Medical ozone induces proliferation and migration inhibition through ROS accumulation and PI3K/AKT/NF-κB suppression in human liver cancer cells in vitro.

BACKGROUND: Hepatocellular carcinoma is one of the most common malignancies and leading cancer-associated deaths worldwide. Ozone has been proposed as a promising therapeutic agent in the treatment of various disorders. PURPOSE: The purpose of this paper is to assess the potential anticancer effects of the ozone on liver cancer cells. METHOD: The liver cancer cell line of bel7402 and SMMC7721 was used in this study. Proliferation was evaluated using the CCK-8 and the colony formation assay. Wond healing assay and transwell assay without Matrigel were used to evaluate their migration ability. Flow cytometry was used for cell cycle analysis and reactive oxygen species (ROS) determination. Glutathione detection kit was used for measurement of glutathione level. Protein expression was estimated by western blot analysis. RESULTS: Ozone treatment inhibited liver cancer cell proliferation, colony formation. Ozone induced G2/M phase cell cycle arrest, which could be elucidated by the change of protein levels of p53, p21, Cyclin D1, cyclin B1, cdc2, and CDK4. We also found that ozone treatment inhibited migration ability by inhibiting EMT-relating protein. Ozone also induced ROS accumulation and decreased glutathione level decreased, which contributed to the inactivation of the PI3K/AKT/NF-κB pathway. Finally, we found that pre-treatment of liver cancer cells with N-acetylcysteine resisted ozone-induced effects. CONCLUSIONS: Ozone restrains the proliferation and migration potential and EMT process of liver cancer cells via ROS accumulation and PI3K/AKT/NF-κB suppression.

Effect of Dietary Curcumin on the Antioxidant Status of Laying Hens under High-Temperature Conditions

Heat stress induces oxidative stress, and reduces body antioxidant metabolite levels, which can affect poultry production performance. Dietary antioxidants protect birds against the adverse effects of heat stress. The effects of increasing concentrations of dietary curcumin on the antioxidant parameters of layers maintained under high-temperature conditions for nine weeks were evaluated. Roman laying hens (n = 336, 22 weeks old, 1420 g BW) were divided into three treatment groups. The first group served as a thermoneutral control (kept at 25 ± 1 °C). The second group was exposed to high temperatures (32 ± 1 °C, 6 h/d), given a basal diet. The third group was further divided into five treatment groups (100, 150, 200, 250, 300 mg/kg Curcumin) fed a basal diet (treatments H1, H2, H3, H4, H5) under high temperatures conditions (32 ± 1 °C, 6 hours/day). As a result of this study, total superoxide dismutase activity was significantly higher in H2 and H3 groups, and total antioxidant capacity was higher in H2, H3, and H5 groups. Catalase and glutathione peroxidase activity was significantly higher in the H3 group. Malondialdehyde concentration was lowered in curcumin supplemented hens compared with control groups hens. Laying hens in all curcumin treatment groups had slightly higher activities of CAT, SOD, GSH-Px, and T-AOC in the liver, heart, and lungs, compared with heat stressed control group. It was concluded that dietary curcumin given to laying hens under heat stress may enhance their antioxidant status, and alleviate the detrimental effects of stressful environmental conditions.(AU)

Effect of Dietary Curcumin on the Antioxidant Status of Laying Hens under High-Temperature Conditions

Heat stress induces oxidative stress, and reduces body antioxidant metabolite levels, which can affect poultry production performance. Dietary antioxidants protect birds against the adverse effects of heat stress. The effects of increasing concentrations of dietary curcumin on the antioxidant parameters of layers maintained under high-temperature conditions for nine weeks were evaluated. Roman laying hens (n = 336, 22 weeks old, 1420 g BW) were divided into three treatment groups. The first group served as a thermoneutral control (kept at 25 ± 1 °C). The second group was exposed to high temperatures (32 ± 1 °C, 6 h/d), given a basal diet. The third group was further divided into five treatment groups (100, 150, 200, 250, 300 mg/kg Curcumin) fed a basal diet (treatments H1, H2, H3, H4, H5) under high temperatures conditions (32 ± 1 °C, 6 hours/day). As a result of this study, total superoxide dismutase activity was significantly higher in H2 and H3 groups, and total antioxidant capacity was higher in H2, H3, and H5 groups. Catalase and glutathione peroxidase activity was significantly higher in the H3 group. Malondialdehyde concentration was lowered in curcumin supplemented hens compared with control groups hens. Laying hens in all curcumin treatment groups had slightly higher activities of CAT, SOD, GSH-Px, and T-AOC in the liver, heart, and lungs, compared with heat stressed control group. It was concluded that dietary curcumin given to laying hens under heat stress may enhance their antioxidant status, and alleviate the detrimental effects of stressful environmental conditions.

