The Role Of PERK-CHOP Pathway In Heat Stress-Induced Intestinal Mucosal Injury

ObjectiveThis study explored the mechanism of PERK-CHOP signalling during endoplasmic reticulum(ER)stress caused by heat stress-induced intestinal mucosal injury.We also investigated the effects of ER stress inhibitor 4-phenylbutyric acid(4-PBA)on this pathway in vitro and in vivo.We aimed to provide valuable therapeutic targets for clinical heatstroke prevention and treatment.Methods1.For our in vitro experiments,Caco-2 cells were exposed to heat-stress at 43℃ for 2 hours.At different rewarming time points,we examined cell viability and injury by MTT and lactate dehydrogenase(LDH)assays,apoptosis by flow cytometry,and expression of PERK-CHOP signalling by qRT-PCR and western blot.In addition,pre-treatment with PERK-specific inhibitors before heat stress was used to observe changes in apoptosis.2.We used a heatstroke mouse model that was pre-treated with 4-PBA for our in vivo experiments.We observed the survival of the animals and used haematoxylin and eosin stain to examine the intestinal histopathology and electron microscopy to observe the intestinal ultrastructure.TUNEL assay was employed to detect apoptosis and immunohistochemistry and western blot was used to determine the expression of PERK-CHOP signalling.3.Subsequently,we transfected Caco-2 cells with CHOP-siRNA or CHOP-overexpression plasmid before heat stress.Additionally,we administered 4-PBA or ER stress inducer tunicamycin(TM)before heat stress.MTT and LDH assays were used to detect cell viability and damage levels.Apoptosis was evaluated by Hoechst stain and flow cytometry and qRT-PCR and western blot were used to determine the expression of PERK-CHOP signalling.Results1.Intense heat stress induced apoptosis of intestinal epithelial cells and activates ER stress PERK-CHOP signal pathway.Intense heat stress significantly decreased the viability of Caco-2 cells,which indicating that heat stress has direct cytotoxic effect.The levels of apoptosis and of PERK-CHOP signal pathway related factors were elevated in Caco-2 cells after intense heat stress.After intervention with PERK-specific inhibitors,apoptosis level was reduced after heat stress.2.Intestinal mucosal injury and intestinal epithelial apoptosis were induced by severe heatstroke.Intestinal ER stress PERK-CHOP pathway was activated.ER stress inhibitor 4-PBA could significantly improve intestinal mucosal injury and intestinal epithelial apoptosisSevere heatstroke leads to intestinal mucosal damage and intestinal epithelial apoptosis in mice,and the expression of PERK-CHOP signal pathway-related proteins in mice intestines were increased.4-PBA pretreatment could greatly prolong the survival time of mice,reduce the intestinal injury score and intestinal epithelial apoptosis level induced by high fever,and aslo reduce the expression level of PERK-CHOP signal pathway-related proteins.These results indicated that ER stress participates in the occurrence of heatstroke-induced intestinal mucosal damage and epithelial apoptosis.3.The activation of CHOP played a key role in that apoptosis of intestinal epithelial cells under intense heat stress.4-PBA,as an ER stress inhibitor,has a protective effect on intestinal epithelial cells under intense heat stress.Our experiment showed that compared with control group,CHOP silencing significantly reduced the apoptosis level of Caco-2 cells after heat stress,while CHOP overexpression significantly increased the apoptosis level.This result indicated that CHOP activation plays a key role in heat stress-induced apoptosis of Caco-2 cells.4-PBA pretreatment decreased the mRNA and protein expression of PERK-CHOP signal pathway related factors and significantly reduced the apoptosis level of cells after heat stress.While TM pretreatment increased the mRNA and protein expression of PERK-CHOP signal pathway related factors and aggravated the apoptosis level of cells after heat stress.ConclusionHeat stress mediates intestinal mucosal damage and intestinal epithelial apoptosis by activating ER stress via the PERK-CHOP signalling pathway,in which CHOP activation plays a central role.ER stress inhibitor 4-PBA downregulated the expression of factors involved in PERK-CHOP signalling and significantly reduced intestinal mucosal damage and epithelial cell apoptosis caused by heat stress,providing a novel treatment strategy for severe heatstroke.