Hsp70 mRNA Levels Research Articles

Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass (Lateolabrax maculatus)

The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass (Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress.

Heat stress is exacerbated by global warming and affects human and animal health, leading to heart damage caused by imbalances in reactive oxygen species (ROS) and the antioxidant system, acid-base chemistry, electrolytes and respiratory alkalosis. Vitamin C scavenges excess ROS, and sodium bicarbonate maintains acid-base and electrolyte balance, and alleviates respiratory alkalosis. Herein, we explored the ability of vitamin C alone and in combination with equimolar sodium bicarbonate (Vitamin C-Na) to stimulate endogenous antioxidants and heat shock proteins (HSPs) to relieve heat stress in H9C2 cells. Control, vitamin C (20μg/ml vitamin C for 16h) and vitamin C-Na (20μg/ml vitamin C-Na for 16h) groups were heat-stressed for 1, 3 or 5h. Granular and vacuolar degeneration, karyopyknosis and damage to nuclei and mitochondria were clearly reduced in treatment groups, as were apoptosis, lactate dehydrogenase activity and ROS and malondialdehyde levels, while superoxide dismutase activity was increased. Additionally, CRYAB, Hsp27, Hsp60 and Hsp70 mRNA levels were upregulated at 3h (p < 0.01), and protein levels were increased for CRYAB at 0h (p < 0.05) and 1h (p < 0.01), and for Hsp70 at 3 and 5h (p < 0.01). Thus, pre-treatment with vitamin C or vitamin C-Na might protect H9C2 cells against heat damage by enhancing the antioxidant ability and upregulating CRYAB and Hsp70.

Arsenic and/or copper caused inflammatory response via activation of inducible nitric oxide synthase pathway and triggered heat shock protein responses in testis tissues of chicken.

The aim of this study is to investigate the effects of arsenic (As) and copper (Cu) on the inflammatory response, and the protective roles of heat shock proteins (Hsps) in chicken testes. Seventy-two 1-day-old male Hy-line chickens were treated with 30mg/kg feed of arsenic trioxide (As2O3) and/or 300mg/kg feed of copper sulfate (CuSO4) for 4, 8, and 12weeks. The histological changes, inducible nitric oxide synthase (iNOS) activity, and the expressions of Hsps and inflammatory cytokines were detected. The results showed that slight histology changes were obvious in the testis tissue exposure to treatment groups. The activity and the protein level of iNOS were increased compared to the control group. The mRNA levels of proinflammatory cytokines and inflammatory factors were increased as a whole. However, anti-inflammatory cytokines were inhibited. The mRNA and protein levels of Hsp60, Hsp70, and Hsp90 were upregulated. These results suggested that sub-chronic exposure to As and/or Cu induced testicular poisoning in chickens. Increased Hsps tried to protect chicken testis tissues from tissues damage caused by inflammation. In conclusion, testicular poisoning induced by As and/or Cu caused inflammatory response and heat shock protein response in chicken testis tissues.

Gender-based behavioral and biochemical effects of diphenyl diselenide in Drosophila melanogaster

In the present study we investigated the effects of Diphenyl Diselenide (DPDS) on behavioral and biochemical parameters in Drosophila melanogaster (DM). We also tried to explore the gender differences in response to DPDS treatment. One of the salient features of the present report is that, at the highest tested concentration (3.0 mmol/kg) the survival rate in females was significantly higher (65%) as compared with male subjects (27.5%). However in alive subjects, different results pattern was observed. The locomotor activity of females significantly decreased at all tested concentrations and no statistical difference was observed in males at 0.1 and 1.0 mM. To explore the toxicological pathway of DPDS, different arrays of experiments were performed. DPDS did not cause any significant alteration in total thiol contents (NPSH) in males. While, a significant decrease was observed in females at all tested concentrations (0.1, 1.0 and 3.0 mM; p<0.005). For gender comparison purpose, its important to note that females showed 2.1 and 1.63 folds increase in SOD and CAT m-RNA expression at the highest (3.0 mM) tested concentration (as compared with male subjects). In contrast to m-RNA levels of catalase, the lowest concentration (0.1 mM) of DPDS significantly decreased (P<0.005) the enzymatic activity in females and a noticeable increase (P<0.005) at highest tested concentration (3.0 mM) was observed in male subjects. Statistically no significant change was observed in Glutathione -S- transferase (GST) activity at all tested concentrations in females. However a marked increase at 1.0 and 3.0 mM was observed in males. Furthermore, we also evaluated, HSP70 mRNA levels in females, which were increased by approximately 10.6 fold (P<0.005) as compared to male subjects. These results demonstrated significant gender-differences in terms of acute response to DPDS treatment. Mechanistically thiol oxidase (TOx) potential is one of the proposed pathways of DPDS toxicity, which can not be excluded in the present findings.

