Heat Shock Protein 70 mRNA Research Articles

Effects of chlorogenic acid on productive and reproductive performances, egg quality, antioxidant functions, and intestinal microenvironment in aged breeder laying hens

This study investigated the effects of dietary chlorogenic acid (CGA) on the productive and reproductive performance, egg quality, antioxidant function, and intestinal microenvironment of laying hens. Thus, 162 healthy Hy-Line Brown breeding hens (63 weeks old) were randomly allocated to 3 groups, each receiving a basal diet plus supplementation: 0, 250, and 500 mg/kg CGA, respectively. Per the in vitro test, CGA had obvious inhibitory effects on Salmonella enteritis and avian pathogenic Escherichia coli and strong free radical scavenging ability. Per the breeder laying hen experiment, the CGA diets had no significant influence on egg production or reproductive performance (P < 0.05). Nevertheless, compared with the control diet, 250 mg/kg CGA significantly increased eggshell thickness, egg weight, yolk color, and Haugh unit (P < 0.05). Compared with the control diet and 500 mg/kg CGA, 250 mg/kg CGA significantly (P < 0.05) elevated antioxidant capacity by reducing serum malondialdehyde content, upregulating heme oxygenase-1, and downregulating heat shock proteins mRNA levels in the ileum. Compared with the control diet and 500 mg/kg CGA, 250 mg/kg CGA (P < 0.05) enhanced intestinal barrier function, shown by the upregulation of ileal Occludin and Mucin-2 mRNA levels; furthermore, 250 mg/kg CGA (P < 0.05) increased anti-apoptotic capacity by increasing B-cell leukemia/lymphoma 2 gene expression and downregulated Bcl2 Associated X mRNA levels in the liver and ileum of late breeder laying hens (P < 0.05). Lastly, 250 mg/kg CGA (P < 0.05) increased cecal g_CHKCI001 and short-chain fatty acid-producing bacteria g_Prevotellaceae UCG-001, positively related to gut health, and in the cecum, 500 mg/kg CGA significantly (P < 0.05) increased g_Shuttleworthia abundance, negatively related to gut health. Our findings suggest that dietary inclusion of 250 mg/kg CGA promotes egg quality, intestinal microbial composition, gut barrier integrity, and the antioxidant capacity of aged breeder laying hens.

Heat shock protein 90 and prolyl hydroxylase 2 co-regulate hypoxia-inducible factor-1α expression in porcine small intestinal epithelial cells under heat stress

Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature–humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.

Melatonin injection and red light irradiation affect the antioxidant response and cell damage in disk abalone (Haliotis discus hannai) exposed to high water temperatures.

The effects of red light-emitting diode (LED) light irradiation (630 nm, 0.5 W/m2) and melatonin (10-8 and 10-7 M) on oxidative stress and physiological responses in abalones exposed to high temperatures (28°C) were investigated. Changes in messenger RNA (mRNA) expressions of melatonin receptor (MT-R), heat shock protein 70 (HSP70), and antioxidant enzymes, as well as alterations in H2O2 levels in the hemolymph, were examined. The results revealed that high-temperature-stressed abalones treated with melatonin injections or exposed to red LED light showed a significant increase in MT-R mRNA expression, while HSP70 mRNA expression decreased. Notably, HSP70 mRNA expression levels in the red LED light-irradiated group were similar to those in the group injected with 10-8 M melatonin after 24 h exposure. Abalones treated with melatonin at 20°C or irradiated with red LED light exhibited decreased H2O2 levels and reduced antioxidant enzyme mRNA expression compared with those of the control group. However, the high-temperature environment induced oxidative stress in abalones, leading to increased antioxidant enzyme mRNA expression compared with that under 20°C conditions. Moreover, abalones exposed to high-temperature stress exhibited hepatopancreatic DNA damage, which was attenuated by melatonin treatment or red LED light irradiation. Hence, red LED light reduces oxidative stress, boosts antioxidant enzymes, and alleviates DNA damage in high-temperature-stressed abalones, akin to 10-8 M melatonin treatment. Therefore, considering the practical challenges of continuous melatonin administration to abalones, utilizing red LED light emerges as a practical, effective alternative to protect abalones from oxidative stress compared to 10-8 M melatonin treatment.

