Molecular Mechanism Of Pyroptosis Induced By Deoxynivalenol In IPEC-J2 Cells Based On The Endoplasmic Reticulum Stress Pathway

Deoxynivalenol(DON)is a mycotoxin produced mainly by Fusarium spp.commonly found in grains such as wheat,it is also known as Vomitoxin.Pigs are the animals with the highest sensitivity to DON,and DON can damage the intestine,and the small intestine is the primary site of toxin action.Therefore,in this study,we cultured piglet small intestinal epithelial cells(IPEC-J2)in vitro to examine the molecular mechanism of DON induced pyroptosis of IPEC-J2 cells through endoplasmic reticulum stress.The experiment was divided into four groups: control group,DON group(500 ng/ m L DON),4-PBA+DON group(1μmol/ m L 4-PBA+500 ng/ m L DON),4-PBA group(1μmol/ m L 4-PBA).CCK-8kit was used to detect cell viability;The growth of cells was observed by inverted microscope.The ultrastructure of cells was observed by transmission electron microscopy.The activities and contents of IL-18,IL-1β,SOD,GSH-Px,CAT and MDA in the cells were detected by ELISA.The relative gene expression levels and protein expression levels of endoplasmic reticulum stress pathway and key molecules of cell pyroapoptosis were detected by q RT-PCR and WB.ROS content was measured by flow cytometry.These results showed that:1.Cell survival rate was significantly reduced when DON concentration reached 125ng/m L(P<0.01),and with the increase of DON supplemental level in cell culture medium,the survival rate of IPEC-J2 cells showed a downward trend as a whole,and DON concentration of 500 ng/m L was about 50% lethal concentration.When the concentration reached 1000 ng/m L,the cell viability did not decrease significantly when the dosage of toxin was increased.In the DON group,the gap between cells was widened,the cell density was extremely reduced,and more floating cells were found.Endoplasmic reticulum dilatation and mitochondrial vacuolation were also observed.In the 4+D group,the number of cells increased,the degree of cell fusion increased,the intercellular connection was closer,the number of floating cells decreased,and the state of organelles was more normal.2.DON can induce oxidative damage in cells,and inhibition of endoplasmic reticulum stress can indirectly inhibit oxidative stress in cells.Compared with the control group,the contents of ROS and MDA in DON group were significantly increased(P<0.01),ROS and MDA contents in 4+D group were significantly decreased(P<0.01);Compared with the control group,the contents of SOD,GSH-Px and CAT in DON group were decreased(P<0.05),while the contents of SOD,GSH-Px and CAT in 4+D group were increased.3.DON can activate cells to enter endoplasmic reticulum stress state.The m RNA relative expression levels of ER stress related proteins GRP78,ATF6 and IRE1 were significantly increased after DON addition(P<0.01),and the m RNA relative expression level in 4+D group was significantly down-regulated(P<0.01),which was basically consistent with GRP78,ATF6 and IRE1 protein expression.4.DON can cause Caspase-1-dependent cell pyroptosis.Inhibition of endoplasmic reticulum stress can down-regulate the expression levels of pyrodeath related genes and proteins as well as the content of pyrodeath related inflammatory factors.Compared with the control group,the contents of IL-1β and IL-18 in DON group were significantly increased after the addition of DON(P<0.01),the contents of IL-1β and IL-18 in 4+D group were significantly decreased;Compared with the control group,the relative gene expression levels of GSDMD and Caspase-1 in DON group were significantly increased(P<0.01),in the 4+D group,the m RNA relative expression levels of GSDMD and Caspase-1 could be decreased to different degrees,which was basically consistent with the protein expression.In conclusion,DON can reduce cell viability,change cell morphology,destroy cell ultrastructure,induce oxidative damage,activate cells to enter the endoplasmic reticulum stress state,and also lead to cell pyrolysis.The addition of ER stress inhibitor 4-PBA can better alleviate the changes of cell viability and morphology caused by DON,reduce the damage caused by oxidative stress,and alleviate the pyroptosis induced by DON.