their roles in cell injury or survival are not well known. Thus, we have investigated the possible relationship between ubiquitin and HSP expressions in relation to cell injury in healthy animals, or following nanoparticle (NP) intoxication in SCI animals. A focal SCI was inflicted on the T10-11 segments over the right dorsal horn; animals were allowed to survive from 5 to 8 h after trauma. Separate groups of rats were exposed to SiO2, Ag, or Cu NPs for 7 days and subjected to SCI on the eighth day. A marked increase in ubiquitin or HSP immunoreactive cells occurred in the T9 to T12 segments 5 h after the injury, which further extended to the T4 and L5 after 8 h of survival. At this time, a marked increase in blood-spinal cord barrier (BSCB) permeability to Evans blue and radioiodine, accompanied by an intense edema formation, was observed. Changes were further exacerbated in NP-treated traumatized rats. The most marked expressions of ubiquitin and HSP in SCI were seen in rats treated with SiO2, followed by Ag, and Cu NPs. Treatment with H-290/51 (50 mg/kg p.o., 30 to 60 min after injury) or carfilzomib (1 mg/kg, i.v., 30 to 60 min after trauma) significantly reduced the ubiquitin or HSP expressions, as well as the BSCB breakdown, the edema formation, and the cell injury in the cord both 5 and 8 h after the injury, in normal animals. However, a double dose of H-290/51 (100 mg/kg) or carfilzomib (2 mg/kg) is needed to reduce cord pathology or ubiquitin and HSP expressions in traumatized animals treated with NPs. H-290/51 showed superior beneficial effects in reducing cord pathology in SCI than carfilzomib. These observations are the first to demonstrate that (i) NP-treated traumatized animals induce a widespread BSCB leakage, edema formation, and cord pathology as well as an overexpression of ubiquitin and HSP, (ii) high doses of antioxidant compounds or proteasome inhibitors are required for neuroprotection in the NP-exposed traumatized group, and (iii) ubiquitin and HSP expressions play a key role in neuronal injury in SCI, not reported earlier.

Neuroprotective effects of hydroalcoholic extract of Ocimum sanctum against H2O2 induced neuronal cell damage in SH-SY5Y cells via its antioxidative defence mechanism.

Oxidative stress mediates the cell damage in several ailments including neurodegenerative conditions. Ocimum sanctum is widely used in Indian ayurvedic medications to cure various ailments. The present study was carried out to investigate the antioxidant activity and neuroprotective effects of hydroalcoholic extract of O. sanctum (OSE) on hydrogen peroxide (H2O2)-induced oxidative challenge in SH-SY5Y human neuronal cells. The extract exhibited strong antioxidant activity against DPPH, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical and hydroxyl radicals with IC50 values of 395 ± 16.2, 241 ± 11.5 and 188.6 ± 12.2 µg/ml respectively, which could be due to high amount of polyphenols and flavonoids. The observed data demonstrates 41.5% cell survival with 100 µM H2O2 challenge for 24 h, which was restored to 73% by pre-treatment with OSE for 2 h. It also decreased the lactate dehydrogenase leakage and preserved the cellular morphology. Similarly OSE inhibited lipid peroxidation, DNA damage, reactive oxygen species generation and depolarization of mitochondrial membrane. The extract restored superoxide dismutase and catalase enzyme/protein levels and further downregulated HSP-70 over-expression. These findings suggest that OSE ameliorates H2O2 induced neuronal damage via its antioxidant defence mechanism and might be used to treat oxidative stress mediated neuronal disorders.

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

The effect of the hyperbaric environment on heat shock protein 72 expression in vivo.

Heat shock protein 72 (HSP72) is expressed in response to stress and has been demonstrated to follow a diurnal expression pattern within monocytes and is sensitive to changes in core temperature. Numerous studies have shown changes in HSP72 expression within cell lines exposed to hyperbaric conditions. No studies have investigated changes in HSP72 expression in vivo. Six males participated in the study and were exposed to hyperbaric air and hyperbaric oxygen a week apart. Monocyte HSP72 was analyzed by flow cytometry at 09:00, 13:00, 17:00, 21:00 with hyperbaric oxygen or hyperbaric air breathing commencing at 15:00 for 78 min at a pressure of 2.8 ATA. HSP72 under normoxia followed the established trend; however, following the hyperbaric air or oxygen exposure a reduction in detectable HSP72 was observed at 17:00 and 21:00. No changes in core temperature were observed between 13:00 and 21:00 for any condition. The data show that HSP72 expression is impaired following hyperbaric air (HA) exposure, when compared with control or hyperbaric oxygen (HO) exposure.

