HSP70 mRNA Expression Levels Research Articles

Assessment of the therapeutic potential of Hsp70 activator against prion diseases using in vitro and in vivo models.

Prion diseases are deadly neurodegenerative disorders in both animals and humans, causing the destruction of neural tissue and inducing behavioral manifestations. Heat shock proteins (Hsps), act as molecular chaperones by supporting the appropriate folding of proteins and eliminating the misfolded proteins as well as playing a vital role in cell signaling transduction, cell cycle, and apoptosis control. SW02 is a potent activator of Hsp 70kDa (Hsp70). In the current study, the protective effects of SW02 against prion protein 106-126 (PrP106-126)-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) were investigated. In addition, the therapeutic effects of SW02 in ME7 scrapie-infected mice were evaluated. The results showed that SW02 treatment significantly increased Hsp70 mRNA expression levels and Hsp70 ATPase activity (p < 0.01). SW02 also significantly inhibited cytotoxicity and apoptosis induced by PrP106-126 (p < 0.01) and promoted neurite extension. In vivo, intraperitoneal administration of SW02 did not show a statistically significant difference in survival time (p = 0.16); however, the SW02-treated group exhibited a longer survival time of 223.6 ± 6.0days compared with the untreated control group survival time of 217.6 ± 5.4days. In addition, SW02 reduced the PrPSc accumulation in ME7 scrapie-infected mice at 5months post-injection (p < 0.05). A significant difference was not observed in GFAP expression, an astrocyte marker, between the treated and untreated groups. In conclusion, the potential therapeutic role of the Hsp70 activator SW02 was determined in the present study and may be a novel and effective drug to mitigate the pathologies of prion diseases and other neurodegenerative diseases. Further studies using a combination of two pharmacological activators of Hsp70 are required to maximize the effectiveness of each intervention.

Effects of high-molecular-weight polyvinyl chloride on Xenopus laevis adults and embryos: the mRNA expression profiles of Myf5, Esr1, Bmp4, Pax6, and Hsp70 genes during early embryonic development.

Microplastics and associated adverse effects have been on the global agenda in recent years. Because of its importance as a model organism for studies on developmental biology, Xenopus laevis has been chosen as the study animal in in vitro teratogenesis studies. FETAX test uses early-stage embryos of X. laevis to measure the potential of substances to cause mortality, malformation, and growth inhibition in developing embryos. The aim of this study was to examine the effects of high molecular weight polyvinyl chloride (HMW-PVC) on parental X. laevis frogs and their embryos using the FETAX test. To this purpose, a HMW-PVC dose of 1% of body weight/twice each week was provided to frogs by oral gavage throughout 6 weeks. After the procedure, oocytes and sperms of HMW-PVC-exposed frogs were fertilized and FETAX was applied to selected embryos. After the completion of a 96-h incubation period, tadpoles were examined, their live/dead status were determined, their lengths were measured, and their anomalies were photographed. Besides, excised organs of the parental frogs were referred to histopathology examination. On the other hand, the mRNA expression levels of Hsp70, Myf5, Bmp4, Pax6, and Esr1 genes were determined by applying real-time quantitative PCR method to cDNA which was synthesized from the total RNA of embryos. The results showed that treatment with HMW-PVC dose of 1% of body weight/twice each week caused malformations and decreased viability. Hsp70 and Pax6 gene expression levels significantly decreased in all assay groups, as compared with controls. Lung and intestine tissues showed normal appearance in histopatological examination. Further research is required to explain the whole effects of HMW-PVC exposure on X. laevis embryos.

HSP70-mediated neuroprotection by combined treatment of valproic acid with hypothermia in a rat asphyxial cardiac arrest model.