Behavioural and physiological responses of juvenile geoduck (Panopea zelandica) following acute thermal stress.

Climate extremes, such as heatwaves, are expected to become more intense and of longer duration in the near future. These climatic conditions may have a significant impact on the prospects of establishing a new aquaculture industry for the endemic New Zealand geoduck, Panopea zelandica. This study focused on characterising animal behaviour, haemocytes , and heat shock protein (HSP70 & HSP90) mRNA expression following exposure to elevated temperatures, such as those encountered during marine heatwaves around 20°C and an extreme scenario of 25°C, contrasted to an ambient temperature of 17°C. After 24h of heat challenge, P. zelandica were found to be significantly influenced by the thermal changes, as there were differences recorded in all the responses examined. With increasing temperatures, juvenile geoduck were observed to fully emerge from the sediment a behaviour that has not previously been quantified nor associated with stress in this species. The ability of P. zelandica juveniles to re-bury still warrants further investigation, as adults are unable to do so. Haemocyte analyses revealed an increase in the abundance of granulocytes, cellular aggregations, and size of these aggregations at the highest temperature exposure. Increased expression of the hsp70 gene in the haemolymph after exposure at 25°C for 24h was detected and attributed to attempts to mitigate protein denaturation caused by thermal stress. The inter-individual variability in the response of heat shock proteins recorded could aid in future selective breeding programs if it is reflected in net thermotolerance. P. zelandica shows great potential for growing in subtidal habitats around New Zealand, and this study highlights the importance of temperature considerations when selecting potential farm and reseeding locations.

Association of heat-shock protein 70.1 gene with physiological and physical performance of Bali cattle.

Global warming challenges cattle productivity and welfare since it affects heat stress and scarce feed. The heat-shock protein 70 (HSP70) gene is essential in cytoprotection against stressors, protecting cells from dysregulated gene expression and apoptosis. This study aimed to identify significant genetic markers of the HSP70.1 gene that can be leveraged genetically to enhance thermotolerance and production in Bali cattle further. Animals were sampled from three different rearing systems. In this study, 83 healthy adult male Bali cattle without abnormalities were utilized. Single-nucleotide polymorphism (SNP) diversity associated with the physiological and physical traits of Bali cattle was assessed using SNPStat online software. Gene expression for putative SNPs and their genotypic groups was further evaluated. There were 15 polymorphic SNPs (c.-185G>A, c.-69T>G, c.10G>C, c.19A>G, c.45C>T, c.101INS, c.115T>C, c.130T>C, c.136G>T, c.159G>C, c.164G>T, c.234G>A, c.303G>A, c.333C>A, and c.456C>T) identified, of which 12 were associated with the assessed trait. Nine SNPs were associated with physiological traits, while eight were with physical traits. The c.136G>T as a novel, high minor allele frequency, and associative SNP was selected for HSP70 gene expression. Individuals with the TT genotype have a trim physique, susceptible physiology, and high HSP70 mRNA expression. On the other hand, the GG genotype was significantly associated with larger physique, lower physiology, and low HSP70 mRNA expression. The higher expression may indicate that HSP70.1 is involved in mitigating the deleterious effects of stress. As a result, the animal experienced negative energy balance, decreasing body size. Single-nucleotide polymorphism c.136G>T is a candidate biomarker for heat resistance traits in Bali cattle.