Angiotensin-(1-7) relieved renal injury induced by chronic intermittent hypoxia in rats by reducing inflammation, oxidative stress and fibrosis.

We aimed to study the renal injury and hypertension induced by chronic intermittent hypoxia (CIH) and the protective effects mediated by angiotensin 1-7 [Ang(1-7)]. We randomly assigned 32 male Sprague-Dawley rats (body weight 180-200 g) to normoxia control, CIH, Ang(1-7)-treated normoxia, and Ang(1-7)-treated CIH groups. Systolic blood pressure (SBP) was monitored at the start and end of each week. Renal sympathetic nerve activity (RSNA) was recorded. CTGF and TGF-ß were detected by immunohistochemistry and western blotting. Tissue parameters of oxidative stress were also determined. In addition, renal levels of interleukin-6, tumor necrosis factor-α, nitrotyrosine, and hypoxia-inducible factor-1α were determined by immunohistochemistry, immunoblotting, and ELISA. TUNEL assay results and cleaved caspase 3 and 12 were also determined. Ang(1-7) induced a reduction in SBP together with a restoration of RSNA in the rat model of CIH. Ang(1-7) treatment also suppressed the production of reactive oxygen species, reduced renal tissue inflammation, ameliorated mesangial expansion, and decreased renal fibrosis. Thus, Ang(1-7) treatment exerted renoprotective effects on CIH-induced renal injury and was associated with a reduction of oxidative stress, inflammation and fibrosis. Ang(1-7) might therefore represent a promising therapy for obstructive sleep apnea-related hypertension and renal injury.

Angiotensin-(1-7) relieved renal injury induced by chronic intermittent hypoxia in rats by reducing inflammation, oxidative stress and fibrosis

We aimed to study the renal injury and hypertension induced by chronic intermittent hypoxia (CIH) and the protective effects mediated by angiotensin 1-7 [Ang(1-7)]. We randomly assigned 32 male Sprague-Dawley rats (body weight 180-200 g) to normoxia control, CIH, Ang(1-7)-treated normoxia, and Ang(1-7)-treated CIH groups. Systolic blood pressure (SBP) was monitored at the start and end of each week. Renal sympathetic nerve activity (RSNA) was recorded. CTGF and TGF-β were detected by immunohistochemistry and western blotting. Tissue parameters of oxidative stress were also determined. In addition, renal levels of interleukin-6, tumor necrosis factor-α, nitrotyrosine, and hypoxia-inducible factor-1α were determined by immunohistochemistry, immunoblotting, and ELISA. TUNEL assay results and cleaved caspase 3 and 12 were also determined. Ang(1-7) induced a reduction in SBP together with a restoration of RSNA in the rat model of CIH. Ang(1-7) treatment also suppressed the production of reactive oxygen species, reduced renal tissue inflammation, ameliorated mesangial expansion, and decreased renal fibrosis. Thus, Ang(1-7) treatment exerted renoprotective effects on CIH-induced renal injury and was associated with a reduction of oxidative stress, inflammation and fibrosis. Ang(1-7) might therefore represent a promising therapy for obstructive sleep apnea-related hypertension and renal injury.

Hypoxia facilitates epithelial-mesenchymal transition-mediated rectal cancer progress.