Heat-induced oxidative stress and inflammation involve in cadmium pollution history in the spleen of zebrafish

Zebrafish were exposed to 0, 2.5 and 5 μg/L cadmium (Cd) for 10 weeks, and then each group was exposed to 26 °C(control) and 32 °C (high temperature) for 7 days. 22 indicators were compared between 26 °C and 32 °C in the spleen, including body weight, LPO and NO levels, activity levels of Cu/Zn-SOD, CAT and iNOS, MTs protein levels, and mRNA levels of Nrf2, Cu/Zn-SOD, CAT, HSF1, HSF2, HSP70, MTF-1, MTs, IL-6, IL-10, IL-1β, TNF-α, iNOS and NF-κB. Most indicators were not significantly affected by heat in fish from no Cd pollution. However, almost all of indicators were responsive to heat in fish pre-exposed to Cd. Several indicators were sensitive to heat in fish pre-exposed to 2.5 μg/L Cd such as iNOS activities, and mRNA levels of iNOS and IL-10. Most other indicators were sensitive to heat in fish pre-exposed to 5 μg/L. The mRNA levels of HSP70 and MTF-1 were up-regulated by heat in fish pre-exposed to 0, 2.5 and 5 μg/L Cd. However, the magnitude of increase was the greatest in fish pre-exposed to 5 μg/L Cd. These differences between control and high temperature would serve as biomarkers to distinguish healthy from Cd-polluted group. The findings imply that metal pollution history should be carefully considered when screening heat biomarkers in fish.

Effect of dietary astaxanthin on the growth performance, non-specific immunity, and antioxidant capacity of pufferfish (Takifugu obscurus) under high temperature stress.

The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production, and immune-related gene expressions of the pufferfish (Takifugu obscurus) under high temperature stress. The experimental basal diets supplemented with astaxanthin at the rates of 0 (control), 20, 40, 80, 160, and 320mgkg-1 were fed to fish for 8weeks. The results showed that the fish fed diet with 80, 160, and 320mgkg-1 astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed the moderate dietary astaxanthin increased plasma alkaline phosphatase activities, and decrease plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, the fish were exposed to high temperature stress for 48h. The results shown that astaxanthin could suppress ROS production induced by high temperature stress. Meanwhile, compared with the control group, the astaxanthin groups increased SOD, CAT, and HSP70 mRNA levels under high temperature stress. These results showed that the basal diet supplemented with 80-320mgkg-1 astaxanthin could enhance growth, nonspecific immune responses, and antioxidant defense system and improve resistance against high temperature stress in pufferfish.

Effects of dietary hawthorn extract on growth performance, immune responses, growth- and immune-related genes expression of juvenile golden pompano (Trachinotus ovatus) and its susceptibility to Vibrio harveyi infection

The present study was conducted to investigate the effects of dietary hawthorn extract (HTE) supplementation on growth performance, immune responses, hepatic antioxidant abilities, growth- and immune-related and heat shock protein genes expression and resistance to the pathogen Vibrio harveyi in Trachinotus ovatus. A basal diet supplemented with HTE at 0 (Diet 1), 0.50 (Diet 2), 1.00 (Diet 3), 2.00 (Diet 4), 4.00 (Diet 5) and 10.00 (Diet 6) g kg−1 were fed to golden pompano for 8 weeks. The highest final body weight, weight gain rate, specific growth rate, feed efficiency ratio and protein efficiency rate were observed in fish fed Diet 2 (P < 0.05). Dietary HTE significantly increased plasma complement 3, complement 4 and immunoglobulin M content (P < 0.05). Hepatic antioxidant enzymes (SOD, T-AOC, CAT, GPx, GR) significantly increased (P < 0.05), whereas MDA content decreased first and then increased in fish fed HTE supplement. After challenge with Vibrio harveyi, significant higher post-challenge survival was observed in fish fed Diet 2 and Diet 3 than the control group (P < 0.05). Transcription levels of growth-related genes (IGF-I and IGF-II) were significantly up-regulated in fish fed HTE supplement (P < 0.05), whereas HSP70 and HSP90 mRNA levels were significantly down-regulated (P < 0.05). With respect to immune-related genes, such as tumour necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) and inhibitor protein κBα (IκB-α), upregulation was observed in the liver of fish fed with the diet supplemented with HTE. In contrast, the expression of antioxidant enzyme genes (CAT, GPx, MnSOD and Keap1) and cytokines (IL-10, TGF-β1 and TOR) was downregulated. These results indicated that golden pompano fed a diet supplemented with 0.50 g kg−1 HTE could significantly promote growth performance and growth-related genes expression, strengthen immunity, and improve hepatic antioxidative abilities and resistance to Vibrio harveyi infection.