An autocatalytic multicomponent DNAzyme nanomachine for tumor-specific photothermal therapy sensitization in pancreatic cancer

Multicomponent deoxyribozymes (MNAzymes) have great potential in gene therapy, but their ability to recognize disease tissue and further achieve synergistic gene regulation has rarely been studied. Herein, Arginylglycylaspartic acid (RGD)-modified Distearyl acylphosphatidyl ethanolamine (DSPE)-polyethylene glycol (PEG) (DSPE-PEG-RGD) micelle is prepared with a DSPE hydrophobic core to load the photothermal therapy (PTT) dye IR780 and the calcium efflux pump inhibitor curcumin. Then, the MNAzyme is distributed into the hydrophilic PEG layer and sealed with calcium phosphate through biomineralization. Moreover, RGD is attached to the outer tail of PEG for tumor targeting. The constructed nanomachine can release MNAzyme and the cofactor Ca2+ under acidic conditions and self-assemble into an active mode to cleave heat shock protein (HSP) mRNA by consuming the oncogene miRNA-21. Silencing miRNA-21 enhances the expression of the tumor suppressor gene PTEN, leading to PTT sensitization. Meanwhile, curcumin maintains high intracellular Ca2+ to further suppress HSP-chaperone ATP by disrupting mitochondrial Ca2+ homeostasis. Therefore, pancreatic cancer is triple-sensitized to IR780-mediated PTT. The in vitro and in vivo results show that the MNAzyme-based nanomachine can strongly regulate HSP and PTEN expression and lead to significant pancreatic tumor inhibition under laser irradiation.

Quercetagetin alleviates zearalenone-induced liver injury in rabbits through Keap1/Nrf2/ARE signaling pathway.

Introduction: This study aimed to assess the alleviative effect of quercetagetin (QG) on zearalenone (ZEN)-induced liver injury in rabbits. Methods: Ninety 41-day-old healthy Hyla rabbits were randomly assigned into three groups, including a control (fed with basic diet), ZEN addition group (fed with basic diet + 600μg/kg ZEN), and ZEN + QG addition group (fed with basic diet + 600μg/kg ZEN + 100mg/kg QG), with 30 rabbits per group. The duration of the experiment was 28days. Results: The results revealed no significant differences in the average daily gain, average daily feed intake, the gain to feed ratio and the liver, kidney and spleen organ indexes (p > 0.05) between the rabbits across the three groups. However, the sacculus rotundus index of the rabbits in the control group was significantly higher than that in the ZEN + QG group (p < 0.05). The intake of ZEN-contaminated diet also significantly increased the activities or levels of alanine transaminase, alkaline phosphatase, total bile acid (TBA), total bilirubin, malondialdehyde, and interleukin-4 (IL-4) and enhanced the abundance of kelch-like ECH-associated protein 1 (Keap1), heat shock protein 70 (HSP70) and cysteine-aspartic acid protease-3 (Caspase-3) mRNA in the blood or liver tissue in ZEN group, compared to the control group (p < 0.05). On the contrary, the activities or levels of immunoglobulin A, complement 3, total antioxidant capacity, glutathione peroxidase (GSH-Px), superoxide dismutase, interleukin-10, and the abundance of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) mRNA were significantly decreased (p < 0.05). Supplementing the diet with QG still maintained significantly higher levels of TBA and IL-4, and the abundance of GSH-Px, HSP70, IL-4, and Caspase-3 mRNA in the blood and liver of rabbits in the ZEN + QG group than in the control group (p < 0.05). At the same time, the other indicators were restored to levels in the control group (p > 0.05). Discussion: In conclusion, QG alleviated the ZEN-induced oxidative damage and liver injury caused by inflammatory reaction through the Keap1-Nrf2-antioxidant response element (ARE) signal pathway, which protected the liver. This study revealed the alleviative effect of QG on the hepatotoxicity of ZEN in rabbits for the first time, providing a new perspective for applying QG and developing a ZEN antidote.

Anti-HSP70 alleviates cell migration and proliferation in colorectal cancer cells (CRC) by targeting CXCR4 (in vitro study).

