The Mechanism Of Heat Shock Proteins Expressions And Stressing Damages Of Tissues And Cells During Stress

The intensive management systems of the swine industry in China are resulting more and more long distance requirement of pig transportation, and increasing serious economic losses due to the transporting. Therefore, it is very important to study the characteristics of transport-induced injury for reduce the stress injury in pig industry. Heat shock proteins (HSPs) are a group of highly conserved proteins, which play important roles in the protection of cells and stabilization of internal envrionment. The main purpose of this study was to investigate the transcription and expression of HSPs and their corresponding mRNAs in the tissues of transport stressed pigs, and to correlate these HSPs levels with tissue damages at various stressing times, through the establishment of transport stress model in vivo; and to study on the mechanism of cytoprotective functions of HSPs, through establishment of heat stress model by primary culturing myocardial cells of rats in vitro.Twenty hybrid pigs from Erhualian and Pietrain strains were selected and raised in individual pens (2.5×3.0 m2). The pigs were randomly placed into 4 groups of 5 pigs each. The mean weight of the pigs was approximately 50±2 Kg (mean±SD). On the day of the transport trial, one group was maintained under normal housing conditions and served as control (not transported animals), while the other three groups were transported for 1,2 or 4 h, respectively. The route included an equivalent mix of local roads, including town traffic, state roads and highways. The average speed was 30 to 40 km per hour. Immediately after the end of the transport, all animals were euthanized by jugular injection with 10 mg/kg of 3% sodium pentobarbital while on the truck or in the animal house. Blood samples for serum enzyme activities were frozen at-20℃until further analysis. Tissue specimens were taken from heart, liver, stomach and M.longissimusdorsi (LD) and fixed in paraformaldehyde solution for histopathological examination. Additional samples were placed into 1.5 mL tubes and frozen in liquid nitrogen for later evaluation of HSPs expressions and their corresponding mRNA transcriptions. Primary cultured rat myocardial cells were divided into 9 groups (control and heat stress 10 min,20 min,40 min,60 min, 120 min,240 min,360 min and 480 min) after 72 h culture. After heat stress, the supernatants were obtained for enzyme detection. The myocardial cells were taken from each group for the experiments of immunohistochemistry, western blot, real-time quantitative PCR, and flow cytometry.A significant increase of ALT, AST, CK、LDH and Cr in the blood serum and acute parenchyma cell lesions characterized by acute degenerations in the heart and liver were observed. Hsp27 expression levels increased significantly in the heart after 2 h and in the liver after 4 h of transportation accompanying with the hsp27 mRNA increasing significantly in the heart and liver after 1 h of transportation. aB-crystallin expression levels were fluctuant (not significantly) in the heart and liver during transporting, however, aB-crystallin mRNA increase notably in the heart after 1 h and decrease significantly in the liver at 1 h and 2 h of transportation. In conclusion, the cellular damage to the heart and liver is highest after 1 h of transportation, Hsp27 and aB-crystallin play dissimilar roles and show tissue-specific response in different tissues during transportation.Pathological examination of all transported pigs showed that exfoliation of chief cells from the gastric surface occurred in pigs during transportation. These results imply that integrity of the gastric mucosa was compromised by damage occurring during the 4 h of transportation, despite the fact that gastric ulcers were not present. Levels of Hsp90 expression in stomach tissues were significantly decreased (P<0.01) after 2h of transportation, but Hsp70 levels increased significantly (P<0.05) after 1,2, and 4 h of transportation. Hsp27 levels remained relatively stable independent of the length of transport. Levels of aB-crystallin expression in the stomach were significantly increased (P <0.05) after 4 h of transportation. Variations in Hsp90, Hsp70, Hsp27, and aB-crystallin levels suggest that distinct protective functions are modulated by different HSPs in stomach tissues during transportation. Alterations in Hsp70 and aB-crystallin expression appear to be associated with protective functions, as no apparent gastric ulcers were present in pigs that underwent 4 h of transportation. Levels of HSF-1, which regulates the expression of HSPs, remained relatively stable independent of the transportation period. This observation indicates that the change of the HSF-1 expression levels is not the only factor which regluates the expressions of HSPs in pigs during transportation.The LD meat from 1 h and 2 h transported pigs had lower initial and ultimate pH values (pHi and pHu, respectively), higher drip loss and L* values compared to controls, indicating a higher likelihood of pale, soft and exudative (PSE) meat. Meat quality was lower after 2h compared to 1 h or 4 h of transport. All four HSPs tested (alpha-B-crystalline, Hsp27, Hsp70 and Hsp90) by ELISA in the LD tissue of pigs tended to decrease after transportation. One possible mechanism resulting in poor meat quality in the LD after transport seems to be a decline in Hsp expression.Levels of ALT, LDH, CK and CKMB in the medium were increased in different degrees. The results of immunofluorescence show that Hsp27 distributes in the cytoplasm and nucleus dynamically before and after heat stress. However, HSF-1 distributes in nucleus stably. Hsp27 protein level was increased after 8 h heat stress significantly, HSF-1 protein levels were decreased at 10 min,40 min,60 min, and 120 min after heat stress, and increased after 240 min stress significantly. The results of FQ-RT PCR show that hsp27 mRNA and hsf-1 mRNA levels were increased significantly after 10 min heat stress. The results indicating that the myocardial cells of rats were injuried during heat stressing, Hsp27 play a protective function during heat stressing. Hsp27 and HSF-1 protein levels were not coincides with their corresponding mRNA levels may be caused by phosphorylation.The result of flow cytometry shows that only at 40 min and 480 min are extremely significantly (p<0.01) and significantly (p<0.05) higher than that of the corresponding control groups. Suggesting that the myocardial cells start some kinds of self-protection mechanisms to adapt to the environment. Western blot results show that the levels of cleaved caspase-3 were increased significantly after 20 min, and continued to 480 min. The changes in levels of cytochrome C are similar to that of cleaved caspase-3, suggesting the two proteins are closely related during heat stress.