Heat indicators of oxidative stress, inflammation and metal transport show dependence of cadmium pollution history in the liver of female zebrafish

Environmental stressors such as high temperature and metal exposure may occur sequentially, simultaneously, previously in aquatic ecosystems. However, information about whether responses to high temperature depend on Cd exposure history is still unknown in fish. Zebrafish were exposed to 0 (group 1), 2.5 (group 2) and 5μg/L (group 3) cadmium (Cd) for 10 weeks, and then each group was subjected to Cd-free water maintained at 26°C and 32°C for 7days respectively. 26 indicators were used to compare differences between 26°C and 32°C in the liver of female zebrafish, including 5 biochemical indicators (activity of Cu/Zn-SOD, CAT and iNOS; LPO; MT protein), 8 molecular indicators of oxidative stress (mRNA levels of Nrf2, Cu/Zn-SOD, CAT, HSF1, HSF2, HSP70, MTF-1 and MT), 5 molecular indicators of inflammation (mRNA levels of IL-6, IL-1β, TNF-α, iNOS and NF-κB), 8 molecular indicators of metal transport (mRNA levels of, ZnT1, ZnT5, ZIP8, ZIP10, ATP7A, ATP7B and CTR1). All biochemical indicators were unchanged in group 1 and changed in group 2 and 3. Contrarily, differences were observed in almost all of molecular indicators of inflammation and metal transport in group 1, about half in group 2, and few in group 3. We also found that all molecular indicators of oxidative stress in group 2 and fewer in group 1 and 3 were significantly affected by heat. Our data indicated that heat indicators of oxidative stress, inflammation and metal transport showed dependence of previous cadmium exposure in the liver of zebrafish, emphasizing metal pollution history should be carefully considered when evaluating heat stress in fish.

Fluoride exposure changed the structure and the expressions of HSP related genes in testes of pubertal rats

Previous studies have indicated that fluoride exposure damaged the male reproductive function; however, the cellular mechanism of fluoride-induced testicular toxicity is still unclear. In this study, twenty-two female pregnant Wistar rats were allotted randomly to two groups: control (deionized water) and sodium fluoride (NaF, contain F−: 67.86 mg/L) groups. After delivery, the dosage was continued for 15 weeks for puppies. Twelve rats in each group were tested at 6 and 9 (pubertal); 12 and 15 (mature) weeks of age. Our results suggested that organ coefficient of epididymis was significantly decreased in the mature (12 and 15 week-old) rats. Epididymal sperm abnormality and femur fluoride concentration were increased with the concomitant decrease in sperm motility and concentration in these experimental periods. Compared to the control, in the NaF group, the seminiferous tubules of each age were reduced in terms of diameter and thickness. The sperm cells were lost and shedding and finally disappeared after 9 weeks. mRNA and protein levels of HSP27 and 90 were decreased with a concomitant increase in HSP70 and HSF mRNA and protein levels in NaF exposed rats. The mRNA and protein levels of HSP27 and HSF (only mRNA) were significantly increased in NaF treated rats at 9 and 15 weeks of age, respectively. In summary, these results emphasize that NaF induces testicular and sperm abnormalities through the involvement of HSPs especially during the pubertal period.

Glutamine promotes Hsp70 and inhibits α-Synuclein accumulation in pheochromocytoma PC12 cells.