Rectal cancer is a commonly observed tumor in clinics, and epithelial-mesenchymal transition (EMT) is very important for tumor invasion and metastasis. We established a rectal cancer HCT-116 cell hypoxia model and detected cell proliferation, invasion, and EMT-related protein expression in this model, aiming to analyze the effect of hypoxia on rectal cancer cell EMT. Rectal cancer cell line HCT-116 was cultured in normoxic, hypoxic, or anaerobic environment, and hypoxia-inducible factor-1α (HIF-1α) mRNA expression was detected in the cells by real-time PCR. Cell proliferation was tested by MTT assay; cell invasion was determined by transwell assay, and HIF-1α, epithelial-cadherin, and Snail protein levels were evaluated by western blot analysis. HIF-1α mRNA level significantly increased in the anaerobic group compared to that in the normoxic and hypoxic groups (P < 0.05). HCT-116 cell proliferation in the anaerobic group was obviously higher than that in the other two groups, with the hypoxic group showing stronger proliferative ability than the normoxic group (P < 0.05). Compared to the normoxic group, the HCT-116 cells demonstrated enhanced cell invasion and migration in hypoxic and anaerobic groups. HIF-1α and Snail expressions were upregulated, whereas epithelial-cadherin expression had declined in the hypoxic and anaerobic groups, compared to those in the normal control (P < 0.05). Therefore, hypoxia promoted rectal cancer cell progress by increasing HIF-1α to induce EMT.

Genetic variability of Myzus persicae nicotianae densovirus based on partial NS and VP gene sequences.

We previously described a novel densovirus [Myzus persicae nicotianae densovirus (MpnDV)] infecting M. persicae nicotianae (Hemiptera: Aphididae) with 34% prevalence. This single-stranded DNA virus has a 5480-nucleotide ambisense genome and belongs to the Densovirinae subfamily within the family Parvoviridae. In the present study, we estimated the genetic diversity of MpnDV using partial nonstructural protein (NS) and capsid protein (VP) gene sequences from 10 locations in China. First, we identified MpnDV-positive samples by amplifying a 445-bp fragment with primers MpDVF/MpDVR. Subsequently, we amplified and sequenced COI genes with primers MpCOIF/ MpCOIR, and partial NS and VP sequences with primers MpnDVF1/MpnDVR1. The respective 655-, 1461-, and 423-bp COI, NS, and VP fragments were used to analyze the genetic diversity of MpnDV using MEGA 6.0 and DnaSP 5.0. The high level of identity shared by all COI sequences (>99%) suggested that the aphids sampled were of the same species, and indicated population homogeneity across the 10 locations investigated. The nucleotide diversity of MpnDV sequences (0.0020 ± 0.0025) was significantly higher than that of the COI genes (0.0002 ± 0.0005). The pairwise fixation index for MpnDV was 0.832, and the total gene flow was 0.05. Phylogenetic analysis revealed that the MpnDV haplotypes clustered according to geographical location, except for those from the Liaoning and Shanxi provinces. In conclusion, MpnDV demonstrated a low level of gene flow and high genetic diversity, suggesting that it is vertically transmitted, and implying that endosymbiotic viruses could be used as markers in studies of insect population genetics.

Characterization and gene cloning of the rice (Oryza sativa L.) dwarf and narrow-leaf mutant dnl3.

The dwarf and narrow-leaf rice (Oryza sativa L.) mutant dnl3 was isolated from the Japonica cultivar Zhonghua 11 (wild-type). dnl3 exhibited pleiotropic developmental defects. The narrow-leaf phenotype resulted from a marked reduction in the number of vascular bundles, while the dwarf stature was caused by the formation of foreshortened internodes and a reduced number of parenchyma cells. The suggestion that cell division is impaired in the mutant was consistent with the transcriptional behavior of various genes associated with cell division. The mutant was less responsive to exogenously supplied gibberellic acid than the wild-type, and profiling the transcription of genes involved in gibberellin synthesis and response revealed that a lesion in the mutant affected gibberellin signal transduction. The dnl3 phenotype was inherited as a single-dominant gene, mapping within a 19.1-kb region of chromosome 12, which was found to harbor three open reading frames. Resequencing the open reading frames revealed that the mutant carried an allele at one of the three genes that differed from the wild-type sequence by 2-bp deletions; this gene encoded a cellulose synthase-like D4 (CSLD4) protein. Therefore, OsCSLD4 is a candidate gene for DNL3. DNL3 was expressed in all of the rice organs tested at the heading stage, particularly in the leaves, roots, and culms. These results suggest that DNL3 plays important roles in rice leaf morphogenesis and vegetative development.

