Role And Mechanism Of Lonp1 In Ursolic Acid Protecting Ochratoxin A-induced Oxidative Stress In HEK293T Cells

Objective: Lonp1 is one of the important proteins involved in OTA induced nephrotoxicity.Based on UA-based renal protection,this study explores the relationship between Lonp1 and renal supportive renal function and its possible mechanism by inhibiting the expression of Lonp1.Methods: 1 ? Adopting semi-adherent cell culture method,HEK293 T cells were independently treated with OTA and UA respectively according to experimental grouping.The morphological changes of cells were observed by inverted microscope and the cell viability was determined by CCK-8 assay.The working concentrations of OTA and UA were determined.2? The following experimental groups were set up: blank control group(group C),UA control group(group U);OTA control group(group O);UA pre-treatment group(group U +),OTA and UA co-treatment group :(group U & O);UA post-treatment group(group O +).HEK293 T cells were treated with OTA and UA in order of exposure time to determine the renal protection mode of UA.3? Based on the best renal protection effect of UA in our experiment,the following experimental groups were set up: inhibitor control group(group C-)and CDDO-me treatment group:(group CD)to determine the relationship between the expression of Lonp1 and the renal function of UA.4?The synthesis of 2,3 in the experimental group,DCFH-DA kit was used to detect intracellular ROS content,Western Blot detection of Lonp1,Aco2,Hsp75,p-m TOR and LC3 protein expression,And to explore the possible mechanism of Lon expression in renal protection of UA.Results:1? The results of CCK-8 showed that after treated with different concentrations of OTA for 24 h,the cell viability decreased with the increase of OTA concentration,and the cell death rate was 50.6% in the 8 ?M OTA group,which reached the half maximal inhibitory concentration.Compared with the control group Compared with the statistical differences,and the relevant literature confirmed that the concentration of mitochondria damage cells,thus selecting 8 ?M OTA for follow-up experiments.Different concentration of UA treated cells for 2 h,the cell survival rate of each concentration decreased with the increase of OTA concentration,compared with the control group,1 ?M UA showed a strong role in promoting proliferation,so select 1 ?M UA for follow-up experiments.2? The U +,O + and U + O groups showed different degrees of protection compared with the O group,in which O + group had a more significant 36% increase in cell viability than the U + and U + O groups.The results of inverted microscope showed that the cell growth of group C and group U was dense,the body cell was full,well differentiated,and the synapse was fusiform;Compared with group C and U,the growth of cells in group O,group U +,group O + and group U&O was blocked,Synapses disappear,cell body shrinkage to form a large number of vacuoles,cell debris in the culture matrix.3? DCFH-DA kit was used to detect the level of ROS production in different treatment groups.Compared with the C group and the O group,8 ?M OTA induced extremely high level of ROS in the cells,suggesting that OTA may induce nephrotoxicity through the oxidative stress pathway.WB detection of different treatment groups of cells in the expression of the protein results,phosphorylation of m TOR as a negative signal protein,the level of autophagy increased by The expression of LC3 also verified this.The expressions of Lonp1,Aco2 and Hsp75 were basically the same.Compared with the normal environment and the weak stress condition,their expressions were relatively lower under the condition of strong stress,suggesting that Lonp1 can exert its hydrolase activity in OTA-induced cell oxidative damage and mate function.Conclusion:1?The best kidney protection mode of UA is the pre-processing protection mode.2? Lonp1 expression and the role of renal protection of UA.3? In UA-protected OTA-induced renal cell oxidative damage,Lonp1 may participate in the protective effect of UA on OTA-induced cell injury through its hydrolase and chaperone function,and its substrate Aco2 and Hsp75,involved in cell autophagy and apoptosis process,play a role in this protection model.