| Higher temperature in living environment and higher stocking density for animal often cause the stressing damage. Heat stress response is a whole systematical response. It has an important effect on nerve and endocrine system, blood circulation, digestion and absorption, substance metabolism, immune system, urinary and reproductive system. Heat stress can cause animals tissue injury, reducing production, even death, cause serious economic losses in livestock. Therefore, it is very important to study the characteristics of heat stress induced injury for reduce the stress influences in livestock. Heat shock proteins (HSPs) are a group of highly conserved proteins and widespread in the organism from yeast to mammalian. They can overexpress stably in the organism under physiological conditions or stress conditions. HSPs play important roles in the protection of cells and stabilization of internal envrionment. HSP70 protein family which is the most widely expressed protein in HSPs, because it has to promote protein synthesis, folding, transport and removal of denatured proteins, which has become the most widely studied HSPs. The main purpose of this study was to investigate the transcription and expression of HSP70 and their corresponding mRNA of the heat stress rat myocardial cells in vivo and in vitro, and to correlate the levels of HSP70 with tissue damages during heat stress, through the establishment of heat stress model in vivo and in vitro, and to study on the mechanism of cytoprotective functions of HSP70. Through RNA interference in H9c2 suppress Hsc70 protein expression, to explore the role of Hsc70 anti-apoptosis in H9c2 myocardial cells.220 ± 20 g of SD rats (n=60) were randomly divided into six groups (10 rats per group) in a controlled climate chamber. In order to reduce the inifluence of environmental stressor, the rats were given a 20-day period to acclimate to their new surroundings. After 20 days of feeding adaptation, control group still placed in room environment (22±1℃). Heat stress group temperature was suddenly increased from 22±1℃to 42±1℃ within 30 min and the rats were heat stressed for 20 min,40 min,60 min,80 min and 100 min respectively. On terminating the heat treatment, the plasma samples were obtained from eyeball for subsequent CK, CK-MB and LDH detennination. Following broken neck, the rats were manually eviscerated, and the heart were quickly dissected and fixed in 10% neutral formalin, after 24h change into fresh neutral formalin, for histological analysis and Irnmnunohistochemistric analysis. Further tissues samples were frozen in liquid nitrogen, and then stored at-70℃ for Western Blot detection and realtime RT-PCR analysis. After incubated at 37℃ for 48 h and cell fusion more than 90%, H9c2 myocardial cells were heat stressed in a incubator at 42℃ with a humidified atmosphere of 5%C CO2 and 95% air for 0 min,20 min,40 min,60 min,80 min and 100 min, respectively. After heat stress, the suspension was obtained for enzyme detection. The myocardial eells were taken from each group for the experiments of immunohistochemistry, Westen Blot and real-time quantitative PCR.Significant increases of CK, CK-MB and LDH enzymatic activities in the supernatant of H9c2 myocardial cell were observed after heat stress. It suggestted that the integrity of the myocardial cells was altered. The vivo enzyme results indicated that the concentrations of plasma CK, CK-MB and LDH increased obviously with heat stress, implying damages to myocardial cells were more serious as the time of heat stress prolonged. Histological results showed that after heat stress numerous fine granular particles in the cytoplasm and enlarged cellular size were found in myocardial cell in vitro. After heat stress, the gap between the myocardial fibers was widened, numerous fine granular particles in the cytoplasm and enlarged cellular size in vivo. The died rats myocardial fibers arranged disorder, a large number of muscle fibers rupture, blood capillary was expanded and filled with erythrocytes. Enzyme results and histopathological results implied that myocardial damages were closely related to the detected plasma enzyme activities in vitro and in vivo. The concentration of plasma enzyme can indirectly reflect stress-induced cell injury in vitro and in vivo.Immunohistochemistry results showed that Hsc70 can express and distribute in the control group and the heat stress myocardial cells in vitro and in vivo, and mainly distributed in the cytoplasm. The difference is Hsc70 positive signals in H9c2 cells were weaked after heat stress, however in myocardial fibers Hsc70 positive signals were enhances after heat stress then weakened. Hsp72 positive signals only exist in heat stress myocardial cells in vitro and in vivo, control group do not exist. In dead rats Hsc70 mainly distributes in the cytoplasm and unevenly distributed, sparse intracytoplasmic and intranuclear labeling due to the presence of small granular Hsp72 particles was observed. The results of the Western Blot displayed that H9c2 cardiomyocytes showed expression of Hsc70 was significantly decreased after 60 min heat stress and the levels of Hsc70 was lower than the control group during heat stress in vitro, while the expression of Hsc70 were significantly increased after 40 min heat stress, with the prolonged heat stress began to decrease and at the time of 100 min heat stress it was significantly lower than the control group. Levels of Hsp72 almost can not be deteted in the initial heat stress and significantly increased after heat stress in vivo and in vitro, however expression of Hsp72 in vitro was earlier than in vivo. The results of the FQ-RT PCR showed that hsp70 mRNA levels were significantly increased after heat stress in vivo and in vitro, however hysteresis is involved in mRNA expression after heat stress in vivo compared with in vitro. The results showed that the rats myocardial cells were injuried during heat stress in vivo and in vitro; expression of Hsp70 gene and protein significant changes in myocardial cells to reveal that Hsp70 was involved in the potential acquisition of thermo tolerance after heat stress. Therefore, Hsp70 might play a fundamental role in opposing and alleviating the stress damages caused by hyperthermic stress in primary myocardial cells.H9c2 cells were cultured and were treatmented as three groups:control group, heat stress group and siRNA+ heat stress group. In order to achieve optimum condition of siRNA transfection, siRNA gene sequences, and transfection time were tested. The expression of Hsc70 and apoptosis signaling pathway were determined by Western Blot. The results indicated that the best transfection conditions were with 80 nmol/L FAM-siRNA+1.5μL Lipofectamine 2000, harvest at 48 h after transfection and with the best siRNA gene of oligo 3. Flow cytometric results showed that apoptosis was significantly decreased compared to the control group after 120 min of heat stress (P< 0.01), And apoptosis was decreased in the group of heat stress siRNA+heat stress compared to heat stress group after 240 min of heat stress (P< 0.05). After heat stress, the viability of H9c2 cells was decreased. Western Blot results showed that the level of cytoplasm Hsc70 in heat stress group was decreased, the level of cytoplasm Hsc70 in heat stress group was significantly decreased. The level of Hsp72 in heat stress+ siRNA group was significantly increased than that of heat stress group. After heat stress, the apoptotic genes such as cleaved PARP, AIF, cleaved caspase-3 and Bax expression were significantly increased, while the anti-apoptotic genes such as Bcl-2 expression was decreased. The expression of GRP78 reduced, the expression of caspase-12 was increased. The expression of Fas and FADD did not have significant changes. Compared with heat stress group, the expression of cleaved PARP, AIF, cleaved caspase-3, Bax and GRP78 in siRNA+ heat stress group did not change significantly, However, caspase-12 was significantly decreased, And Fas and FADD were significantly increased. The level of Hsc70 in nucleus was significantly increased in heat stress and siRNA+ heat stress groups, however it was decreased after 120 min of heat stress. The level of Hsc70 in nucleus in siRNA group was significantly higher than that of heat stress group. In nucleus the expression levels of Hsp72, AIF and cleaved PARP were increased. The results indicate that apoptosis cells are significantly increased after Hsc70 gene keep silence. Hsc70 plays protective role on apoptosis induced by heat stress through the endoplasmic reticulum pathway and the death receptor pathway. |
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