Angiotensin-(1-7) inhibits inflammation and oxidative stress to relieve lung injury induced by chronic intermittent hypoxia in rats.

Obstructive sleep apnea is associated with inflammation and oxidative stress in lung tissues and can lead to metabolic abnormalities. We investigated the effects of angiotensin1-7 [Ang-(1-7)] on lung injury in rats induced by chronic intermittent hypoxia (CIH). We randomly assigned 32 male Sprague-Dawley rats (180-200 g) to normoxia control (NC), CIH-untreated (uCIH), Ang-(1-7)-treated normoxia control (N-A), and Ang-(1-7)-treated CIH (CIH-A) groups. Oxidative stress biomarkers were measured in lung tissues, and expression of NADPH oxidase 4 (Nox4) and Nox subunits (p22phox, and p47phox) was determined by Western blot and reverse transcription-polymerase chain reaction. Pulmonary pathological changes were more evident in the uCIH group than in the other groups. Enzyme-linked immunosorbent assays and immunohistochemical staining showed that inflammatory factor concentrations in serum and lung tissues in the uCIH group were significantly higher than those in the NC and N-A groups. Expression of inflammatory factors was significantly higher in the CIH-A group than in the NC and N-A groups, but was lower than in the uCIH group (P<0.01). Oxidative stress was markedly higher in the uCIH group than in the NC and N-A groups. Expression of Nox4 and its subunits was also increased in the uCIH group. These changes were attenuated upon Ang-(1-7) treatment. In summary, treatment with Ang-(1-7) reversed signs of CIH-induced lung injury via inhibition of inflammation and oxidative stress.

Angiotensin- (1-7) inhibits inflammation and oxidative stress to relieve lung injury induced by chronic intermittent hypoxia in rats

Obstructive sleep apnea is associated with inflammation and oxidative stress in lung tissues and can lead to metabolic abnormalities. We investigated the effects of angiotensin1–7 [Ang-(1–7)] on lung injury in rats induced by chronic intermittent hypoxia (CIH). We randomly assigned 32 male Sprague-Dawley rats (180–200 g) to normoxia control (NC), CIH-untreated (uCIH), Ang-(1–7)-treated normoxia control (N-A), and Ang-(1–7)-treated CIH (CIH-A) groups. Oxidative stress biomarkers were measured in lung tissues, and expression of NADPH oxidase 4 (Nox4) and Nox subunits (p22phox, and p47phox) was determined by Western blot and reverse transcription-polymerase chain reaction. Pulmonary pathological changes were more evident in the uCIH group than in the other groups. Enzyme-linked immunosorbent assays and immunohistochemical staining showed that inflammatory factor concentrations in serum and lung tissues in the uCIH group were significantly higher than those in the NC and N-A groups. Expression of inflammatory factors was significantly higher in the CIH-A group than in the NC and N-A groups, but was lower than in the uCIH group (P<0.01). Oxidative stress was markedly higher in the uCIH group than in the NC and N-A groups. Expression of Nox4 and its subunits was also increased in the uCIH group. These changes were attenuated upon Ang-(1–7) treatment. In summary, treatment with Ang-(1-7) reversed signs of CIH-induced lung injury via inhibition of inflammation and oxidative stress.

Population genetic structure of Myzus persicae nicotianae (Hemiptera: Aphididae) in China by microsatellite analysis.

The tobacco aphid, Myzus persicae nicotianae (Hemiptera: Aphididae), is an important agricultural pest that feeds on host plants and transmits plant viruses in China. To effectively control this pest, we investigated the genetic variation and genetic structure of 54 populations of tobacco aphids collected in China, using five microsatellite loci. An average of 7 alleles with effective number ranging from 1.5 to 6.6 was detected using these five loci, and the average polymorphic information content (PIC) was 0.652, suggesting that the five selected microsatellite loci were polymorphic and suitable for the study of population genetics. The expected heterozygosities in the populations studied ranged from 0.128 and 0.653, with an average value of 0.464. However, the observed heterozygosities ranged from 0.250 and 0.942 (average = 0.735), revealing a high genetic variability and heterozygosity excess in the Chinese tobacco aphid populations. The global fixation index (F(ST)) and mean gene flow (N(m)) were 0.34 (P < 0.0001) and 0.50, respectively, suggesting the high genetic differentiation among Chinese populations. The 54 populations of tobacco aphids were classified into two groups. The populations did not cluster geographically, as populations from the same provinces were usually present in different clusters. This was also confirmed by the Mantel test, which showed no significant correlation between the genetic distance and geographical distance or altitude. Long distance migration might be responsible for the lack of distance-related isolation.

