Anti-heat Stress Protection And Its Mechnism On Chicken Myocardial Cells By Induced Hsp90 Expression

Organs and tissues (especially for heart) of animals can be seriously damaged by long period of time and intense stress. Numerous stressors influence the production and reproduction of livestock and poultry. For instance, heat stress can lead the reduction of chicken growth rate and egg production,even the sudden death,which is closely related to the heat stress-induced heart diseases. When exposed to stress, adaptive defensive reaction,namely stress reaction, is caused in the organism. During the process of heat stress, the body's cells synthesize rapidly a series of heat shock protein (HSPs) under the regulation of various heat shock factor (HSF). HSPs can protect effectively cells from various stress damage and apoptosis, and HSP90 family is one of the most important heat shock protein family. Hsp90 can help its client proteins to maintain mature and stability of molecular structure, and most of these client proteins are the key proteins for cellular pro-survival/anti-apoptotic signal transduction pathways. Aspirin can reduce the body temperature threshold and induce more rapidly self-protective reaction, such as the induction of Hsp70 high expression during exposure to heat stress. In the present study, on the basis of successful establishment of the heat-stress models in chicken myocardial cells in vivo and in vitro, the effects of ASA on Hsp90 expression of chicken myocardial cell in the process of heat stress and its effect on the resistance to cell pathological damage/apoptosis were studied, and the mechanism of regulation and protection of ASA was also investigated through the detection of HSFs dynamic curve and Hsp90 related signal transduction pathways. At the same time, with ASA as Hsp90 inducer and geldanamycin as inhibitor, the effects of Hsp90 expression on resisting heat-stress damage/apoptosis and its mechanism in the primary chicken myocardial cells was also systematically studied.The heat-stress model of the chicken myocardial cells in vivo and in vitro was firstly established with Specific Pathogen Free (SPF) chicken of 21 day age and chicken primary myocardial cells, respectively, and randomly divided into three groups, namely heat stress group (HS), aspirin+heat stress group (ASA+HS) and aspirin group (ASA). ASA (1 mg/kg body weight) was oral fed to the chicken in group ASA+HS and ASA 2 h prior to heat stress, meanwhile, the primary cells were treated with ASA at 1 mg/mL in vitro. The exposure of heat stress for HS and ASA+HS group was executed at 42?. 10 chickens/ 3 cell plates were selected randomly at 0 h,1 h, 2 h,3 h,5 h, 7 h,10 h,15 h and 24 h respectively, and sampled. Blood and cells culture supernatants were used to detect CK,AST and LDH activities, and heart tissues/primary myocardial cells were collected and fixed in 4% paraformaldehyde solution. The results showed that after exposure to heat stress, mouth breathing, increased breathing rate, increase of water quantity and decrease of food intake was observed in the chicken in HS group, and body temperatures of several chickens were increased to 43 ?. The clinical symptoms in ASA+HS were more mild.Heat stress induced the increases of plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After ASA administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. In addition, in the in vitro trial, significant induction of enzymes levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterized by acute degeneration, karyopyknosis and karyorrhexis were observed in the HS group, compared with the non-treated cells.However, the lesions of cells treated with ASA were milder,characterized by earlier recovery of enzyme levels to the level of the non-treated cells and no obvious heat-stress related cellular necrosis. In conclusion, ASA could weaken significantly the damage degree of heat stress on chicken myocardial cells in vivo and in vitro.broiler chickens were administered ASA (1 mg/kg body weight) 2 h before exposure to heat stress, and heat stressed at 42 ? for 0, 1, 2, 3, 5, 7, 10, 15 and 24 h. With the immunohistochemical detection and molecular biological detection method, the expression changes of hsp90 mRNA, Hsp90 and HSF during exposure to heat stress were observed to study whether ASA weakened the pathological injury of myocardial cells through its regulation of Hsp90 expression. The results showed that the induction and consumption of HSF-1, and reductions of HSF-2 and HSF-3 induced by heat stress led to a delay in hsp90 mRNA transcription. Administration of ASA changed the expression patterns of HSF-1, 2 and 3 such that significant expression of hsp90 mRNA was stimulated throughout the experiment, and rapid and significant synthesis of Hsp90 was induced before and at the initial phase of heat stress when compared with cells exposed to heat stress alone, A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with ASA, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. The residence time of Hsp90 in aortic endothelial cell was also increased. The present study implied that specific pre-induction of Hsp90 and its distribution changes in cardiovascular tissue through the regulation of ASA on HSFs may be association with resisting heat-stress damage.The transcription levels of hsp90 mRNA, the expression levels of Hsp90, the expression and activation of anti-apbptosis key protein Akt and the apoptosis rate in the chicken heart tissues after ASA oral administration following exposure to high temperature for varying times were observed and studied, by means of RT-PCR, western blot, ELISA and TUNEL methods. The results showed that after exposure to heat stress, hsp90 mRNA transcription and Hsp90 expression was delayed, which in turn inhibited the synthesis and activation of Akt,which resulted in a significant