It has been reported that valproic acid (VPA) combined with therapeutic hypothermia can improve survival and neurologic outcomes in a rat asphyxial cardiac arrest model. However, neuroprotective mechanisms of such combined treatment of valproic acid with hypothermia remains unclear. We hypothesized that epigenetic regulation of HSP70 by histone acetylation could increase HSP70-mediated neuroprotection suppressed under hypothermia. Male Sprague-Dawley rats that achieved return of spontaneous circulation (ROSC) from asphyxial cardiac arrest were randomized to four groups: normothermia (37°C ± 1°C), hypothermia (33°C ± 1°C), normothermia + VPA (300 mg/kg IV initiated 5 minutes post-ROSC and infused over 20 min), and hypothermia + VPA. Three hours after ROSC, acetyl-histone H3 was highly expressed in VPA-administered groups (normothermia + VPA, hypothermia + VPA). Four hours after ROSC, HSP70 mRNA expression levels were significantly higher in normothermic groups (normothermia, normothermia + VPA) than in hypothermic groups (hypothermia, hypothermia + VPA). The hypothermia + VPA group showed significantly higher HSP70 mRNA expression than the hypothermia group. Similarly, at five hours after ROSC, HSP70 protein levels were significantly higher in normothermic groups than in hypothermic groups. HSP70 levels were significantly higher in the hypothermia + VPA group than in the hypothermia group. Only the hypothermia + VPA group showed significantly attenuated cleaved caspase-9 levels than the normothermia group. Hypothermia can attenuate the expression of HSP70 at transcriptional level. However, VPA administration can induce hyperacetylation of histone H3, leading to epigenetic transcriptional activation of HSP70 even in a hypothermic status. Combining VPA treatment with hypothermia may compensate for reduced activation of HSP70-mediated anti-apoptotic pathway.

Effects of Low Temperature Stress on the Morphology and hsp70 and hsp90 Gene Expression of Phascolosoma esculenta

Phascolosoma esculenta is an intertidal organism that has recently attracted attention because of its ability to survive at relatively low temperatures. However, the gene regulation in P. esculenta in relation to its response to low temperatures is unclear. To explore the low temperature adaptability of P. esculenta, this study analyzed the changes in the morphology and hsp70 and hsp90 gene expression of P. esculenta exposed to a low temperature gradient. At 5°C, P. esculenta stretched and softened, and some individuals moved apart from the group. Histological analysis revealed cuticle breaches, myofiber scattering, disruption of the body wall, and epithelial layer dispersion and muscle fiber rupturing in the nephridium. Furthermore, the mRNA expression levels of hsp70 and hsp90 increased under acute low temperature stress, suggesting that these genes function in low temperature tolerance. Overall, low temperature stress causes morphological changes and histological damage in P. esculenta, and hsp70 and hsp90 potentially function in the low temperature adaptability of P. esculenta. Our results provide new insights into the adaptive strategies of P. esculenta under low temperature environments.

Acute Heat Stress Induces the Differential Expression of Heat Shock Proteins in Different Sections of the Small Intestine of Chickens Based on Exposure Duration.

Simple SummaryThe small intestine is a pivotal organ in the alimentary tract of chickens. Approximately 90% of digestion and absorption occurs in the small intestine. The expression of heat shock proteins (HSPs) in different sections of the small intestine upon varying durations of heat exposure is unclear. In this study, we evaluated the protein and mRNA expression levels of HSP70, HSP60, and HSP47 in the duodenum, jejunum, and ileum, on acute heat exposure. The protein and gene expression of the HSPs in the different sections of the small intestine of chicken increased at different stages of acute heat stress and were reduced after the induction of heat tolerance. However, acute heat stress damaged the integrity of the different sections of the small intestine and liver in chickens.In this study, we examined the protein and gene expression of heat shock proteins (HSPs) in different sections of the small intestine of chickens. In total, 300 one-day-old Ross 308 broiler chicks were randomly allocated to the control and treatment groups. The treatment group was divided into four subgroups, according to the duration of acute heat exposure (3, 6, 12, and 24 h). The influence of heat stress on the protein and gene expression of HSP70, HSP60, and HSP47 in different sections of the small intestine of chickens was determined. The protein expression of HSP70 and HSP60 was significantly higher at 6 h in the duodenum and jejunum and 12 h in the ileum. The HSP47 protein expression was significantly higher at 3 h in the duodenum and ileum and at 6 h in the jejunum. The gene expression levels of HSP70, HSP60, and HSP47 were significantly higher at the 3 h treatment group than the control group in the duodenum, jejunum, and ileum. The glutamate pyruvate transaminase and glutamate oxaloacetate transaminase levels were significantly higher at 12 and 24 h in the serum of the blood. Acute heat stress affected the expression of intestinal proteins and genes in chickens, until the induction of heat tolerance.