Colorectal cancer (CRC), the third most common cancer in men and women, accounts for 8% of new cancer cases in the US and 8 to 9% of the nation's anticipated cancer mortality in 2014. In the pathophysiology of colon cancer, heat shock protein 70 (HSP70) and CXCR4 are essential. In this research, we concentrated on the connection between CXCR4 expression and HSP70 inhibitor activity in the development of colorectal cancer. The HSP70 inhibitor's effect on cell proliferation was also evaluated. Samples were obtained from patients with CRC; the surrounding marginal tissues were considered healthy. The One CRC cell lines (HCA-7) were divided into two groups based on untreated and treated with anti-HSP70. HSP70 and CXCR4 mRNA expression and migration (Wound healing assay) were measured in these groups. Also, we evaluated the expression levels of HSP70 and CXCR4 in thirty CRC and healthy non-cancerous samples (Using Real-time PCR and Western Blotting). Moreover, we examined the viability of CRC cells in untreated and treated groups with anti-HSP70. Higher expression levels of CXCR4 (p < 0.0001) and HSP70 (p = 0.002) mRNA were observed in patients who had CRC. In contrast, lower mRNA expressions of HSP70 (p < 0.0001) and CXCR4 (P < 0.0001) were detected in the CRC cell line (HCA-7) after being treated with anti-HSP70. Moreover, the viability and migration of cancer cells were remarkably reduced in CRC cells treated with anti-HSP70. Our study's innovation was the in vitro demonstration of inhibiting HSP70 in the CRC cancer cell line drastically reduced CXCR4 expression, viability, and cancer cell migration. These findings may pave the way for additional studies on CRC cancer treatment and be examined in vivo in studies, given that the primary goal of therapy is to decrease the viability and spread of cancer cells.

Eco-friendly postharvest irradiation strategy with 131I isotope for environmental management of populations of migratory locust, Locusta migratoria

Purpose Irradiation of food is promising for control of pests to minimize postharvest losses of yields and thus improvement of food safety, shelf life of produce. It is a method of choice that induces a series of lethal biochemical and molecular changes culminating into the engagement of a downstream cascade to cause abnormalities in irradiated pests. In this study, the effects of iodine-131 (131I) isotope radiation on the male gonad development of the migratory locust, Locusta migratoria, were evaluated. Materials and methods Newly emerged adult male locusts, less than one-day-old, were divided into two groups, control and irradiated. Locusts in the control group (n = 20 insects) didn’t drink irradiated water and were reared under normal environmental conditions for one week. Locusts in the irradiated group (n = 20 insects) were exposed to irradiated water at a dose of 30 mCi and they were subsequently observed until they drank the whole quantity. Results At the end of the experiment, scanning and electron microscopic examination of testes obtained from irradiated locusts revealed several major abnormalities, including malformed nuclei of spermatozoa, irregular plasma membranes, shrinkage of testicular follicles, vacuolated cytoplasm, disintegrated nebenkern and agglutinations of spermatids. Flow cytometry analysis revealed that 131I radiation induced both early and late apoptosis, but not necrosis, in testicular tissues. Testes of irradiated insects also exhibited a burst in reactive oxygen species (ROS), as indicated by significant elevation in amounts of malondialdehyde (MDA), a marker for peroxidation of lipids. In contrast, irradiation coincided with significant reductions in activities of enzymatic antioxidant biomarkers. Relative to controls, a three-fold upregulation of expression of mRNA of heat shock protein, Hsp90, was observed in testicular tissue of irradiated locusts. 131I-irradiated insects exhibited genotoxicity, as indicated by significant increases in various indicators of DNA damage by the comet assay, including tail length (7.80 ± 0.80 µm; p < .01), olive tail moment (40.37 ± 8.08; p < .01) and tail DNA intensity % (5.1 ± 0.51; p < .01), in testicular cells compared to the controls. Conclusion This is the first report on elucidation of I131-irradiation-mediated histopathological, biochemical and molecular mechanisms in gonads of male L. migratoria. Herein, the findings underscore the utility of 131I radiation as an eco-friendly postharvest strategy for management of insect pests and in particular for control of populations of L. migratoria.

