| Stress factors including long-distance transportation for livestock trade and higher temperature in living environment for poultry often cause the stressing damage, PSE (pale, soft, exudative) meat, and even to death in the effected animals. It is necessary to study the relation between stress and its damage to stressed animals and provide reasonable methods to reduce the stress influences to the farming industry. Heat shock proteins (Hsps), widespread in the organism from yeast to mammalian, are some endogenous proteins which have protective role. Furthermore, they can overexpress stably in the organism under physiological conditions or stress conditions, and act as the role of molecular chaperone. Hsp110is one of the most important of heat shock proteins family (HSPs) and can protect cells or targeted protein against stressed damages. The overexpression of Hsp110can indentify the denatured proteins and make them keep in a stable and soluble state, and then restore the activities of the denatured protein. In view of consideration that sudden death of stressed animal might be the damage of myocardial cells and heart failure, the protective role and mechanism of Hsp110against stressing damages during heat stress were studied through establishing the mode of myocardial cells stressed by heat in vitro, observing the expression of Hsp110and transcription of hsp110mRNA as well as the correlations between the variations of Hsp110expression and stressing damages of myocardial cells in vitro.The optimizations of the culture conditions of in vitro myocardial cells were carried out by using cell culture techniques, cell growing on coverslips technology, immunofluorescence technique, histological techniques and MTT assay etc. The results showed that the purity of the myocardial cells incubated in vitro were up to94%and met the test requirements under the following conditions, the cells quantities of the immunofluorescence test were plated at a total of1.8-2.0×106cells/mL, the cells quantities of western blot test were plated at a total of2.2-2.3×106cells/mL, the cells quantities of the MTT test were plated at a total of0.25-1x106cells/mL. The results also showed that it was necessary to obtain optimal primary myocardial cells by selecting2-day-old SD neonatal rats, digesting heart tissue with collagenase I for14h, blowing gently heart tissue mass7-9times with the pipette, making the cells adherent and differential for60min, adding0.1-0.3M BRDU into the cells after cell adherence, seeding cells into the dishes with point by point.After incubated at37℃for72h, myocardial cells were heat stressed in a incubator at42℃with a humidified atmosphere of5%CO2and95%air for0min,10min,20min,40min,60min,120min,240min,360min, and480min, respectively. The correlations between the cell damage and the variation of Hsp110expression were carried out by using the histological examination, special myocardial enzyme detections, the immunofluorescence technology, real time PCR technology as well as western blot technology. Significant increases of AST, LDH, and CK enzymatic activities in the suspension of myocardial cell were observed during the heat stress, suggesting that the integrity of the myocardial cells was altered. The results of HE staining showed that acute cell swelling characterized by a number of red granules in the cytoplasm deeply stained and concentrated nuclei were observed during heat stress from40min to120min. The results of the western blot test displayed that the overall levels of Hsp110expression increased significantly after20min and the Hsp110levels were approximately1.2-fold higher than that of the control after240min of heat stress. Immunocytochemical analysis revealed that the expressed Hsp110was constitutively localized in the cytoplasm with small amounts in the nuclei characterized by a granular pattern, nuclear Hsp110levels increased significantly after240min of heat stress in comparison with the control. The results of real time PCR proved that increasing levels of hsp110mRNA were observed after20min of heat stress, and the levels peaked with a10-fold increase after240min of heat stress. These results above indicate that the expression of Hsp110in primary myocardial cells in vitro is sensitive to hyperthermic stress by increasing both hsp110mRNA transcription and Hspl10expression levels, Hsp110is involved in the potential acquisition of thermotolerance after heat stress. Therefore, Hsp110might play a fundamental role in opposing and alleviating the stress damages caused by hyperthermic stress in primary myocardial cells.After incubated at37℃for72h, myocardial cells were heat stressed in an incubator at42℃with a humidified atmosphere of5%CO2and95%air for0min,10min,20min, 40min,60min,120min,240min,360min, and480min, respectively. The results of the immuofluorescence test showed that Hsp110positive staining distributed with enrichment in the cytoplasm and small amount in the nuclei, however, Hsp110increased both in the cytoplasm and the nuclei of myocardial cells after4h of heat stress. In contrast to Hsp110, HSF-1staining was only observed in the nuclei of primary rat myocardial cells. Both of Hsp110and HSF-1increased in the heat-stressed myocardial cells, meanwhile the increasing of hsp110mRNA and HSF-1mRNA transcript were observed after120min of heat stress at42℃. The results showed that Hsp110and HSF-1were sensitive to heat stress in a time-dependent manner. The characteristics of Hsp110inductions during heat stress, nuclear HSF-1expression initial decrease and then significant increase between240and480min, suggesting that both Hsp110and HSF-1have different roles in protecting myocardial cells from heat stress damage.Rat primary myocardial cells in vitro were pretreated for1h with N-acetyl-L-cysteine (NAC) and then heat-stressed. The proteins associated with mitochondrial apoptosis signaling pathway, nuclear transcription factor (NF-κB) signaling pathway as well as the expression levels of Hsp110in the heat stressed myocardial cells were studied. The results showed that NAC inhibited significantly myocardial cells apoptosis induced by heat stress within1h, and the role became weaker with the elongation of heat stress. However NAC inhibited significantly the expression levels of NF-κB belonging to NF-κB signaling pathway.After incubated at37℃with lipofectamine2000for48h and Hsp110siRNA primer for24h, then continued to incubate the cells until84h with or without transfection mixture solution including Hsp110siRNA primer and lipofectamine2000at37℃, and then the transfected myocardial cells were harvested at24h,36h,48h,60h,72h,84h, respectively. The correlations among the expression Hsp110, the transfection methods, silencing time as well as with or without serum in the media were studied in the rat primary myocardial cells silenced by Hsp110siRNA primer in vitro. The results showed that the relative expression levels of Hsp110in the myocardial cells between the negative control and the test groups reached the peak level from36to48h and decreased gradually after discarding the transfection mixture solution. The silencing efficiency within36to72h was about40%as well as the silencing efficiency at84h was about82%; Hsp110expression in the myocardial cells of the negative control reached the peak level at72h and declined gradually, however, Hsp110expression of the test groups went up to the top level at36h and declined gradually with the transfection mixture solution and arrived the lowest levels at84h. The silencing efficiency of Hsp110in the test group from72to84h was about from86%to95%. The results indicate that the effect of transfection reagent and Hsp110siRNA on the transfection and silencing efficiency is significant. Furthermore, the optimal silencing efficiency of Hsp110could be obtained through silencing the cells from72to84h with the transfection solution containing40-120nM Hsp110siRNA primer.The myocardial cells incubated at37℃for48h were transfected with Hsp110siRNA primer and continued to be incubated for60h at37℃. After changed the medium with DMEM medium containing15%FBS and continued to incubate the cells for12h at37℃, the myocardial cells were stressed in a incubator at42℃with a humidified atmosphere of5%CO2and95%air for4h. The comparative study of the correlations among the expression levels of Hsp110, the levels of the related enzyme including AST, CK, CK-MB, LDH in the suspension of myocardial cell, as well as the apoptosis of the myocardial cells were carried out by using Western blot test, E1ISA test, flow cytometry test. The results showed that the expression levels of Hsp110reduced significantly in the myocardial cells of the test groups. The levels of enzyme AST, CK, CK-MB which indicating myocardial cells damages rose without significant difference, as well as the early and late apoptosis rate reduced without significant difference, suggesting that the present results are different from the former reports, the protective role and mechanism of Hsp110during heat stress still need to be verified further. |
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