Effect of Glutamine on Antioxidant Capacity and Lipid Peroxidation in the Breast Muscle of Heat-stressed Broilers via Antioxidant Genes and HSP70 Pathway.

Simple SummaryBroilers are heat stress sensitive animals. Heat stress can reduce the meat quality and increase the oxidative stress of broilers. The aim of this study was to investigate the effect of dietary supplementation with Glutamine (Gln) on meat quality, antioxidant capacity, lipid peroxidation, antioxidant genes, and the HSP70 gene and protein expression in broilers exposed to cyclic heat stress. Results showed that Gln improved the meat quality in heat-stressed broilers. In conclusion, our study suggested that dietary Gln supplementation alleviated antioxidant capacity and lipid peroxidation in the breast muscle of heat-stressed broilers by antioxidant genes and the HSP70 pathway.This study investigated whether Glutamine (Gln) could be used as an additive to improve antioxidant capacity in the breast muscle of heat-stressed broilers. Two hundred and forty 22-day-old Arbor Acres broilers in the G1, G2, G3, and G4 groups (n = 60 each) were housed in a cyclic hot environment and fed the basal diet with 0%, 0.5%, 1.0%, and 1.5% Gln, respectively. Compared with the G1 group, dietary 1.5% Gln increased (p < 0.05) pH and b* values, but decreased (p < 0.05) L* cooking loss, drip loss, and water loss rate in breast meat of heat-stressed broilers. Malondialdehyde levels in the breast muscle were lower (p < 0.05) in 1.0% and 1.5% Gln groups than that of the heat-stress group. Compared with the G1 group, dietary 1.5% Gln increased (p < 0.05) catalase (CAT), glutathione, glutathione peroxidase (GSH-Px,) and total antioxidant capacity in the breast muscle of heat-stressed broilers. Furthermore, the CAT, GSH-Px, HSP70 mRNA expression levels, and HSP70 protein expression levels were increased (p < 0.05) in the G3 and G4 groups compared with the G1 group. In sum, Gln alleviated antioxidant capacity and lipid peroxidation in the breast muscle of heat-stressed broilers through antioxidant genes and HSP70 pathways.

Dietary soybean antigen impairs growth and health through stress-induced non-specific immune responses in Pacific white shrimp, Litopenaeus vannamei

In order to compare the effect of substituting fish meal with fermented soybean meal and soybean meal, and confirmed whether the benefit from the two feed materials was related to the content of inclusive soybean antigen protein, two experiments were designed. In experiment 1, one of the two practical diets contained 24.9% soybean meal (SBM), the other one containing 8% fermented soybean meal and 16.95% soybean meal (FSBM); in experiment 2, two semi-purified diets were included with high antigen protein (SPD1) and low antigen protein (SPD2) approximately equal to SBM and FSBM group respectively in experiment 1. Diets were fed to Litopenaeus vannamei (initial weight: 7.48 ± 0.24 g) for 60 days. The results showed that in experiment 1, growth performance was not significantly different between two groups, the enzyme activity (AKP, AST, ALT, SOD and LZM) and mRNA expression levels of TLR, LZM, IMD and HSP70 were significantly higher in the SBM group; In experiment 2, weight gain and specific growth rate were significantly higher in the SPD2 group, while higher activities of AKP, ALT and LZM, lower expression levels of TLRmRNA, LZMmRNA and IMDmRNA and higher expression level of HSP70mRNA were found in SPD1 group. These results implied SBM was more likely to induce stress reaction in shrimp than FSBM, which were closely related to the antigen protein in SBM.

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.

Effects of Taurine on ACE, ACE2 and HSP70 Expression of Hypothalamic-Pituitary-Adrenal Axis in Stress-Induced Hypertensive Rats.