461-P: Treatment with Mild Electrical Stimulation + Heat Shock (MET) Suppresses the Progression of Atherosclerosis in ApoE-Deficient Mice

[Purpose] We have previously reported that mild electrical stimulation + heat shock (MET) treatment promotes the expression of heat shock protein 72 (HSP72), which works as molecular chaperone to suppresses oxidative stress, JNK activity and mitochondrial dysfunction, and thus ameliorates insulin resistance and hyperglycemia. Macrophage polarity and atherogensis with MET were investigated.[Methods] RAW264.7 and mouse bone marrow-derived macrophages (BMM) were treated with MET (42°C, 55 pulses/sec, 0.1 ms duration) and LPS 1 ng/mL. Six-week-old HSP+/+/ApoE-/- and HSP-/-/ApoE-/- mice were given a high-fat diet (HFD) for 14 weeks, and Sham/MET treatment was started from 10 weeks of age onwards. Glucose tolerance, biochemical parameters were examined, and the degree of arteriosclerosis (AS) formation and oxidative stress were evaluated histologically in aorta. [Results] After MET treatment in RAW264.7, HSP72 mRNA was increased, and LPS-stimulated MCP-1 and iNOS expression and JNK phosphorylation were all decreased. Experiment using BMM showed similar results with RAW264.7. There were no significant differences in physiologocal and metabolic parameters between Sham and MET groups after HFD. Compared with the MET group of HSP-/-/ApoE-/- mice, the degree of AS formation, necrotic core and fibrous capsule thickness were all significantly decreased in the MET group of HSP+/+/ApoE-/- mice. However, no beneficial effects were observed either in Sham or MET in HSP-/-/ApoE-/- mice. Immunostaining of the plaque showed significant suppression of 4-HNE and F4/80 in the HSP+/+/ApoE-/- mice treated with MET compared to those with Sham. Neither Sham nor MET had an inhibitory effect of 4-HNE and F4/80 in HSP-/-/ApoE-/- mice.[Conclusion] The enhancement of HSP72 expression by MET showed anti-inflammatory effect by polarity changes in macrophages and suppression of oxidative stress, which may lead to the suppression of atherosclerosis. Disclosure R.Yoshizumi: None. T.Kondo: None. T.Watanabe: None. N.Miyakawa: None.

Elucidation on potential utilization of Forsythia suspensa fruits in freshwater bivalve (Hyriopsis cumingii) culture against Aeromonas veronii infection

Plant extracts have been implicated in biological studies for their antibacterial, antioxidant, and anti-inflammatory properties. The curative efficacy of fruit extracts (Forsythia suspensa and Prunus mume) and three common antibiotics against Aeromonas veronii (alone or in extract-antibiotic combination) were clarified in vitro by microplate assays and checkerboard methods. Then the extracts with high antibacterial activity (F. suspensa) in vitro were exposed to Hyriopsis cumingii with A. veronii infection for in vivo assessments of their protective efficacy. Subsequently, oxidative stress markers, immune enzymes, and mRNA expression of immune genes in H. cumingii were investigated. Finally, principal component analysis of the physiological biomarkers and genetic markers that respond to A. veronii infection and extract exposures was conducted. The FICI values in vitro implied the following: P. mume combined with cephalexin was antagonistic, as opposed to other combinations whose effects were commutative or indifferent. In vivo results demonstrated that A. veronii caused lipid oxidative damage led to significant decreases in antioxidant defenses and induced immune responses in H. cumingii. F. suspensa significantly enhanced alanine transaminase activities and reduced malondialdehyde levels caused by A. veronii infection in gills and hepatopancreas. It also promoted antioxidant defenses, including superoxide dismutase activities and peroxidase contents, inhibiting increased alkaline phosphatase and acid phosphatase activities. Notably, 0.6% of F. suspensa extract (dry fruits: wet H. cumingii, w/w) reversed the upregulation of glutathione S-transferase and small heat shock protein mRNA levels in hepatopancreas and remarkably improved the survival rate of H. cumingii after the infection. Building on the research, F. suspensa may represent a promising substitute for offering effective protection to H. cumingii against bacterial diseases and contribute to the transformation and upgrading of the freshwater pearl culture industry.