increase in caspase-3 and caspase-9 activities and the heat-stressed apoptosis of the myocardial cells. The pre-administration of ASA up-regulated significantly the expression of hsp90 gene and Hsp90 protein (P<0.01 for the gene expression and by 2.3 - 4.1 times compared with the control for the protein).The resultant increase in Akt expression and activation inhibited caspase-3 and caspase-9 activities and reduced the myocardial cells apoptosis rate (by 2.14 - 2.56 times). The results demonstrated that ASA administration could inhibit heat-stressed apoptosis of myocardial cells in vivo and maybe closely associated with its promotion of heat stress response of chicken hearts, especially Hsp90 expression.With the techniques of RT-PCR, Western blot, immunocytochemistry and cytological detection, the pathological damage, the transcription level hsp90 mRNA and the induction of Hsp90 expression of ASA-treated primary chicken myocardial cells exposed to heat stress were detected for studying the mechanism of ASA inducing Hsp90 expression and remitting heat-stress damage. In the present study, primary chicken myocardial cells were treated with ASA (1 mg/mL) 2 h in advance, and exposed to heat stress. To understand the induction effect of ASA on Hsp90 in the chicken primary myocardial cells, and its potential protection against heat-stress damage, we investigated the enzyme activities related to stress damages, the cytopathological changes, the expressions and distributions of Hsp90,and hsp90 mRNA levels in the myocardial cells exposed to heat stress with/without ASA administration (1 mg/mL, 2 h prior) in vitro. Results show that ASA at the concentration of 1 mg/mL does not affect the viability of myocardial cell, and could significantly induce the high-expression of Hsp90. After exposure to heat stress at 42 ? for different durations,immunofluorescence showed that stronger positive signals in the cytoplasm and longer signal retention of Hsp90 in nuclei were observed in ASA-treated myocardial cells than those of only heat-stressed cells. Hsp90 level in the myocardial cells treated by ASA was 11.1-fold higher than the control level, and remained relative high at early stage of heat stress, while it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to low level. Meanwhile, the overexpression of hsp90 mRNA in ASA-treated cells was observed throughout the experiment,while hsp90 mRNA transcription level decreased significantly in only heat-stressed cells. The present results indicated that ASA treatment could induce significantly the high expression of Hsp90, and the pre-induction of Hsp90 over-expression with ASA ensured and maintained a higher level of Hsp90 in the chicken primary myocardial cells during the exposure to heat stress, especially in the nucleus, which may be closely related to the anti-heat stress capacity of cells.Based on the the induction effects of ASA and the inhibition effects of geldanamycin(GA) on Hsp90 expression, the expression levels of Hsp90, the cell viabilities, apoptosis and the levels of reactive oxygen species (ROS) in the myocardial cells after heat stress were evaluated, by using of the western blot, MTT method, flow cytometry and ROS detection. The results showed that in initial period of heat stress, the cell viability of myocardial cells, apoptotic rate and oxidative stress level was affected significantly, and with heat stress, the damage degree was increased. GA treatment at 0.1 ?M and above could effectively restrain the expression level and function of Hsp90 in chicken primary myocardial cells. ASA treatment could significantly (P<0.05) stimulate Hsp90 expression of tested cells exposed to heat stress, and the viability of myocardial cells was better, while the apoptotic rate and oxidative stress level is lower; GA could down-regulate (P<0.05) the Hsp90 expression after exposure to heat stress, accompanied with the lower viability of myocardial cells, and the higher apoptosis rate and oxidative stress level; Additionally,compared with those of only GA and heat-stress treated myocardial cell, ASA after GA treatment partially increased the Hsp90 expression, which was connected with same cell viability and relatively lower apoptosis rate and ROS level. Therefore, Hsp90 expression has a positive connection with the cell viability,apoptosis rate and oxidative stress level,in other words, the capacity of anti-heat stress injury of chicken primary myocardial cells.The chicken primary myocardial cells were treated with the Hsp90 expression promoter, ASA, and the Hsp90 inhibitor, geldanamycin (GA), before heat stress treatment.The caspase activities in the heat-stressed myocardial cells,the key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected and analysized in this section. The results showed that the expressions of Akt, STAT-3 and p-IKK?/? were effectively increased after ASA treatment. The colocalization of Akt and STAT-3 with Hsp90 during heat stress were increased,accompanied with the lower activities of caspases in the heat stressed myocardial cells. The levels of Akt and p-IKK?/? were inhibited significantly, and the level of STAT-3 was highly induced after GA treatment. The colocalization of Akt and STAT-3 with Hsp90 was weakened, accompanied with higher caspases activities. ASA administration after GA treatment, the caspases activities in the tested cells were partially inhibited, the expression of Akt and colocalization was recoveryed. The level of STAT-3 (including its co-localization with Hsp90) and p-IKK?/?were decreased. It is indicated that the different level of Hsp90 expression has an important effects on the cellular capacity to resist heat stress damages and apoptosis via the regulation of its client proteins. The protective effects against stress damage and apoptosis partially attenuated if Hsp90 expression is inhibited before stress.