Effect Of Oleanolic Acid On Autophagy And Apoptosis Induced By Ochratoxin A In HEK293T Cells

Objective: In this study,human embryonic kidney epithelial cells(HEK293T)were selected as research subjects to study the effects of Ochratoxin A(OTA)on cell morphology,reactive oxygen species,autophagy-related proteins,and apoptosis-related proteins.The effect of different interventions of Oleanolic acid(OA)on autophagy and apoptosis of HEK293 T cells treated by OTA was explored.Methods: 1.Cell Culture: Human embryonic kidney epithelial cells(HEK293T)were cultured,adherent growth was performed,and logarithmic growth phase cells were passaged for follow-up experiments.2.Group dosing treatment: The experiment was divided into 6 groups: control group,OTA group,OA group,OA pretreatment group(OA treatment + OTA post-treatment,P group),OA and OTA treatment group(M group),OA Post-treatment group(OTA first processed +OA post-treatment,ie,L group).Cells were treated with OTA for 24 hours and cells were treated with OA for 2 hours.After the treatment is completed,relevant indicators for cell detection are collected.3.Detection of reactive oxygen species by flow cytometry: After group treatment,cells were collected,added with DCFH-DA probe,serum-free medium was washed three times,and flow cytometry was used.4.Western blot: extract cell proteins after group treatment,determine the concentration by BCA method,and detect the expression of related proteins.5.Data processing: SPSS17.0 software was used.The results were expressed as mean±standard deviation((?)±s).When the mean number of multiple groups was compared,the homogeneity of variance test was satisfied.One-way analysis of variance was used and SNK was used.The law compares the two.p<0.05 indicates that the difference was statistically significant.Results: 1.Observed under an inverted microscope,compared with the control group,OTA(8 μM)treatment for 24 hours after the cell morphology changes,some cells from the shuttle to a circular shape,the edge is not clear,the cells shrink.Compared with the OTA group,the morphology of cell changes in the OA post-treatment group was improved and shrinkage was reduced.2.The results of CCK8 method showed that compared with the control group,OA(1 μΜ)significantly promoted the proliferation of HEK293 T cells(P<0.05).In group treatment,compared with OTA group,the survival rate of OA pretreatment group(P group),OA and OTA simultaneous treatment group(M group)and OA post-treatment group(L group)increased(P<0.05).3.Flow cytometry results suggested that the level of ROS in OTA group was higher than that in control group(P<0.05).Compared with the OTA group,the ROS content in the P group and the L group was significantly decreased(P<0.05),and the ROS content in the M group was decreased but there was no significant difference(P>0.05).4.Western blot results showed that compared with the control group,the phosphorylation levels of p-m TOR and p-p70s6 k in HEK293 T cells were decreased in OTA group,and the levels of LC3-II and P-Beclin1 were increased(P<0.05).Compared with the OTA group,phosphorylation of p-m TOR and p-p70s6 k increased after OA intervention,and the expression of LC3 B and p-Beclin1 decreased(P<0.05).Among them,the OA post-processing method works well.5.Western blot results showed that compared with the control group,the expression of Caspase-9,Bak,Bax and Cyt C protein in OTA group increased(P<0.05).Compared with OTA group,the expression of Bak and Cyt C in P group,M group and L group was decreased(P<0.05),Bax expression was decreased in M group and L group(P<0.05).Conclusion: 1.OA can reduce OTA-induced oxidative stress and apoptosis in HEK293 T cells.2.OA reduces OTA-induced autophagy by p-m TOR/p-p70s6k/p-Beclin1/LC3 B signaling,OA post-processing effect is better.3.OTA-induced autophagy inhibited the excessive apoptosis of cells to some extent.