Correlation between interleukin-6 expression in articular cartilage bone and osteoarthritis.

This study aimed to investigate the expressional profile of interleukin-6 (IL-6) in articular cartilage bone of osteoarthritis (OA) patients and its correlation with OA. A total of 30 articular cartilage bone samples from knee OA patients, which were collected by knee arthroscopy or articular surgery, comprised the study group, and 30 samples of normal articular cartilage tissue comprised the control group. Both mRNA (messenger ribonucleic acid) and protein levels of IL-6 and matrix metalloproteinase-9 (MMP-9) were measured and compared, and a correlation analysis was performed between the two. The integral optical density (IOD) values of MMP-9 and IL-6 proteins in the study group were 9.21 ± 3.22 and 8.94 ± 3.17, respectively; these were significantly higher (P < 0.05) than those in the control group at 3.14 ± 1.48 and 6.64 ± 1.53, respectively. The IOD values of mRNA transcripts for MMP-9 and IL-6 in the study group were 8.31 ± 2.28 and 8.78 ± 3.43, respectively; these were significantly higher than the values in the control group at 3.52 ± 1.37 and 5.21 ± 1.72 (P < 0.05), respectively. Further, the correlation analysis revealed significantly positive relationships for both protein (r = 0.434, P = 0.001) and mRNA (r = 0.413, P = 0.002) levels between MMP-9 and IL-6. In conclusion, articular cartilage tissues in knee OA patients have higher levels of MMP-9 and IL-6 expression, and these may play a synergistic role in OA pathogenesis.

Analysis of the gene-protein interaction network in glioma.

Glioma is the most aggressive type of brain tumor. Great progress has been achieved in glioma treatment, but the protein-protein interaction networks underlining glioma are poorly understood. We identified the protein-protein interaction network for glioma based on gene expression and predicted biological pathways underlying the molecular complexes in the network. Genes involved in glioma were selected from the Online Mendelian Inheritance in Man (OMIM) database. A literature search was performed using the Agilent Literature Search plugin, and Cytoscape was used to establish a protein-protein interaction network. The molecular complexes in the network were detected using the Clusterviz plugin, and pathway enrichment of molecular complexes was performed using DAVID online. There were 378 glioma genes in the OMIM database. The protein-protein interaction network in glioma contained 1814 nodes, 6471 edges, and 8 molecular complexes. There were 17 pathways (false discovery rate <1), which were related to cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, chemokine signaling pathway, oocyte meiosis, progesterone-mediated oocyte maturation, transmembrane transport of small molecules, metabolism of amino acids, and notch signaling pathway, among others. Our results provide a bioinformatic foundation for further studies of the mechanisms of glioma.

Mutation analysis of PVRL1 in patients with non-syndromic cleft of the lip and/or palate in Guangdong.

Non-syndromic cleft of the lip and/or palate (NSCLP) is a very common birth defect; the poliovirus receptor-like 1 gene (PVRL1) has been identified as a genetic risk factor for NSCLP in patients from Norway, the Philippines, and South America. Given the considerable variation in allele frequencies across these geographical regions, this study explored the relationship between NSCLP and mutations of PVRL1 in patients from Guangdong, China. We recruited 171 NSCLP patients and 100 volunteers, and divided our samples into 2 groups: a sequencing group and a mass spectrometry group. In the sequencing group, we screened for mutations in exons 2 and 5 of PVRL1 by polymerase chain reaction and direct sequencing in 71 NSCLP patients and 100 volunteers. In the mass spectrometry group, we screened for amino acid mutations in α-spliced transcript codons 112, 131, and 395, and in the ß-spliced transcript codon 1082 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis in 100 NSCLP patients and 100 volunteers. No mutations were detected in either PVRL1 exons 2 or 5 in the 71 NSCLP patients and 100 volunteers, nor did we find mutations of α-spliced transcript codons 112, 131, 395 and the ß-spliced transcript codon 1082 in any of the 100 NSCLP patients and 100 volunteers. Thus, mutations in exons 2 and 5 of PVRL1, and T334A, A391T, G1183A in the α-spliced transcript, and G1082T in the ß-spliced transcript do not participate in the development of NSCLP in patients from Guangdong.