Impacts of Habitat Quality on the Physiology, Ecology, and Economical Value of Mud Crab Scylla sp.: A Comprehensive Review

The water of the mangrove ecosystem and surrounding coastal areas are gradually shrinking due to the intense destruction. Therefore, the effects of the physicochemical properties of the habitat water on the in-habitant species must be studied. Scylla sp. is involved in the food chain and bioturbation structure formation in mangrove forests. Five major electronic databases, such as PubMed, Scopus, Web of Science, AGRICOLA, and Google Scholar, were systematically searched to review the cause and effects of influencing abiotic factors, mainly physicochemical properties of habitat water, including water pollution on Scylla sp. Responses of mud crabs at biochemical, molecular, physiological, growth, reproduction, and production level were independently reviewed or in relation to physicochemical properties of habitat water, pathogens, heavy metals, and harmful chemicals present in their habitat water. Review results suggest that these crabs are mostly under threats of overfishing, varied physicochemical properties of habitat water, pathogens, heavy metals, and chemical toxicants in water, etc. At low temperatures, the expression of calreticulin and heat shock protein-70 mRNA expression is elevated. Like melatonin, the hormone serotonin in mud crabs controls ecdysteroids and methyl farnesoate at 24 °C, 26 ppt salinity, and pH 7.2 of habitat water, facilitating their reproduction physiology. Xenobiotics in habitat water induce toxicity and oxidative stress in mud crabs. These crabs are prone to infection by white spot and rust spot diseases during the winter and spring seasons with varied water temperatures of 10–30 °C. However, elevated (65%) weight gain with higher molting at the juvenile stage can be achieved if crabs are cultured in water and kept in the dark. Their larvae grow better at 30 ± 2 °C with salinity 35 ppt and 12 hL/12 hD day length. So, monitoring habitat water quality is important for crab culture.

Overexpression of heat shock protein 90 through the downregulation of miR-9-5p in extramammary Paget's disease.

Heat shock protein 90 (HSP90) facilitates diverse cellular processes by interacting process with more than 200 client proteins. Overexpression of HSP90 contributes to the pathogenesis of various malignant tumors, and HSP90 inhibitors attenuate the progression of malignant tumors in vitro/vivo. Numerous clinical trials have used HSP90 inhibitors to treat several cancers, and pimitespib (an HSP90 inhibitor) is covered by insurance for advanced gastrointestinal stromal tumor in Japan. In this study, we investigated the expression pattern of HSP90 and analyzed its clinical significance in extramammary Paget's disease (EMPD). All 77 EMPD tissues investigated were positive for HSP90 expression. The immunoreactivity of HSP90 in fetal cases due to EMPD tended to be highly stained. Although there was no significant difference in HSP90 mRNA levels between 24 paired lesional and nonlesional tissues, microRNA-inhibiting HSP90 levels in tumor tissues were significantly decreased compared with those in normal tissues. Thus, HSP90 may play an important role in the pathogenesis of EMPD and may be a novel therapeutic target for EMPD.

