Mechanism Of Ferulic Acid Protects NRK-52E Cells Against Anoxia/Reoxygenation Injury Mediated By AMPKα1

Objective:Anoxia/reoxygenation(A/R)damage causes renal tubular epithelial cell(NRK-52E)dysfunction,which may be related to excessive production of reactive oxygen species(ROS),and ultimately leads to cell apoptosis.Ferulic acid(FA)belongs to the family of phenolic acid,which is very abundant in fruits and vegetables.It has the ability to scavenge free radicals and activate cell protection system to enhance cell resistance to stress.However,it has not been studied whether FA can protect NRK-52 E cells against A/R damage and its protective mechanism remains unclear.Therefore,in this study,we verified whether FA can resist A/R damage in NRK-52 E.The AMPK inhibitor,Compound C,plus FA pretreat NRK-52 E cells to explore further whether the mechanism of FA resistance to A/R damage is anti-apoptosis mediated by upregulating expression of AMPKα1.Methods:NRK-52 E cells was used as the research object to establish acute A/R injury model.The NRK-52 E cells were randomly divided into 4 groups,and each group was repeated 3 times:(1)normal control group(Control);(2)A/R group;(3)75μM FA+A/R group;(4)75μM FA+5μM Compound C +A/R group.The cell viability was detected by the CCK-8,the lactate dehydrogenase(LDH)activity was detected by spectrophotometry to determine the optimal protective concentration of FA;then the Glutathione Peroxide enzyme(GSH-Px),Catalase(CAT),Superoxide Dismutase(SOD)activity,Malondialdehyde(MDA)content,mitochondrial permeability transition pore(m PTP)opening and ATP/AMP ratio were mearsured;cells were detected by fluorescence-labeled flow cytometry intracellular ROS level,mitochondrial membrane potential(MMP)and apoptosis level;Western blotting detected AMPKα1,p-AMPK,Bcl2,Bax,Cleaved-caspase 3 and mitochondrial/cytoplasmic Cytochrome c expression;TUNEL detect cell apoptosis.Results:Compared with the A/R group,the cell viability and LDH showed that 75μM FA pretreatment had the best protective effect.Compared with the A/R group,75μM FA pretreatment elevated the expression of AMPKα,AMPKα1 and p-AMPK,and reduced the ratio of AMP/ATP.75μM FA pretreatment significantly inhibited the production of intracellular ROS,stabilized MMP,thereby inhibited the open of m PTP,and prevented the release of Cyt c from mitochondria to the cytoplasm,reduced the expression of Cleaved-caspase 3,and finally alleviated cell apoptosis.However,when NRK-52 E cells were pretreated with 75μM+5μM Compound C,Compound C inhibited the expression of AMPKα1 and p-AMPK,thereby significantly abolished the protective effect of FA on NRK-52 E cells.Conclusion:FA pretreatment can protect NRK-52 E cells against hypoxia/reoxygenation injury through upregulating AMPKα1 expression,and may be a potential drug for the treatment of renal ischemia-reperfusion injury.