Hsp70 regulates α-Synuclein (α-Syn) degeneration in Parkinson's disease (PD), indicating that Hsp70 promotion may be able to prevent or reverse α-Syn-induced toxicity in PD. Additionally, it has been demonstrated that glutamine (Gln) enhances Hsp70 expression. In the present study, Gln-induced Hsp70 promotion in pheochromocytoma was investigated with reverse transcription- quantitative polymerase chain reaction and western blotting methods. Then it was observed whether heat shock factor (HSF)-1 was required for this phenomenon with an RNA interference strategy. The regulatory role of Gln on α-Syn degeneration was also determined in the α-Syn-overexpressed PC12 [PC12 (α-Syn+)] cells, which were treated with or without the proteasomal inhibitor lactacystin (Lac). The results demonstrated that treatment with ≥10 mM Gln significantly increased Hsp70 mRNA and protein levels (P<0.05) and that this promotion was HSF-1-dependent, as HSF-1 knockout with HSF-1-specific small interfering RNA abrogated Hsp70 promotion in PC12 (α-Syn+) cells. Furthermore, Gln treatment markedly upregulated α-Syn degeneration in PC12 (α-Syn+) cells, which was significantly reduced (P<0.05) in the presence of Lac. Therefore, the present study suggests that Gln is able to induce the promotion of Hsp70 expression in PC12 cells in an HSF-1-dependent manner and that Gln-mediated Hsp70 promotion may increase α-Syn degradation even in the presence of proteasomal inhibitor. Thus, glutamine may be a potential therapeutic agent to prevent α-Syn aggregation in PD.

Hsp70 protects human trabecular meshwork cells injury induced by UVB through Smad pathway.

Glaucoma is a universal eye disease which is commonly associated with increased intraocular pressure caused by impaired aqueous humor (AH) drainage. This study aimed to investigate the effects of Hsp70 on trabecular meshwork (TM) injury induced by UVB. Real-time quantitative PCR (qRT-PCR) was used to examine the mRNA levels of Hsp70. siRNA was used to downregulate Hsp70 expression in the TM cells to inspect changes in cell proliferation and apoptosis. Cell proliferation was assessed by a Cell Counting Kit-8 (CCK-8) assay and the number of apoptotic cells was assessed using annexin V-FITC/PI apoptosis detection kit. The Smad signaling pathway was investigated using western blotting analyses. The overexpression of Hsp70 promoted cell proliferation and suppressed apoptosis. What's more, the overexpression of Hsp70 suppressed the expression of Smad-2, Smad-3 and Smad-7. Hsp70 might improve cell viability and inhibit TM apoptosis by inhibition of the Smad pathway. Hsp70 is a potential therapeutic target for the treatment of glaucoma.

Bio-effect-monitoring of long-term thermal wastes on the oyster, Crassostrea gigas, using heat shock proteins

We bio-monitored the stress of oyster, Crassostrea gigas, for possible long term effects of thermal waste from a power plant. The expression level of its heat shock proteins (HSPs) was measured by real time-reverse transcript PCR along with their density and growth in the field. Oyster size varied in a distance dependent pattern. Physics modeling for evaluation of spreading of the thermal effluent revealed that station A is affected by the thermal effluents abundance, and the size of C. gigas showed a negative relationship with distance to the power plant. The abundance and size of C. gigas were smallest at station A, which was closest to the thermal effluent outlet. The kinetics of changes in the hsp70 and hsp90 mRNA levels in the mantle of C. gigas were also investigated. Regardless of the higher expression level of hsp70 mRNA than hsp90, both hsp70 and hsp90 mRNA levels were significantly higher at station A. The expression levels decreased inversely with distance from the thermal effluent outlet, with expression of hsp70 mRNA at station A being approximately 7-fold higher than at station B and 15-fold higher than at station C. Similarly, expression of hsp90 mRNA at station A was approximately 14-fold higher than at station B and 22-fold higher than at station C. The present findings provide new insights on biological correlation among the growth of individuals and population size and the molecular index in C. gigas following thermal effects.

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

ABSTRACTThe 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.

High-sucrose diet induces diabetic-like phenotypes and oxidative stress in Drosophila melanogaster: Protective role of Syzygium cumini and Bauhinia forficata.