Phosphorylated heat shock protein 27improves the bone formation ability of osteoblasts and bone marrow stem cells from patients with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a scoliotic deformity of unknown etiology that occurs during adolescent development. Abnormal bone metabolism is closely related to AIS, but the cause is uncertain. Recent studies have shown that heat shock protein 27 (HSP27) and its phosphorylation (pHSP27) play important roles in bone metabolism. However, whether HSP27 and pHSP27 are involved in abnormal bone metabolism in AIS is unclear. Osteoblasts (OBs) and bone marrow stem cells (BMSCs) were extracted from the facet joints and bone marrow of AIS patients and controls who underwent posterior spinal fusion surgery. The expression levels of HSP27 and pHSP27, as well as the expression levels of bone formation markers in OBs from AIS patients and controls, were examined by quantitative real-time PCR (qRT-PCR) and Western blotting. The mineralization ability of OBs from AIS patients and controls was analyzed by alizarin red staining after osteogenic differentiation. Heat shock and thiolutin were used to increase the levels of pHSP27 in OBs, and the levels of bone formation markers were also investigated. In addition, the levels of pHSP27 and the bone formation ability of BMSCs from AIS patients and controls were investigated after heat shock treatment. Lower pHSP27 levels and impaired osteogenic differentiation abilities were observed in the OBs of AIS patients than in those of controls. Thiolutin increased HSP27 phosphorylation and increased the mRNA levels of SPP1 and ALPL in OBs from AIS patients. Heat shock treatment increased SPP1 and HSP27 mRNA expression, pHSP27 levels, OCN expression, and mineralization ability of both OBs and BMSCs from AIS patients. Heat shock treatment and thiolutin can increase the levels of pHSP27 and further promote the bone formation of OBs and BMSCs from AIS patients. Therefore, decreased pHSP27 levels may be associated with abnormal bone metabolism in AIS patients.

N-butyrate increases heat shock protein 70 through heat shock factor 1 and AMP-activated protein kinase pathways in human intestinal Caco-2 cells

Impaired integrity of the intestinal epithelium is a cause of intestinal and extraintestinal diseases. Heat shock protein 70 (HSP70), a cytoprotective protein, plays an important role in maintaining intestinal homeostasis. The intestinal expression of HSP70 is linked with the local microbiota. The present study investigated the molecular mechanisms underlying the upregulation of HSP70 by n-butyrate, a major metabolite of the intestinal microbiota in human intestinal Caco-2 cells. Treatment of Caco-2 cells with n-butyrate upregulated HSP70 protein and mRNA levels in a dose-dependent manner. Using luciferase reporter assay, it was found that n-butyrate enhanced the transcriptional activity of HSP70. These effects were sensitive to the inhibition of heat shock factor 1 (HSF1), a transcription factor, and AMP-activated protein kinase (AMPK). N-butyrate increased the phosphorylation (activity) of HSF1 and AMPK. Taken together, this study shows that n-butyrate is partly involved in the microbiota-dependent intestinal expression of HSP70, and the effect is exerted through the HSF1 and AMPK pathways.

Mifepristone inhibits hepatoma growth by enhancing the GR-HSP60-survivin interaction to facilitate survivin degradation.

Silencing of heat shock protein 60 (HSP60) suppresses the growth of hepatocellular carcinoma (HCC). Mifepristone inhibits HSP60 mRNA expression in Chlamydophila-infected epithelial cells. The aim of this study was to determine whether mifepristone could inhibit the growth of HCC cells by affecting the functions of HSP60. The effect of mifepristone on cell viability was examined by flow cytometry and a cell proliferation assay. Protein-protein interactions were examined using the immunoprecipitation assay. The anti-tumor effect of mifepristone was evaluated using a xenograft model. Our results indicated that mifepristone induces cell cycle arrest at the G1 phase and early-stage apoptosis in HCC cells. Instead of reducing the total amount of HSP60, mifepristone induced the release of mitochondrial HSP60 into the cytosol by causing a loss of ΔΨm, thereby enhancing glucocorticoid receptor (GR)-HSP60-survivin complex formation as well as survivin degradation. Animal models have confirmed the growth inhibitory effects of mifepristone on HCC, including changes in the abundance of HSP60 in mitochondria and cytosol, decreased survivin and Ki-67-positive cells, as well as increased cell apoptosis. In conclusion, the inhibition of HCC growth by mifepristone may be achieved by altering the subcellular distribution of HSP60 to enhance the formation of cytosolic GR-HSP60-survivin complexes in the cells, leading to the degradation of survivin.