Hyperthermia protects mice against chronic unpredictable stress-induced anxiety-like behaviour and hippocampal CA3 cell apoptosis.

PURPOSE: It is widely accepted that chronic stress can induce anxiety; however, the cellular and molecular mechanisms of stress-induced anxiety are far from being elucidated. Hyperthermia has been shown to induce expression of heat shock proteins (HSPs) to provide protection against a variety of stresses. To our knowledge, the effect of hyperthermia on the development of chronic unpredictable stress (CUS)-induced anxiety has not been studied. This study was to determine the relationship between hyperthermia induced Hsp72 and CUS related anxiety. MATERIALS AND METHODS: Heat shock factor 1 knockout (hsf1(-/-)) and wild-type (hsf1(+/+)) mice were subjected to CUS with or without hyperthermia treatment. Anxiety-like behaviours were evaluated by elevated plus maze and open field tests. Apoptosis in the hippocampal CA3 area was detected by TUNEL staining. Hsp72 protein level in the hippocampus was measured by Western blot. RESULTS: CUS caused significant apoptosis in hippocampal CA3 cells in both hsf1(-/-) and hsf1(+/+) mice, which significantly correlated with anxiety-like behaviours. Hyperthermia induced Hsp72 expression in hsf1(+/+) mice, but not in hsf1(-/-) mice. Importantly, hyperthermia protected hsf1(+/+) mice against developing CUS-related anxiety-like behaviours and reduced CUS-induced apoptosis in hippocampal CA3 cells. In contrast, hyperthermia exhibited no protective role in hsf1(-/-) mice. CONCLUSIONS: Apoptosis of hippocampal CA3 cells is involved in the development of anxiety-like behaviours underlying CUS. Hsp72 protein is a crucial player in the protective effect of hyperthermia against CUS-induced apoptosis and development of anxiety-like behaviours. Our study suggests hyperthermia is an effective treatment for CUS-induced mood disorders.

Warm SPA-induced hyperthermia confers protection to rats against airway inflammation evoked by capsaicin and substance P.

Solus par aqua (SPA) is a traditional health care therapy. Warm SPA may enhance immunity and cellular defense to protect body against diseases. The present study investigated whether the warm SPA could confer protection to neurogenic inflammation in rats. The rats were immersed in water where the body core temperatures were maintained at hyperthermia (41.5 degrees C) or normothermia (37 degrees C) for a period of 15min. After SPA for 1 or 6 days, neurogenic inflammation was induced by intravenous injection of capsaicin (90microg/kg) or substance P (SP; 3microg/kg). The plasma leakage and arterial pressures in rats after neurogenic inflammation were monitored. The extent of capsaicin- or SP-induced plasma leakage and hypotension was significantly attenuated in rats on day 1 after SPA hyperthermia. However, such resistance to neurogenic inflammation was not found on day 6 after hyperthermia. Western blotting analysis showed that the expression of heat shock protein 72 (HSP 72) in the trachea on days 1 and 2 after hyperthermia was 9.61-fold and 6.66-fold, respectively, of that in normothermia. Afterwards, the hyperthermia-induced HSP 72 upregulation gradually declined in a time-dependent manner. Thus, SPA hyperthermia may protect rats against neurogenic inflammation through modulation of HSP expression.

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis.

AIMS: The aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats. MAIN METHODS: Expression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 degrees C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation. KEY FINDINGS: Expression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-alpha and IL-1beta in esophageal mucosa was also suppressed. SIGNIFICANCE: These results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.

Heat stress attenuates ATP-depletion and pH-decrease during cardioplegic arrest.

BACKGROUND: The capacity of heat stress induction to improve myocardial tolerance against ischemia is well known. We investigated cardiac energy metabolism after hsp 72(+)/73(+) induction in isolated perfused neonatal rabbit hearts subjected to prolonged cold cardioplegic ischemia. METHODS: Hearts from neonatal rabbits were excised, isolated perfused and arrested by 2-h cold cardioplegic ischemia. Rectal temperature of eight neonatal rabbits was raised to 42.0 to 42.5 degrees C for heat shock protein expression in a whole body water bath for 15 min before the onset of arrest. Another set of eight rabbits without hyperthermia pretreatment served as control. Recovery of left ventricle function was assessed by aortic flow, cardiac output, and max dP/dt. Status of high-energy phosphates was measured by (31)phosphorus nuclear magnetic resonance-spectroscopy. RESULTS: Immunoblot analysis revealed clear hsp 72+/73+ induction after a brief period of systemic hyperthermia. Heat stress pretreatment resulted in a better recovery of left ventricular function (aortic flow and cardiac output improvement P < 0.05, max dP/dt P < 0.01) than in controls at 60 min after reperfusion. During ischemia and reperfusion, myocardial energy metabolism was better preserved in hearts after hsp induction as a consequence of increased gamma-, alpha-, and beta-ATP as well as phosphocreatine-values over controls. The ischemia-induced pH-decrease was attenuated. CONCLUSION: These data contribute to the evidence of heat stress mediated beneficial effects on functional myocardial recovery and improved cardiac energy metabolism after prolonged cold cardioplegic ischemia. More importantly, the attenuation of ischemic pH reduction and better restoration suggest an involvement of mitochondrial membrane potential alterations.