Neuroprotective effect of ketamine on acute spinal cord injury in rats.

The aim of this study was to investigate the neuroprotective effects of ketamine during acute spinal cord injury in rats. Sprague Dawley (SD) rats (N = 70) were randomly divided into three groups: sham-operated (N = 10), control (N = 30), and treatment (N = 30) groups. The moderate spinal cord injury model was established. After injury, the sham-operated group received no drug, the treatment group received intraperitoneal ketamine injections, and the control group received intraperitoneal normal saline injections. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and spinal cord malondialdehyde (MDA) were assessed, and nerve cell apoptosis was evaluated in each group at varying time points. After spinal cord injury, TNF-α, IL-6, and MDA levels, and the number of TUNEL-positive cells among 2500 cells significantly increased (P < 0.05). Further, compared with the control group, the treatment group showed significantly lower TNF-α, IL-6, and MDA levels, and fewer TUNEL-positive cells among 2500 cells at each time point (P < 0.05). Our data indicate that ketamine exerts a neuroprotective effect on injured spinal cord.

Association of heat shock protein 70 expression with rat myocardial cell damage during heat stress in vitro and in vivo.

To investigate the mechanism of sudden death as a result of stress-induced damage to heart tissue and myocardial cells and to investigate the cardioprotective role of Hsp70 during heat stress, the distribution and expression of Hsp70 was evaluated in the heart cells of heat-stressed rats in vivo and heat-stressed H9c2 cells in vitro. After exposure to heat stress at 42°C for different durations, we observed a significant induction of CK, CK-MB, and LDH as well as pathologic lesions characterized by acute degeneration, suggesting that cell damage occurs from the onset of heat stress. Immunocytochemistry showed that Hsp70 was distributed mainly in the cytoplasm of myocardial cells in vivo and in vitro. Hsp70-positive signals in the cytoplasm were more prominent in intact areas than in degenerated areas after 60 min of heat stress. Hsp70 protein levels in myocardial cells in vitro decreased from the beginning to the end of heat stress. Hsp70 protein levels in rat heart tissues in vivo decreased gradually with prolonged heat stress, with a slight increase at the beginning of heat stress. These results indicate that Hsp70 plays a role in the response of cardiac cells to heat stress and that decreased Hsp70 levels are associated with damage to rat myocardial cells in vitro and in vivo. Significant differences were found in hsp70 mRNA, which began to increase after 20 min of heat stress in vitro and after 40 min in vivo. This indicates that hysteresis is involved in mRNA expression after heat stress in vivo.

Association of Hsp60 expression with damage to rat myocardial cells exposed to heat stress in vivo and in vitro.

To investigate the protective role of Hsp60 against stress damage and its role in the sudden death of stressed animals, changes in the levels of Hsp60 protein and hsp60 mRNA of myocardial cells in vivo and in vitro were studied. In addition, the relationship between Hsp60 expression and heat-induced damage was also studied. Rats were exposed to a temperature of 42° ± 1°C for 0, 20, 40, 60, 80, or 100 min. More than 50% of the rats died suddenly within 100 min. With increasing heat stress duration, hsp60 mRNA levels significantly increased in both in vivo and in vitro rat myocardial cells; however, a similar trend was not observed for Hsp60 protein levels. Although the changes observed in Hsp60 expression in myocardial cells in vitro were inconsistent with those of rat heart tissues in vivo, Hsp60 expression levels were consistent with the histopathological damage observed in myocardial cells both in vivo and in vitro. Differences in Hsp60 expression may reflect the degree of injury sustained by myocardial cells in vivo and in vitro. As a mitochondrial protein, Hsp60 represents a potential biomarker of heat stress, and may protect against heat stress induced myocardial cellular damage both in vivo and in vitro.

Evaluation of Hsp47 expression in heat-stressed rat myocardial cells in vitro and in vivo.

