The Protective Effect And Mechanism Of Astragalosides Ⅳ On Free Fatty Acids-induced Renal Tubular Epithelial Cells Injury

Part One ObjectiveTo study the mechanism of Astragaloside Ⅳ inhibiting HK-2 cell apoptosis,and further explore the role of oxidative stress and endoplasmic reticulum stress in PA-induced apoptosis of HK-2 cells.MethodsHuman renal tubular epithelial cells were cultured and grouped as follows: Bovine serum albumin(BSA)control,Palmitic acid(PA)group,different doses of ASIV(10,20,40 μmol/L).The activity of HK-2 cells was determined by MTT,Oil red staining observed lipid deposition.The intracellular lipid transport was observed by BODIPY FLC16.The apoptosis of cells was determined using Hoechst-33258 staining and Annexin-V/FITC/PI dual-labeled fluorescence microscopy.The protein levels of Bax,Bcl-2,Cleaved-caspase3 and Nuclear Nrf2 were determined by Western blot.ResultsThe growth of HK-2 cells was not significantly affected by ASIV(0-100 μmol/L).The results showed that PA at 200 μmol/L could induce apoptosis of HK-2 cells,increase intracellular lipid deposition,induce oxidative stress and endoplasmic reticulum stress,and ASIV(10,20,40 μmol/L)could significantly reduce the number of apoptotic cells induced by PA,decrease intracellular lipid deposition and reduce the content of reactive oxygen species.After PA induction,the expression of endoplasmic reticulum stress-related proteins ATF4,CHOP,Bax and Cleaved-caspase 3 increased,the content of oxidative stress-related proteins p-Nrf2 decreased,while the content of ATF4 and CHOP decreased and the content of p-Nrf2 increased when Astragaloside was given to HK-2 cells.ConclusionASIV can inhibit the apoptosis of human renal tubular epithelial cells induced by free fatty acids.The mechanism may be that ASIV could reduce the expression of apoptotic proteins Bax and Cleaved-caspase 3,reduce the content of intracellular reactive oxygen species and increase the expression of Bcl-2 and Nrf2 in HK-2 cells.Part Two ObjectiveTo study the specific mechanism of ASIV in protecting HK-2 cells from apoptosis.To further explore the role of oxidative stress and endoplasmic reticulum stress in apoptosis of HK-2 cells.MethodsHuman renal tubular epithelial cells were cultured and grouped as follows: Bovine serum albumin(BSA)control,Palmitic acid(PA),different doses of ASIV(10,20,40 μmol/L).The activity of HK-2 Cells was determined by MTT,Oil red staining corrosion.The apoptosis of cells was determined using Hoechst-33258 staining and Annexin-V/ FITC/PI dual-labeled fluorescence microscopy.The protein levels of Bax,Bcl-2,Cleaved-Caspase 3,Nuclear Nrf2 were determined by Western blot analysis.ResultsTempol or 4-PBA can improve cell apoptosis and reduce the expression of apoptosis-related proteins.Tempol,an oxidative stress inhibitor,can not only reduce the content of ROS and increase the content of p-Nrf2 protein in cells,but also change the content of ER stress protein and reduce the expression of endoplasmic reticulum apoptotic protein ATF4 and CHOP.Similarly,we found that endoplasmic reticulum stress inhibitor 4-PBA can not only reduce the content of endoplasmic reticulum stress-related protein,but also improve intracellular oxygen.ConclusionThe apoptotic mechanism of HK-2 cells induced by PA may be related to oxidative stress and endoplasmic reticulum stress.ASIV can protect HK-2 cells from PA-induced injury.The protective mechanism of ASIV may be that ASIV can reduce oxidative stress and endoplasmic reticulum stress-related responses,thus playing a protective role in HK-2 cells.