The experiment was to elucidate protective mechanism of taurine against stress-induced hypertension. Thirty two male Wistar rats were randomly divided into four groups. Normal control group and stress control group were intragastrically administered saline; β-alanine stress group were fed with β-alanine (200 mg/kg/day) and taurine stress group with taurine (200 mg/kg/day). The hypertensive model was established by giving rats stress for 3 weeks.Results showed that significant expression levels of angiotensin converting enzyme (ACE) in the hypothalamus, pituitary and adrenal were observed in β-alanine stress group and stress control group (P < 0.05), but significant mRNA expression levels of angiotensin-converting enzyme 2 (ACE2) in taurine stress group and normal control group (P < 0.05). All the groups showed no significant differences in HSP70 mRNA expression levels in hypothalamus (P > 0.05), while taurine stress group exhibited the highest HSP70 mRNA expression levels both in pituitary and in adrenal (P < 0.05). It was also found that β-alanine stress group and stress control group had significantly higher protein expression levels of ACE in hypothalamus, pituitary and adrenal (P < 0.05), but significantly lower protein expression of ACE2 compared to taurine stress group and control groups (P < 0.05). The results indicated that taurine regulated the hypothalamus pituitary adrenal (HPA) axis of the renin-angiotensin-aldosterone system (RAAS) by inhibiting ACE gene and protein expressions and promoting ACE2 and HSP70 protein expressions, thereby contributing to the prevention of stress-induced hypertension.

Adequate dietary protein level enhances stress resistance and immune status of blunt snout bream (Megalobrama amblycephalaYih, 1955)

This study hypothesized that an optimum dietary protein level might play an important role in improving stress tolerance, enhancing an immune function, and ultimately minimizing temperature stress. For this purpose, the present study conducted a 10-week feeding trial followed by a 7-day stress experiment to evaluate the effects of dietary protein levels (28%–36%) on the physiological performances and hepatic Hsp70 gene expression in blunt snout bream Megalobrama amblycephala fry under a high temperature challenge. Fish fry (initial weight, 16.08 ± 0.03 g, n = 25) were fed with their respective diets to apparent satiation, and samples taken once before stress and four times during high temperature stress days (0.125, 0.5, 2 and 7 days). Serum total protein and cholesterol contents before stress were affected by dietary protein levels; during stress these parameters showed no significant changes up until day 2, showing some changes thereafter. Regardless of 0.125, 0.5, 2 or 7 days during stress, cortisol and aspartate aminotransferase levels in the group fed 32% dietary protein were significantly lower than in the other groups. At 0.125, 0.5 or 2 days during stress, the complement component 3 (C3) and 4 (C4) levels increased significantly up to certain levels, declining thereafter. The expression level of Hsp70 mRNA before stress was not affected by dietary protein levels, but increased significantly from 0.125 to 0.5 days during stress, and was reduced thereafter. Overall, low cortisol and high C3, C4 and Hsp70 mRNA levels were found in the group fed 32% protein throughout the stress experiment, which indicated that an adequate protein level enhances stress resistance and immunity in fish.

Triptolide reduces the viability of osteosarcoma cells by reducing MKP-1 and Hsp70 expression.

Osteosarcoma is the most common type of malignant bone tumor found in adolescents and young adults. The aim of the present study was to determine whether triptolide, a diterpene epoxide extracted from the Tripterygium plant, was able effectively decrease the viability of osteosarcoma cells. The underlying molecular mechanisms are also investigated. The human osteosarcoma cell lines U-2 OS and MG-63 were used in this study. The U-2 OS and MG-63 cells were treated with 0, 5, 10, 25 or 50 nM triptolide. Cells treated with dimethyl sulfoxide only were used as the no drug treatment control. A commercial MTT kit was used to determine the effects of triptolide on cells. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is frequently overexpressed in tumor tissues, possibly related to the failure of a number of chemotherapeutics. Heat shock protein 70 (Hsp70) is a chaperone molecule that is able to increase drug resistance. The protein expression levels of MKP-1 and Hsp70 were determined using western blot analysis. The results indicate that triptolide effectively reduced the viability of the osteosarcoma cells. Furthermore, triptolide was found to effectively reduce MKP-1 expression and Hsp70 levels. Further analysis showed that triptolide reduced MKP-1 mRNA expression in the U-2 OS and MG-63 cells. Triptolide reduced Hsp70 mRNA expression levels in U-2 OS and MG-63 cells. These results suggest that triptolide effectively decreases the viability of osteosarcoma cells. These effects may be associated with the decreased expression of MKP-1 and Hsp70 levels. These results suggest that triptolide may be used in the treatments of osteosarcoma.

Toxicity of Mycotoxins from Contaminated Corn with or withoutYeast Cell Wall Adsorbent on Broiler Chickens.