Manganese alleviates heat stress of primary cultured chick embryonic myocardial cells via enhancing manganese superoxide dismutase expression and attenuating heat shock response

Manganese (Mn) is an essential trace element that has been shown to attenuate the adverse effects of heat stress in the heart of broiler breeders and embryos. However, the underlying molecular mechanisms involving this process remain unclear. Therefore, two experiments were conducted to investigate the possible protective mechanisms of Mn on primary cultured chick embryonic myocardial cells exposed to heat challenge. In experiment 1, the myocardial cells were exposed to 40 °C (normal temperature, NT) and 44 °C (high temperature, HT) for 1, 2, 4, 6 or 8 h. In experiment 2, the myocardial cells were preincubated with no Mn supplementation (CON), 1 mmol/L of Mn as the inorganic MnCl2 (iMn) or organic Mn proteinate (oMn) under NT for 48 h, and then continuously incubated under NT or HT for another 2 or 4 h. The results from experiment 1 showed that the myocardial cells incubated for 2 or 4 h had the highest (P < 0.0001) heat-shock protein 70 (HSP70) or HSP90 mRNA levels than those incubated for other incubation times under HT. In experiment 2, HT increased (P < 0.05) the heat-shock factor 1 (HSF1) and HSF2 mRNA levels as well as Mn superoxide dismutase (MnSOD) activity of myocardial cells compared with NT. Furthermore, supplemental iMn and oMn increased (P < 0.02) HSF2 mRNA level and MnSOD activity of myocardial cells compared with the CON. Under HT, the HSP70 and HSP90 mRNA levels were lower (P < 0.03) in iMn group than in the CON group, in oMn group than in iMn group; and the MnSOD mRNA and protein levels were higher (P < 0.05) in oMn group than in the CON and iMn groups. These results from the present study indicate that supplemental Mn, especially oMn, could enhance the MnSOD expression and attenuate heat shock response to protect against heat challenge in primary cultured chick embryonic myocardial cells.

Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA.

Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.

Effect of intermittent mild cold stimulation on intestinal immune function and the anti-stress ability of broilers

A total of 240 healthy 1-day-old Ross 308 male broilers were randomly divided into 3 groups (CS0 group, CS3 group, and CS6 group), with 5 replicates in each group and 16 broilers in each replicate, in order to evaluate the effects of intermittent mild cold stimulation (IMCS) on the intestinal immune function and anti-cold stress ability of broilers after acute cold stress. The mRNA expression levels of cytokines and Toll-like receptors (TLRs) in the duodenum and jejunum were detected at the end of cold stimulation (36 d), 2 wk after recovery (50 d), and after acute cold stress (Y6). In addition, the mRNA and protein expression levels of heat shock proteins (HSPs) were measured before and after acute cold stress. The experimental data were statistically processed using 1-way ANOVA and Duncan's multiple comparisons. The results showed that the mRNA expression levels of IL2, IL8, IFN γ, TLR7, and TLR21 in the duodenum and IL2 and IFNγ in jejunum were significantly higher in the CS6 group than in the CS0 and CS3 groups at 36 d (P < 0.05). All TLR levels in the jejunum were significantly lower in the CS3 group than in the CS0 and CS6 groups at 36 d (P < 0.05). After 6 h of acute cold stress, in the duodenum, the mRNA expression levels of IL6 and IL8 were significantly decreased in the CS0 and CS6 groups compared to levels at 50 d (P < 0.05), while levels in the CS3 group remained stable (P > 0.05). Compared with 50 d, the expression level of HSP mRNA in the jejunum in the CS3 group was relatively stable compared to that in the CS0 and CS6 groups after acute cold stress (P > 0.05). At the protein level, the HSP60 expression level in the duodenum and HSP40, HSP60, and HSP70 expression levels in the jejunum were significantly higher in the CS3 group than in the CS0 and CS6 groups after acute cold stress (P < 0.05). In conclusion, cold stimulation training at 3℃/3 h lower than the conventional feeding temperature can improve the intestinal immune function and anti-stress ability of broilers.

Upstream Flanking Sequence Assists Folding of an RNA Thermometer

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5’UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5’UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.