Diet is a key component for development and longevity of organisms. Here, the fruit fly was used to evaluate the detrimental effects caused by consumption of high-sucrose diets (HSD), namely phenotypic responses linked to insulin signaling and oxidative stress. The protective effects of extracts from medicinal plants Syzygium cumini and Bauhinia forficata were investigated. HSD intake (15% and 30%) delayed the time to pupation and reduced the number of white pupae. In adult flies, the intake of diets was associated with mortality and increased levels of glucose+trehalose, triacylglycerols and hydrogen peroxide. Indeed, 30% HSD induced body-weight loss, mitochondrial dysfunction and changes in acetylcholinesterase, δ-aminolevulinate dehydratase and antioxidant enzymes activity. Catalase, superoxide dismutase, keap1, HSP70, dILP-5 and Insulin receptor mRNA levels were over-expressed in flies emerged from 30% HSD. The extract treatments blunted the developmental alterations elicited by diets. Syzygium cumini extract was more efficient than B. forficata in reducing hyperglycaemia, redox disturbances and the changes in mRNA expression of insulin receptor.

Effects of niacin and betaine on bovine mammary and uterine cells exposed to thermal shock in vitro

The objective of this study was to investigate the direct effects of feed supplements niacin and betaine on the heat shock responses of in vitro cultured cells derived from bovine mammary and uterine tissues. First, we determined the mRNA expression profiles of the niacin receptor (GPR109A) in bovine tissues (liver, skin, uterus, udder, and ovary) and in cells derived from bovine mammary epithelium (mammary alveolar cells, MAC-T; bovine mammary epithelial cells, BMEC) and endometrium (bovine endometrial cells, BEND). We found that GPR109A was distributed in all examined tissues and cells, and the highest expression was in cells from skin and udder. Second, we evaluated the effects of niacin treatment on the mRNA abundance of heat shock proteins 70 and 27 (HSP70 and HSP27) in MAC-T, BMEC, and BEND under thermoneutral conditions and heat stress, and whether these effects were associated with alterations in the mRNA expression of prostaglandin E2 synthesis-related genes, including cyclooxygenase 1 and 2 (COX-1 and COX-2) and microsomal prostaglandin E synthase 1 and 2 (mPGES-1 and mPGES-2). Quantitative PCR data indicated that niacin suppressed HSP70 mRNA expression in BMEC and both HSP70 and HSP27 in BEND under thermoneutral conditions. Only COX-2 expression was downregulated by niacin in BMEC; other prostaglandin E2 synthesis-related genes stayed unaltered in BMEC and BEND. The mRNA abundance of HSP70, COX-1, COX-2, and mPGES-1 were elevated in niacin-treated MAC-T. During heat stress, niacin increased mRNA levels of HSP70 and HSP27 in MAC-T and HSP27 in BEND, but decreased HSP70 in BMEC. Although mPGES-2 was stimulated by niacin in BEND, the mRNA expression of prostaglandin E2 synthesis-related genes were consistent with neither HSP70 nor HSP27 expression patterns in niacin-treated BMEC and MAC-T. These data suggest that the effects of niacin on heat shock protein expression and prostaglandin E2 synthesis were not well coupled in these cells. Finally, we tested the effects of betaine treatment on viability and apoptosis in BMEC. Compared with control cultures, viability was higher in betaine-treated cells at 8 h under thermoneutral conditions and at 16 h in heat stress, and apoptotic rates were lower at 8 h. Our data support a dual role for niacin in regulating heat shock protein expression in normal and heat-shocked cells derived from mammary and uterine tissues, and positive effects of betaine in regulating mammary cell viability during heat stress.

Orexin-A Rescues Chronic Copper-Dependent Behavioral and HSP90 Transcriptional Alterations in the Ornate Wrasse Brain.

Recently, orexin (ORX)ergic system has gained great attention for its major neuroregulatory role on fish motor, circadian, feeding activities and above all during water toxic conditions. Fish are particularly sensitive to acute sublethal copper (Cu) concentrations while little is known about neurobehavioral data after chronic Cu exposure. For this work, the marine teleost ornate wrasse (Thalassoma pavo) was monitored for 21days during exposure to a relatively low CuCl2 concentration (0.25mg/l). In particular, at 1day fish displayed moderate reductions (-32%) of feeding behaviors versus controls that decreased up to -94% at 21days. Swimming activities were also moderately (-44%) reduced at 1day, which diminished even further (~ -75%) from 7 to 21days. The lethargic attitudes seemed to be frequently interrupted by aquatic surface respiration and abnormal motor behaviors such as rapid and abrupt changes of direction. Contextually, an upregulatory trend of HSP90 mRNAs occurred especially in the lateral part of the dorsal telencephalon (Dl; +72%), medial preglomerular nucleus (NPGm; +87%), and torus longitudinalis (TLo; +108%). Moreover, some of these nuclei displayed extensive argyrophilic signals that are typical of notable neurodegenerative events. Interestingly, a single intraperitoneal administration of ORX-A (10ng/g of body weight) at 21days attenuated not only some behavioral impairments but also HSP90 mRNA levels and neurodegenerative events. Behavioral indications of the present results tend to underlie the valuable role of HSP90 transcript together with the ORXergic system as probable rescuing factors operating in marine fish during persistent adverse environmental states with eventual bearings on human health issues.

