ABSTRACT
OBJECTIVE@#To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism.@*METHODS@#Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells.@*RESULTS@#With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure.@*CONCLUSION@#Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.
Subject(s)
Humans , Apoptosis , Cell Line , Epithelial Cells , Pathology , Hot Temperature , Membrane Potential, Mitochondrial , Nuclear Proteins , Genetics , Metabolism , Reactive Oxygen Species , Metabolism , Superoxide Dismutase , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the changes of expression and subcellular location of nuclear growth-induced protein-B(NGFI-B) in cardiomyocytes of stressed rats and its biological effect and to provide scientific evidences for exploring the mechanism underlying myocardium injury induced by stress.</p><p><b>METHODS</b>The cell model of stress-induced cardiomyocyte injury were established. Western blot method and confocal microscopy method were used to investigate the subcellular location of NGFI-B in cardiomyocytes under stress. The flow-cytometry was selected to detect the apoptotic rate in cardiomyocytes in vitro. Western blot method was used to determine the content of cytochrome C protein in mitochondria and cytoplasm respectively.</p><p><b>RESULTS</b>Stress induced the increase of NGFI-B content in the mitochondria of cardiomyocytes and the translocation of NGFI-B from the nucleus to the mitochondria. The translocation of NGFI-B promoted the release of cytochrome C from the mitochondria and the cardiomyocyte apoptosis. Treatment of stressed cardiomyocytes with leptomycin B, a non-specific blocker of nuclear export, resulted in nuclear retention of NGFI-B and abrogated its ability to induce the release of cytochrome C from the mitochondria.</p><p><b>CONCLUSION</b>Stress could induce NGFI-B translocation from the nucleus to the mitochondria in cardiomyocytes, which activated the mitochondrial pathway of cell apoptosis.</p>
Subject(s)
Animals , Male , Rats , Animals, Newborn , Apoptosis , Cells, Cultured , Myocytes, Cardiac , Cell Biology , Metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1 , Metabolism , Rats, Wistar , Stress, PhysiologicalABSTRACT
<p><b>AIM</b>To construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection.</p><p><b>METHODS</b>The specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting.</p><p><b>RESULTS</b>The DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1.</p><p><b>CONCLUSION</b>A RNAi eukaryotic vector containing prohibitin gene was successfully constructed.</p>
Subject(s)
Humans , Genetic Vectors , Genetics , HEK293 Cells , MicroRNAs , Genetics , RNA Interference , RNA, Small Interfering , Genetics , Recombinant Proteins , Genetics , Repressor Proteins , Genetics , TransfectionABSTRACT
<p><b>AIM</b>To study the effects of stress on the cognitive function and physical fitness and its biological mechanisms, provide scientific basis for seeking protective measures to reduce stress-induced damage.</p><p><b>METHODS</b>The model of restraint stress was adopted in our experiment. Step-down test and exhaustive swimming test were used to measure the learning and memory function and physical fitness in mice, respectively. The contents of GC in plasma, hippocampus LTP, ECG, myocardial ultra-structure, cell apoptosis rate and the level of Hsp70 expression of myocardium in rats were detected.</p><p><b>RESULTS</b>Compared with control group, the impairment of learning and memory function and decline of exercise tolerance were observed in restraint stress group. The elevation in plasma GC levels, ECG abnonrmality, and cell apoptosis rate were also observed under restraint stress. Furthermore, myocardial structure was damaged, and myocardial Hsp70 expression and hippocampus LTP were suppressed in restraint stress group than those in the control.</p><p><b>CONCLUSION</b>Stress may cause the neuro-endocrine dysfunction and homeostasis disorder, and then, lead to the cognitive function and physical fitness damage consequently.</p>
Subject(s)
Animals , Male , Mice , Rats , Cognition , Physiology , HSP70 Heat-Shock Proteins , Metabolism , Hippocampus , Physiology , Long-Term Potentiation , Physiology , Myocardium , Metabolism , Physical Fitness , Physiology , Random Allocation , Rats, Wistar , Restraint, Physical , Stress, Physiological , PhysiologyABSTRACT
<p><b>AIM</b>To explore the level of anti-HSP70 antibody in plasma during atherosclerosis procedure induced by high-fat diet in rat and the relationship of them.</p><p><b>METHODS</b>Twenty eight rat were divided into high-fat diet group (H) and control group (C). The total cholesterol (TC), Glyceride (TG), low density lipoprotein cholesterol (LDL-C) in serum, pathology change of rat Arch of the aorta were determined, the level of anti-HSP70 antibody and their Phenotype were evaluated by ELISA.</p><p><b>RESULTS</b>After two weeks, the serum concentrations of TC and LDL-C in rat supplemented by high-fat diet were significantly higher than those in control group (P < 0.01), the serum TG were much lower than those in control group (P < 0.01). Four weeks later the level of anti-HSP70 antibody, IgM, IgG phenotype were significantly higher than those in control group (P < 0.01). There were lipin deposition and mottling formation in rat Arch of the aorta in rat supplemented by high-fat diet in 12th week.</p><p><b>CONCLUSION</b>Atherosclerosis could be induced by high-fat diet in rat. Accompany with the atherosclerosis procession, the level of anti-HSP70 antibody was continuously elevated, the level of anti-HSP70 antibody was related to atherosclerosis. The level of anti-HSP70 antibody was closely associated with atherosclerosis.</p>
Subject(s)
Animals , Male , Rats , Antibodies , Blood , Atherosclerosis , Allergy and Immunology , Diet, High-Fat , Dietary Fats , HSP70 Heat-Shock Proteins , Allergy and Immunology , Immunoglobulin G , Blood , Immunoglobulin M , Blood , Rats, WistarABSTRACT
<p><b>AIM</b>To assay the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.</p><p><b>METHODS</b>The total length of hHSF1 exon was amplified by PCR method and cloned into pcDNA3. 1(+) vector. pcDNA3. 1(+)-hHSF1 and pGI3 prohibitin were co-transfected into HEK293 cells. The luciferase activity was detected by Dual-Luciferase Reporter Assay System to evaluate the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.</p><p><b>RESULTS</b>The pcDNA3.1(+)-hHSF1 vector was constructed successfully. The assay of luciferase activity showed that the transcription of pGL3-prohibitin was dramatically upregulated by hHSF1.</p><p><b>CONCLUSION</b>hHSF1 can transcriptionally regulate prohibitin expression.</p>
Subject(s)
Humans , Base Sequence , DNA-Binding Proteins , Physiology , Gene Expression Regulation , HEK293 Cells , Heat Shock Transcription Factors , Molecular Sequence Data , Promoter Regions, Genetic , Repressor Proteins , Genetics , Transcription Factors , Physiology , Transcription, Genetic , TransfectionABSTRACT
<p><b>AIM</b>To assay the transcriptional activation effect of TR3 and it's deletion mutation in yeast two hybrid system.</p><p><b>METHODS</b>The total length of TR3 and TR3/delta1-690 gene was amplified by PCR method and cloned into pGBKT7 vector. Bait vector of pGBKT7-TR3 and pGBKT7-TR3/delta1-690 was transformed into AH109 competence yeast. Then self activation of the recombination vector was tested by assay the activity of beta-galactosidae.</p><p><b>RESULTS</b>The pGBKT7-TR3 and pGBKT7-TR3/AM 690 vector was successfully constructed. The filter paper containing beta-galactosidae didn't changed to blue showed that the reporter gene wasn't activationed.</p><p><b>CONCLUSION</b>TR3 and TR3/delta1-690 hadn't the activity of transcriptional activation.</p>