Effects of Attachment and Culture Temperatures on Viability, Apoptosis, and HSPs Expression in Broiler Hepatocytes
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Heat stress is an environmental factor that significantly impacts the productivity and welfare of poultry, resulting in severe physiological imbalances. However, most cell culture studies are conducted at the standard mammalian incubation temperature of 37℃, which limits their ability to reflect the physiological characteristics of poultry. This study was conducted to investigate the effects of combining attachment and incubation temperature on cell viability, cell cycle, apoptosis, and heat shock protein (HSP) expression using broiler hepatocytes. Hepatocytes isolated from 16-day-old broiler embryos were attached at 37℃ and 41℃, and then cultured at 37℃, 41℃, and 43℃. As a result, cells attached and cultured at 41℃ exhibited the highest survival rate, while cells cultured at 43℃ exhibited significantly lower survival rates. Cell cycle analysis revealed that the 37-41 group had a higher sub-G1 ratio, leading to apoptosis. The 37-43 group showed an increase in G2/M phase, suggesting a high-temperature-induced DNA damage. Furthermore, apoptosis analysis revealed that the 41-43 group exhibited the highest rate of early apoptosis, suggesting that the rapid temperature increase induced intracellular protein denaturation and mitochondrial dysfunction. HSP expression revealed that HSP90, HSP70, HSP60, HSP40, and HSP27 significantly increased at culture temperatures higher than the attachment temperature, with maximum expression observed at 43℃. These results identify 41℃ as the optimal temperature for maintaining hepatocyte homeostasis and demonstrate that temperatures ≥43℃ trigger strong stress responses and apoptosis. additionally, the attachment temperature was found to be a critical factor influencing subsequent cellular stress sensitivity.
Abstract
Heat stress is an environmental factor that significantly impacts the productivity and welfare of poultry, resulting in severe physiological imbalances. However, most cell culture studies are conducted at the standard mammalian incubation temperature of 37℃, which limits their ability to reflect the physiological characteristics of poultry. This study was conducted to investigate the effects of combining attachment and incubation temperature on cell viability, cell cycle, apoptosis, and heat shock protein (HSP) expression using broiler hepatocytes. Hepatocytes isolated from 16-day-old broiler embryos were attached at 37℃ and 41℃, and then cultured at 37℃, 41℃, and 43℃. As a result, cells attached and cultured at 41℃ exhibited the highest survival rate, while cells cultured at 43℃ exhibited significantly lower survival rates. Cell cycle analysis revealed that the 37-41 group had a higher sub-G1 ratio, leading to apoptosis. The 37-43 group showed an increase in G2/M phase, suggesting a high-temperature-induced DNA damage. Furthermore, apoptosis analysis revealed that the 41-43 group exhibited the highest rate of early apoptosis, suggesting that the rapid temperature increase induced intracellular protein denaturation and mitochondrial dysfunction. HSP expression revealed that HSP90, HSP70, HSP60, HSP40, and HSP27 significantly increased at culture temperatures higher than the attachment temperature, with maximum expression observed at 43℃. These results identify 41℃ as the optimal temperature for maintaining hepatocyte homeostasis and demonstrate that temperatures ≥43℃ trigger strong stress responses and apoptosis. additionally, the attachment temperature was found to be a critical factor influencing subsequent cellular stress sensitivity.