T-helper 2 cytokine-induced heat shock protein 70 secretion and its potential association with allergic rhinitis.

Various inflammatory mediators have been found to be involved in the pathogenesis of allergic disease (AR). The role of heat shock proteins in AR has not been studied. The aim of this study was to investigate the levels of heat shock protein 70 (Hsp70) in the nasal lavage fluids of AR patients and controls to elucidate the role of Hsp70 in the pathogenesis of AR. Using an enzyme-linked immunosorbent assay, the levels of Hsp70, Hsp90, interleukin (IL)-4, IL-13, and IL-8 in nasal lavage fluid from patients were measured and statistically analyzed. Primary human nasal epithelial cells were culturedin vitroand T-helper 2 (Th2) cytokines (IL-4, IL-13) were added to the culture medium. We evaluated the mRNA and protein expression levels of Hsp70 using real-time polymerase chain reaction and western blot assay. Hsp70 was easily detected in nasal lavage fluid and the levels of Hsp70 were higher in AR patients than in healthy controls. Other clinical characteristics of subjects were not significantly associated with Hsp70 levels. Furthermore, we found that treatment with IL-4 and IL-13 induced the secretion of Hsp70 in human nasal epithelial cells. We found that Hsp70 was abundant and positively detected in nasal lavage fluid samples from all subjects, and that Hsp70 levels were significantly higher in AR patients. We demonstrated, bothin vivoandin vitro,that Hsp70 could play an important role in the pathogenesis of AR, and we suggest that Hsp70 can be used as a disease marker for AR.

Experimental Study on the Protection of Agrimony Extracts from Different Extracting Methods against Cerebral Ischemia-Reperfusion Injury.

Objective To study the protective effect of agrimony extracts from different extracting methods on cerebral ischemia-reperfusion injury in rats, in order to optimize the extraction scheme of agrimony. Methods Male rats were randomly assigned into seven groups: 1. Sham-operated group, 2. Untreated MCAO group (MCAO), 3. Petroleum ether extract of Agrimonia pilosa treated MCAO group (PEA), 4. Ethyl acetate extract of Agrimonia pilosa treated MCAO group (EAEA), 5. Ethanol extract of Agrimonia pilosa treated MCAO group (EEA), 6. Water extract of Agrimonia pilosa treated MCAO group (WEA), 7. Nimodipine treated MCAO group (NP). Intragastrical drug administration (i.g) was performed at 0 and 6 hours after MCAO. Neurological function tests were performed after reperfusion for 24 hours, then the brain was removed for the evaluations of the cerebral infarction volume (percentage of total brain volume) by immunohistochemistry, histological changes (hematoxylin-eosin staining), Na+/K+-ATPase, Ca2+-ATPase (modified method of Svoboda and Mosinger), mRNA expression of Tumor suppressor gene (P53) and hot shock protein (HSP70) (quantitative real-time PCR). Results The neurological function of MCAO group had significantly higher scores than the sham group (P<0.01). The WEA group showed a significantly lower neurological score than the MCAO group (P<0.05), indicating the protective effect of WEA on neurological deficits. The mean infarction volumes of WEA (13.5±6.6%, F=4.75, P<0.01), EEA (19.90±6.90%, F=5.23, P<0.01), PEA (20.40±5.30%, F=4.68, P<0.01) and EAEA (22.50±10.50%, F=6.25, P<0.05) group were all significantly smaller than that of MCAO group (29.40±6.50%). HE staining demonstrated that, compared to the treated groups, the infarcted cerebral tissue of MCAO group had more swelling neural cells, lighter stained nucleus, fewer and irregularly distributed neurons. The activity of Na+/K+-ATPase and Ca2+-ATPase reduced in the MCAO group (3.67±0.48 U/mg, 1.28±0.26 U/mg, respectively), and were significantly higher in WEA group (7.56±0.85 U/mg, F=12.65, P=0.010; 3.59±0.22 U/mg, F=8.32, P=0.041, respectively). The MCAO group showed significantly elevated P53 and HSP70 mRNA expressions compared to the sham group (P<0.01, P<0.05). P53 mRNA expressions in Agrimony extracts treated groups were significantly lower than that of the MCAO group (all P<0.01), with the WEA group showing the greatest difference from MCAO group. The HSP70 mRNA level of the treated groups were not significantly different from that of the MCAO group. Conclusions Treatment using water extracts of agrimony can promote the best functional and metabolic recovery for rat model of cerebral ischemia-reperfusion injury, which maybe relate with the upregulation of energy metabolism in nerve cells after MCAO.