This study investigated the effects of feeds naturally contaminated with mycotoxins on growth performance, serum biochemical parameters, carcass traits, and splenic heat shock protein 70 (Hsp70) mRNA expression levels in broiler chickens. The efficacy of yeast cell wall (YCW) adsorbent in preventing mycotoxicosis was also evaluated. Three hundred 1-d-old Arbor Acres broiler chicks were randomly allotted to 3 treatments in completely randomized design for 42 d. Each treatment group had 5 replicate pens with 20 birds. The treatments were as follows: i) basal diet (control), ii) naturally contaminated diet (NCD), and iii) NCD+0.2% YCW adsorbent (NCDD). The NCD decreased average daily gain (ADG) (p<0.01) of 0 to 21 d, 22 to 42 d, and 0 to 42 d, and increased feed conversion ratio (p<0.01) of 22 to 42 d and 0 to 42 d. Both the breast meat percentage and thigh meat percentage of the NCD group were significantly higher (p<0.01) than that of the control group on d 21. The NCD group showed significantly increased levels of triglycerides (p<0.05) and cholesterol (p<0.05) on both d 21 and d 42 compared to the control group. However, the NCD significantly reduced (p<0.01) the high-density lipoprotein (HDL) on d 42 compared to controls. Compared with the NCD, supplementation with YCW significantly improved (p<0.01) the ADG of 0 to 21 d and 0 to 42 d, and increased (p<0.01) concentrations of HDL on d 42, and on d 21, and triglycerides (p<0.05) on d 21 and d 42. Supplementation with YCW reduced (p<0.01) the breast meat percentage, the thigh meat percentage, the concentrations of cholesterol (p<0.01) and the low-density lipoprotein (p<0.05) on d 21, and improved (p<0.01) the splenic Hsp70 mRNA expression levels compared with the NCD group. The results of this study indicated that feeding NCD for 42 d had adverse effects on broiler chickens, and that YCW might be beneficial in counteracting the effects of mycotoxins.

Metabolomic responses of sea cucumber Apostichopus japonicus to thermal stresses

Elevated water temperature is generally considered as one of the most widespread and problematic stressors on Apostichopus japonicus farming. To date, however, little is known about the intrinsic mechanisms of A. japonicus to cope with the rising temperature at the metabolic level. In the present study, the metabolomic responses were characterized in the muscles of A. japonicus at two thermal points, 20 °C and 25 °C by 1H NMR-based metabolomics. The results showed that both temperatures disturbed energy metabolism and induced osmotic regulation based on the metabolic biomarkers such as elevated threonine, alanine, arginine, glutamate, tyrosine, histidine, glycine, fumarate, glucose, and AMP and decreased ATP, aspartate, hypotaurine, and choline after exposure for 0 days and 3 days. At 7 days of thermal-stress, several metabolites were similarly altered including the increased glutamine, glycine, glucose, AMP, tyrosine, and histidine and the decreased ATP at 20 °C. Surprisingly, a large amount of amino acids (valine, leucine, isoleucine, threonine, arginine, glutamate, phenylalanine) markedly declined at 25 °C. In addition, the mRNA expression levels of HSP70 and ATP synthase as well as SOD activity and MDA content were also investigated, and their expression levels were significantly altered at different exposure conditions. Our present results provided new insights into thermal-stressed effects on A. japonicus at metabolic responses.

Multiple effects of repetitive transcranial magnetic stimulation on neuropsychiatric disorders

Repetitive transcranial magnetic stimulation (rTMS) is a new tool that has been used for the treatment of patients with neuropsychiatric disorders. However, the mechanisms underlying the effects of rTMS are still unclear. We analyzed the changes in mRNA expression in mouse brain that occurred after rTMS with an Affymetrix GeneChip. Following 20days of rTMS, many genes were differentially expressed in the mouse brain. Downregulation of Period 2 and 3 mRNA expression levels and a subsequent decrease in food and water intake were observed. HSP70 mRNA expression levels were upregulated after transient and chronic rTMS. In N2A 150Q cells, an upregulation of HSP70 mRNA and protein levels and subsequent cell-protective effects were observed after chronic rTMS. In addition, dopamine receptor 2 mRNA expression levels were downregulated, and a subsequent decrease in the binding of [3H]raclopride was observed. These results indicated that the modulation of several genes may be involved in the therapeutic mechanisms of chronic rTMS for patients with neuropsychiatric disorders.

Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress

In order to study the effects of dietary emodin, high-dose vitamin C (Vc) and their combination on growth of Wuchang bream (Megalobrama amblycephala Y.) and its resistance to high temperature stress, 1200 healthy Wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. After feeding for 60 days, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to heat stress of 34 °C. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that before stress, compared with the control, the weight gain (WG) and specific growth rate (SGR), serum total protein (TP), lysozyme (LSZ), and alkaline phosphatase (ALP) levels, liver superoxide dismutase (SOD) activity and expression level of HSP70 mRNA significantly increased in emodin and Vc groups while feed conversion rate (FCR), serum cortisol (COR), triglyceride (TG) and liver malondialdehyde (MDA) contents decreased (P < 0.05); liver catalase (CAT) activity also significantly increased in emodin group (P < 0.05). Although serum TP, LSZ, and liver HSP70 mRNA levels significantly increased and liver MDA level decreased in combination group (P < 0.05), no synergism was observed.After heat stress, compared with the control, the serum TP, LSZ, ALP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P < 0.05) while the difference between the combination and control groups was not significant (P > 0.05). Thus, the basal diet supplemented with 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, reduce FCR, increase non-specific immunity of fish, antioxidant capacity, and two HSP70s mRNA expression levels, and enhance resistance to heat stress in fish. However, the combination of emodin and high-dose Vc showed no better effect.

Effect of ulinastatin on the expression of heat shock protein 70 and NF-kappaB in lung tissue in rats with paraquat poisoned

To observe the expression levels of heat shock protein 70 (hsp70) and NF-kappaB p65 mRNA in lung tissue of acute paraquat (PQ) poisoning rats, and intervention effects of ulinastatin (UTI). Seventy-two Sprague-Dawley (SD) rats were randomly divided into three groups: PQ poisoning group, UTI group and control group. The rats were exposed intragastrically to PQ at the dose of 80 mg/kg to establish a model of the rat acute lung injury. The UTI group was intervened by peritoneal injection with 10000 U/kg UTI in 30 minutes. On the 12, 24, 48, 72 h after exposure, myeloperoxidase (MPO) activity in lung tissue were detected. The expression of the NF-kappaB p65 mRNA and hsp70 mRNA in lung tissue was detected by the reverse transcription-PCR (RT-PCR). The lung pathological changes of rats were observed. The degree of lung injury in PQ group and UTI group was higher than that in control group. But in UTI group the degree of lung injury was lower than PQ group. MPO activity in the lung tissues in PQ group was (31.72 +/- 6.42), (56.23 +/- 8.63), (87.21 +/- 10.02) and (107.21 +/- 13.52) micro/g in 12, 24, 48 and 72 h, respectively which was significantly higher than that [(11.38 +/- 1.25) micro/g] in control group (P < 0.01). MPO activity in the lung tissues in UTI group was (15.65 +/- 3.21), (35.98 +/- 5.74), (59.33 +/- 9.65) and (71.25 +/- 10.58) micro/g in 12, 24, 48 and 72 h, respectively which was significantly lower than those in PQ group (P < 0.01). The expression levels of NF-kappaB p65 mRNA of lung tissues in UTI group in 12, 24, 48 and 72 h were 0.3288 +/- 0.0147, 0.5337 +/- 0.0328, 0.7357 +/- 0.0424 and 0.7547 +/- 0.0905, respectively, which were significantly lower that those (0.4185 +/- 0.0294, 0.8532 +/- 0.0841, 0.9554 +/- 0.0975 and 1.0094 +/- 0.0703) in PQ group (P < 0.01). hsp70 mRNA expression levels in 12, 24, 48 and 72 h of the UTI group were 0.5193 +/- 0.0254, 0.8289 +/- 0.0606, 0.7566 +/- 0.0277 and 0.4873 +/- 0.0105, respectively, which were significantly higher than those (0.3897 +/- 0.0125, 0.5904 +/- 0.0186, 0.4007 +/- 0.0237 and 0.2293 +/- 0.0137) in PQ group (P < 0.01). The expression levels of hsp70 mRNA and NF-kappaB p65 mRNA of rats after intoxication increased significantly. UTI can protect the lung tissues by elevating the expression of hsp70 and reducing the expression of NF-kappaB in the lung tissues of rats with acute paraquat poisoning.

