Study On Heat Stress-induced Endoplasmic Reticulum Stress Mediated-apoptosis In IPEC-J2 Cells And Intervention Mechanism Of Trehalose

Heat stress(HS)impairs intestinal barrier function and leads to intestinal damage.Intestinal epithelial cells are rich in endoplasmic reticulum.Under stress,increases in protein misfolding in endoplasmic reticulum(ER)triggered endoplasmic reticulum stress(ERS).Whether heat stress regulates intestinal barrier function by mediating ERS is unclear.Trehalose severs as molecular chaperone and antioxidant.Therefore,trehalose will be used as a nutrient to interfere HS-induced intestinal damages and its underlying mechanisms will be studied.In this study,a heat stress model of porcine small intestinal epithelial cells(IPEC-J2)was established in vitro.The molecular mechanism of cell damage caused by short-term and long-term heat stress and the intervention effect of trehalose were explored through adding ERS inhibitor(4-PBA)and ROS scavenger(NAC)and using transcriptomics,Western blot,immunofluorescence and combined with transmission electron microscopy(TEM)assays.The main results are as follows:1.The mechanisms of ERS mediated apoptosis in IPEC-J2 cell induced by short-term heat stressShort-term heat stress(43?,2h)treatment significantly reduced cell survival,significantly increased apoptosis rate and lactate dehydrogenase(LDH)activity.Transcriptome sequencing and KEGG pathway enrichment analysis showed that short-term heat stress can activate the ERS signaling pathway,trigger an unfolded protein response,and inhibit cell proliferation.Through immunofluorescence and Western blotting,we found that short-term heat stress significantly induced the expression of ERS marker protein(GRP78),ER signaling pathway protein(p-PERK/pe IF2?/ATF4/CHOP)and apoptosis marker protein(Caspase 3,BAX and Cyt c).The results of TEM showed that the number of ribosomes attached to ER decreased and the diameter of ER lumen increased significantly under short-term heat stress.Analysis of the intestinal barrier function found that short-term heat stress destroyed intestinal barrier function,characterized by down-regulated the expression of intestinal tight junction proteins(Claudin3,ZO1 and Occludin).4-PBA pretreatment for 4 h inhibited the increases in expression of ERS marke proteins,apoptosis-related proteins and tight junction proteins,suggesting ERS-mediated apoptosis in heat-stressed cells,which in turn lead to barrier dysfunction.2.The mechanisms of oxidative stress and ERS mediated apoptosis in IPEC-J2 cell induced by long-term heat stressLong-term heat stress(43 ?,12h)significantly reduced cell survival and proliferation,significantly reduced intracellular SOD activity,and extremely significantly increased LDH,ROS,MDA activity and cell apoptosis.It significantly reduced the expression of antioxidation-related proteins(SOD2,CAT),extremely significantly reduces the expression of anti-apoptotic protein(Bcl2),and extremely significantly increases the expression of pro-apoptosis-related proteins(Caspase 3,BAX,Cyt c).The results suggested that heat stress induces oxidative stress and apoptosis in cells.Transcriptome sequencing and KEGG pathway enrichment analysis showed that glutathione metabolism pathway,nicotinic acid and nicotinamide metabolism pathway,peroxidase pathway,Wnt signaling pathway,MAPK signaling pathway and endoplasmic reticulum stress are involved in heat stress response.The results of TEM showed that the inner morphology of mitochondria was blurred,and the diameter of ER lumen became larger under long-term heat stress condition.The expression of endoplasmic reticulum stress marker protein(GRP78),ER signal pathway protein(p-e IF2?/ATF4/GADD34/CHOP)was significantly up-regulated by immunofluorescence and Western Blot assays.NAC pretreatment cells for 12 h protected agains the decreases in expression of antioxidant enzymes(SOD1,SOD2 and CAT).Compared with heat stress,NAC leads to upregulation of GCLM,a rate-limiting enzyme for GSH generation while upregulation of CHAC1,being involved in GSH degradation.NAC pretreatments inhibited the HSinduced increases in expression of ERS-involved moleculars and apoptosis-related proteins.These results suggest HS-induced oxidative stress acconts at least in part for ERS,leading to caspasedependent apoptosis.3.Study on the mechanism of trehalose interfering with heat stress-induced apoptosis in IPEC-J2 CellsThis experimente was divided into 4 groups: CON,cells were treated in 37? condition,HS,cells were subjected to 43? of heat exposure for 12 h;HS+TRE10,cells were pretreated with 10 m M trehalose for 4 h before exposed to heat stress for 12 h.Compared with the heat stress,Trehalose supplementation significantly reduced the LDH activity,increased the SOD activity and increased the expression of SOD2 at m RNA and protein levels.Heat stress decreased the expression of LC3,ATG5,and Beclin1,and trehalose pretreatment increased their expressions when compared with the heat stress.Trehalose pretreatment attenuated the heat-induced expression of ERS-related proteins GRP78,p-e IF2?,ATF4,CHOP at m RNA and protein levels.Trehalose pretreatment can significantly increase the expression of intestinal tight junction proteins(ZO1,Claudin1).In summary,we draw the following conclusions:Short-term heat stress induced barrier dysfunction in IPEC-J2 cells through endoplasmic reticulum stress mediated apoptosis by p-eif2?/CHOP pathway.Long-term heat stress leads to oxidative stress and ER stress.The trehalose pretreatment relieves oxidative stress and ERS induced by heat stress,and maintains the intestinal barrier function.These results clarify the new mechanism of heat stress-induced intestinal barrier dysfunction,and provide insight into nutriental intervention.