Cardioprotection of exercise preconditioning involving heat shock protein 70 and concurrent autophagy: a potential chaperone-assisted selective macroautophagy effect.

It has been confirmed that exercise preconditioning (EP) has a protective effect on acute cardiovascular stress. However, how Hsp70 participates in EP-induced cardioprotection is unknown. EP may involve Hsp70 to repair unfolded proteins or may also stabilize the function of the endoplasmic reticulum via Hsp70-related autophagy to work on a protective formation. Our EP protocol involves four periods of 10min running with 10min recovery intervals. We added a period of exhaustive running to test this protective effect, using histology and molecular biotechnology methods to detect related markers. EP provided cardioprotection at its early and late phases against exhaustive exercise-induced ischemic myocardial injury. Results showed that Hsp70 co-chaperone protein BAG3, ubiquitin adaptor p62 and critical autophagy protein LC3 were significantly upregulated at the early phase. Meanwhile, Hsp70, Hsp70/BAG3 co-localization extent, LC31 and LC3II were significantly upregulated at the late phase. Hsp70 mRNA levels and LC3II/I ratios were also consistent with the extent of myocardial injury following exhaustive exercise. Hsp70 increase was delayed relative to BAG3 and p62 after EP, indicating a pre-synthesized phenomenon of BAG3 and p62 for chaperone-assisted selective autophagy (CASA). The decreased Hsp70, BAG3 and p62 levels and increased Hsp70/BAG3 co-localization extent and LC3 levels induced by exhaustive exercise after EP suggest that EP-induced cardioprotection might associate with CASA. Hsp70 has a cardioprotective role and has a closer link with CASA in LEP. Additionally, EP may not cause exhaustion-dependent excessive autophagy regulation. Collectively, during early and late EP, CASA potentially plays different roles in cardioprotection.

Different Relationship between hsp70 mRNA and hsp70 Levels in the Heat Shock Response of Two Salmonids with Dissimilar Temperature Preference

The heat shock response (HSR) refers to the rapid production of heat shock proteins (hsps) in response to a sudden increase in temperature. Its regulation by heat shock factors is a good example of how gene expression is transcriptionally regulated by environmental stresses. In contrast, little is known about post-transcriptional regulation of the response. The heat shock response is often used to characterize the temperature tolerance of species with the rationale that whenever the response sets on, a species is approaching its lethal temperature. It has commonly been considered that an increase in hsp mRNA gives an accurate indication that the same happens to the protein level, but this need not be the case. With climate change, understanding the effects of temperature on gene expression of especially polar organisms has become imperative to evaluate how both biodiversity and commercially important species respond, since temperature increases are expected to be largest in polar areas. Here we studied the HSR of two phylogenetically related Arctic species, which differ in their temperature tolerance with Arctic charr having lower maximally tolerated temperature than Atlantic salmon. Arctic charr acclimated to 15°C and exposed to 7°C temperature increase for 30 min showed both an increase in hsp70 mRNA and hsp70 whereas in salmon only hsp70 mRNA increased. Our results indicate that the temperature for transcriptional induction of hsp can be different from the one required for a measurable change in inducible hsp level. The species with lower temperature tolerance, Arctic charr, are experiencing temperature stress already at the higher acclimation temperature, 15°C, as their hsp70 mRNA and hsp70 levels were higher, and they grow less than fish at 8°C (whereas for salmon the opposite is true). Consequently, charr experience more drastic heat shock than salmon. Although further studies are needed to establish the temperature range and length of exposure where hsp mRNA and hsp level are disconnected, the observation suggests that by measuring both hsp mRNA and hsp level, one can evaluate if a species is approaching the higher end of its temperature tolerance, and thus evaluate the vulnerability of an organism to the challenges imposed by elevated water temperature.