| Background: zearalenone(ZEA)is a kind of estrogenic mycotoxin,it can destroy animal reproductive and reproductive disorders,and it can cause serious losses in livestock and animal husbandry;proanthocyanidin is a natural antioxidant,which can remove the animal body oxygen free radicals effectively,and reduce the body’s oxidation and apoptosis stress and cell injury.Objective: The present study mainly investigated whether procyanidins could protect zearalenone-induced oxidative damage and apoptosis of mouse intestinal epithelial cells by inhibiting the endoplasmic reticulum stress(ERS)signaling pathway.Methods: by using CCK8 method were measured for zearalenone and proanthocyanidins alone and in combination on MODE-K cell survival rate,ensure the optimum concentration of ZEA and PC;model testing,test group: control group,ZEA group,PC group and combined treatment group by superoxide dismutase enzyme detection test group(SOD),lactate dehydrogenase(LDH),malondialdehyde(MDA),glutathione peroxidase(GSH-px),glutathione(GSH)activity to determine the antioxidant effect of ZEA on oxidative stress and PC;apoptosis by flow cytometry,and the analysis of each treatment group cell gene and protein levels using fluorescence quantitative PCR and Western blot,further to determine the molecular mechanism of drug effects on cells.Result:1,The damaging effects on MODE-K cells were observed by inverted microscope,which showed that cells were shrinkaged,shedding unadherent;CCK8 assay results showed that when the ZEA concentration was higher than 10 μM,the survival rate of MODE-K cells decreased significantly(P<0.01),while the measured half inhibitory concentration on cell ZEA IC50 is 65μM,the optimum proanthocyanidin concentration was 5 μg/mL,however 30 μg/mL PC showed significant cytotoxicity to cells(P<0.01).2,LDH and antioxidant assays showed that LDH increased significantly(P<0.01),MDA increased significantly(P<0.05),and SOD,GSH,and GSH-Px decreased significantly in the ZEA-treated group compared with the control group.(P<0.01);MDA and LDH activity in PC-treated cells was significantly decreased(p<0.01),SOD,GSH,and GSH-px were significantly increased(p<0.01);PC and ZEA combined treatment group compared with ZEN group LDH activity was significantly decreased(P<0.05),MDA was decreased,GSH-px activity was significantly increased(p<0.01),SOD was significantly increased,and GSH was increased.3,comparing with the control group,the results of flow cytometry showed that the apoptosis cells in ZEA group increased significantly(P<0.01),the intestinal epithelial cells of low concentration of procyanidins treatment did not change significantly,and the ZEA combined with PC treatment,PC can reduce the apoptosis(P<0.01).4.The apoptosis related genes and protein expression of endoplasmic reticulum stress were detected by fluorescence quantitative PCR and Western blot.Compared with the control group,in ZEA group,cell apoptosis,endoplasmic reticulum stress related genes Bax,CHOP,GRP78/Bip,JNK and Caspase-12 were significantly up-regulated,anti apoptosis gene Bcl-2 were significantly reduced;apoptosis protein Bax/ Bcl-2 ratio increased,the relative expression associated with endoplasmic reticulum stress protein CHOP,GRP78/Bip,JNK,P-JNK,Caspase-12 volume increased significantly.Conclusion: Proanthocyanidins can protect the oxidative damage and apoptosis of small intestinal epithelial cells induced by zearalenone in maize,the protective mechanism might be that PC could alleviate the apoptosis of mouse intestinal epithelial cells by inhibitition of endoplasmic reticulum stress signaling pathway. |