Using double-stranded RNA to explore the role of heat shock protein genes in heat tolerance in Bemisia tabaci (Gennadius)

The whitefly, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) biotype B, is one of the most destructive invasive pests of field and glasshouse crops, and has a high tolerance to heat. Our previous work found that whitefly females are more heat tolerant than males. In the present study, real-time PCR and double-stranded RNA (dsRNA) methods were used to explore the role of heat shock protein (Hsp) genes in whitefly of both sexes; this provided further evidence of the mechanism underlying the differential heat tolerance abilities of females and males. The results showed that both hsp23 and hsp70 mRNA expression levels were higher in females than in males from 37.5 to 42°C, while at the extreme temperature of 44°C the hsp70 mRNA level was higher in males than in females. There was no significant difference in hsp90 mRNA expression between females and males under heat shock conditions. Furthermore, the survival rate of females fed hsp23 or hsp70 dsRNA significantly decreased following heat shock at 44°C for 1 h, but male survival rate was not significantly affected. Additionally, the survival rate of both females and males showed no significant change after they were fed with hsp90 dsRNA. Collectively, the present study shows that the optimum mRNA expression of Hsp genes in females promotes a higher survival rate under heat shock conditions; hsp23 and hsp70 play a key role for heat tolerance in females but not in males, and hsp90 shows no significant role in heat tolerance in either females or males. Further, our study indicates that feeding with dsRNA is an effective method by which to study gene function, and the simplicity of this approach opens the way for further research on gene function in different sexes and diverse groups of species.

Impaired avoidance learning and increased hsp70 mRNA expression in pentylenetetrazol‐treated zebrafish

The effects of pentylenetetrazol (PTZ), a GABA receptor antagonist, were studied on passive avoidance learning and expression of heat shock protein 70 (hsp70), neuroglobin, and fatty acid binding protein‐7 (fabp‐7) genes. Zebrafish were trained to stay in a dark compartment to avoid a weight dropping in an acryl shuttle box with a central sliding door. In two training sessions of 2 h interval, each consisting of 3 trials, the crossing time was significantly increased from 43.2±14.4s to 149.3±38.5s in the first training session and remained 116.1 ±36.0 s in the first trial of the second training session in the control. In zebrafish treated with PTZ before the first training session, the crossing time was significantly increased neither in the first nor in the second training session. However, the increased crossing time was maintained in the second training session when 10 mM PTZ was treated three times for 10 min at 30 min intervals between the first and second training session. Quantitative real‐time PCR showed that expression level of hsp70 mRNA increased two to eight fold over that of control in the brain at 0–24 h after termination of PTZ treatment. No change in expression of neuroglobin and fabp‐7 mRNA was shown in PTZ‐treated zebrafish. Our studies suggest that PTZ impairs learning ability in avoidance response and also modifies expression of genes related to the neuroprotection.

Polymorphism of the Promoter Region of Hsp70 Gene and Its Relationship with the Expression of HSP70mRNA, HSF1mRNA, Bcl-2mrna and Bax-AMrna in Lymphocytes in Peripheral Blood of Heat Shocked Dairy Cows

The blood samples were collected from dairy cows at the same milking stage. The single-strand conformation polymorphism (PCR-SSCP) method was used to analyze for polymorphism at the 5'-flanking region of the hsp70 gene. The mRNA expression levels of HSP70,HSF1, Bcl-2 and Bax-α at different daily-mean-temperature were analyzed by relative quantitative RT- PCR. The DNA content, cell phase and the ratio of apoptosis of lymphocytes in peripheral blood of dairy cattle at different daily-mean- temperature were determined by FCM. The PCR-SSCP products of primer pair 1 showed polymorphisms and could be divided into four genotypes: aa, ab, ac, cc, with the cis-acting element (CCAAT box) included. Mutations in the hsp70 5'-flanking region (468-752 bp) had different effects on mRNA expression of HSP70, HSF1, Bcl-2 and Bax-α. The ac genotypic cows showed higher expressions of HSP70mRNA, HSF1mRNA and Bcl-2mRNA/Bax-αmRNA and lower ratio of apoptosis. These mutation sites can be used as molecular genetic markers to assist selection for anti-heat stress cows. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 5 : 734-740)