Inhibitory effect of Patrinia scabiosaefolia on acute pancreatitis.

AIM: To investigate the effect of Patrinia scabiosaefolia (PS) on the cholecystokinin (CCK) octapeptide-induced acute pancreatitis (AP) in rats. METHODS: Wistar rats weighing 240-260 g were divided into three groups: (1) Normal saline-treated group; (2) treatment with PS at 100 mg/kg group, in which PS was administered orally, followed by subcutaneous administration of 75 microg/kg CCK octapeptide three times after 1, 3 and 5 h, and this whole procedure was repeated for 5 d; (3) treatment with saline group, in which the protocols were the same as in treatment group with PS. We determined the pancreatic weight/body weight ratio, the levels of pancreatic HSP60, HSP72 and the secretion of pro-inflammatory cytokines. Repeated CCK octapeptide treatment resulted in the typical laboratory findings of experimentally induced pancreatitis. RESULTS: PS reduced the pancreatic weight/body weight ratio, the levels of serum amylase and lipase, and inhibited expressions of pro-inflammatory cytokines in the CCK octapeptide-induced AP. Furthermore, PS pretreatment increased the pancreatic levels of HSP60 and HSP72. CONCLUSION: Pretreatment with PS has an anti-inflammatory effect on CCK octapeptide-induced AP.

Stress proteins (Hsp72/73, Grp94) expression pattern in rat organs following metavanadate administration. Effect of green tea drinking.

Expression pattern of heat shock proteins (Hsp) 72/73 and glucose regulated protein (Grp) 94 was studied in liver, kidney and testis of rats injected with sublethal doses of ammonium metavanadate (5 mg/kg/day). In addition, some batches of animals were given green tea decoction, known to be rich in anti-oxidative compounds, as sole beverage in order to evaluate its protective properties. In control animals, the stress proteins expression was found to be organ-dependent: anti-Grp94 antibody revealed two bands at 96 and 98 kDa in kidney and liver whereas the 98 kDa band only was found in testis; anti-Hsp72/73 antibody revealed that the constitutive Hsp73 was present in all organs whereas the inducible Hsp72 was only present in kidney and testis. In kidney of vanadium-treated rats, Hsp73 was over-expressed by about 50% whereas Hsp72 was down-regulated by 50-80%. No such effects were observed in liver and testis. In liver and kidney of vanadium-treated rats, Grp94 was over-expressed by 50% and 150% respectively whereas no change was found in testis. In rats given green tea as sole beverage, the 96 kDa protein expression level in liver was reduced both in controls and in vanadium-treated animals. However, green tea drinking failed to prevent the vanadium-induced Hsp72 under-expression in kidney of vanadium-treated rats.

[Effect of Xingding Injection on expression of heat shock protein 72 in vascular endothelial cells].

OBJECTIVE: To study the effect of Xingding Injection on the expression of heat shock protein 72 (HSP 72) in vascular endothelial cells after ultraviolet radiation. METHODS: Porcine aortic endothelial cells were cultured for 72 hours in culture mediums with different concentrations (0.1, 0.5, 1.0, 5.0, 10 mg/ml) of Xingding Injection. Ultraviolet radiation was administered to the cultured cells for 30 minutes. Western-blot assay was used to measure the expression of HSP 72 in the vascular endothelial cells. RESULTS: There was no expression of HSP 72 in the cultured vascular endothelial cells without ultraviolet radiation, but there was some expression of HSP 72 after ultraviolet radiation. Xingding Injection of different concentrations could significantly improve the expression of HSP 72. The expression of HSP 72 in the vascular endothelial cells cultured in culture medium with 1.0 mg/ml Xingding Injection was the highest, and there was no more increase of expression when the concentration was higher, instead the expression decreased. CONCLUSION: Xingding Injection can protect the vascular endothelial cells from injury during stress. It may be one of its mechanisms in preventing and treating cardio-cerebrovascular disorders.