The aim of the present study was to identify the correlation between expression of heat shock protein 47 (Hsp47) and stress injury in heat-stressed myocardial cells and to compare variations in Hsp47 expression in rat myocardial cells exposed to different heat stress for varying periods in vitro and in vivo. Exposure to heat stress at 42°C resulted in similar induction patterns of the heart damage-related enzyme aspartate aminotransferase in the supernatants of H9c2 cells and in the serum of rats. Histological analysis revealed that both H9c2 cells and heart tissues displayed cellular degeneration in response to different periods of heat stress. Hsp47 was constitutively expressed in the cytoplasm of H9c2 cells at all time points during heat stress, which was consistent with observations in heart fibers in vivo. Immunoblotting analysis revealed no significant difference between the expression of Hsp47 in H9c2 cells and heart tissue. However, the expression of hsp47 mRNA in response to heat stress was significantly increased in H9c2 cells at 60 min (P < 0.01) and 100 min (P < 0.01), which was comparable to that at 100 min (P < 0.01) in the rat heart. Thus, Hsp47 was elevated significantly after hyperthermia at the mRNA level but not at the protein level both in vitro and in vivo. The results suggest that Hsp47 turnover may increase during heat stress or that Hsp47 consumption exceeds its production.

Expression of heat shock protein 90 alpha (Hsp90α) in primary neonatal rat myocardial cells exposed to various periods of heat stress in vitro.

The objective of this study was to investigate the mechanism of heat shock protein 90 alpha (Hsp90α) protection against heart damage resulting from heat stress by detecting Hsp90α mRNA, Hsp90α protein, protein localization, and cell damage in primary myocardial cells of neonatal rats in response to heat stress in vitro. The cells were heat-stressed at 42°C in an incubator with 95% air and 5% CO2 for different periods. Levels of Hsp90α, protein localization, enzymes, and cytopathological lesions were detected using Western blot, immunocytochemistry enzymatic assays, and cytopathological techniques. Aspartate aminotransferase, lactate dehydrogenase, and creatine kinase enzyme levels were elevated during heat stress, and acute cellular lesions that were characterized by vacuolar degeneration and necrosis were observed. Hsp90α levels decreased between 10 and 60 min of heat stress and increased after 360 and 480 min, while Hsp90α mRNA decreased after 360 min. These results indicate that heat stress might induce irreversible damage in certain myocardial cells. The elevated Hsp90α level at the end of heat stress and its positive signal in the cytoplasm of myocardial cells after heat stress could be associated with its protective role. Additionally, the consumption of Hsp90α exceeded its production in the first period of treatment.

The role of Hsp90α in heat-induced apoptosis and cell damage in primary myocardial cell cultures of neonatal rats.

To understand the mechanism underlying the sudden animal death caused by acute heart failure during heat stress, the relationships among the heat-induced pathological changes and apoptosis and the variations in the levels of protective Hsp90α and its mRNA in the heat-stressed primary myocardial cells of neonatal rats in vitro were studied by cytopathological observation, immunoblotting, RT-PCR, and analysis of the related enzymes. After a period of adaptive cell culture, the myocardial cells were immediately exposed to heat stress at 42°C for 10, 20, 40, 60, 120, 240, 360, and 480 min. Levels of creatine kinase increased from the beginning of heat stress, and the cells exposed to heat stress showed acute cellular lesions characterized by vacuolar degeneration and necrosis after 40 min of heat stress, suggesting that the myocardial cells in vitro were obviously stressed and damaged by higher temperature. The levels of cleaved caspase-3 and cytochrome C, which were related to apoptosis, increased significantly after 40 min of heat stress while the Hsp90α protein level significantly decreased. In contrast, after 6 h of exposure to heat stress, the levels of cleaved caspase-3 and cytochrome C decreased while those of Hsp90α significantly increased, suggesting that early depletion of Hsp90α coincides with a high rate of necrosis and apoptosis in heat-stressed myocardial cells, while the Hsp90α level in surviving cells increases again with significantly less apoptosis after 6 h of heat stress. These findings also indicate that apoptosis of myocardial cells occurs through the activation of the cytochrome C and caspase-3 pathway. The cell repair capacity of Hsp90α is overstrained in the early phase of heat treatment and needs some hours to stabilize. As a result, in the primary myocardial cells in vitro, Hsp90α shows protective activity against damage at the end period of the heat exposure.