Study On The Mechanism Of Dapagliflozin Protective Effect On Diabetic Kidney Disease

ObjectiveExplore the possible mechanism of the protective effect of dapagliflozin on diabetic kidney disease,and detect the effects of dapagliflozin on the cell viability,apoptosis level,mitochondrial function,oxidative stress and autophagy levels of palmitic acid induced renal tubular epithelial cells Changes.Methods(1)Use DMEM/F12 complete culture medium to culture HK-2 cells.Incubate in a constant temperature incubator at 37℃ and 5%CO2 saturated humidity.Passage when the cell growth density reaches 70%-80%.Cell grouping:normal control group:cultured HK-2 cells with complete culture medium;palmitic acid group:cultured HK-2 cells with palmitic acid concentration of 150umol/L;palmitic acid+dapagliflozin group:use palmitic acid Culture medium with a concentration of 150umol/L and 2umol/L of dapagliflozin was used for HK-2 cell culture;dapagliflozin group:HK-2 cells were cultured with a medium containing 2umol/L dapagliflozin.(2)Each group of HK-2 cells were tested separately,and the cell viability was tested by the CCK8 method.Mitochondrial membrane potential detection uses JC-1 fluorescent staining.Intracellular ROS uses DCF immunofluorescence.SOD and MDA kits are used to detect oxidative stress in cells.Western blot detection of apoptosis proteins Bax,Cleaved caspase-3,anti-apoptotic protein Bcl-2,cytoplasmic cytochrome C(Cyto C)and autophagy-related protein expression,Hoechst33258 fluorescent apoptosis staining and Annexin V-FITC cell apoptosis detection experimental technology detects cell apoptosis.Results(1)The CCK-8 method detects the effect of dapagliflozin on the cell proliferation of palmitic acid-treated HK-2 cells:Compared with the CON group,the HK-2 cell viability of the PA group was significantly decreased(P<0.05),and the Dapa group HK-2 The difference in cell viability was not statistically significant(P>0.05).Compared with the PA group,the HK-2 cell viability of the PA+Dapa group increased,and the difference was statistically significant(P<0.05).(2)The effect of dapagliflozin on ROS production in HK-2 cells treated with palmitic acid by DCF immunofluorescence:Compared with the CON group,the ROS production in HK-2 cells in the PA group increased,and the difference was statistically significant(P<0.05),And there was no significant change in ROS in HK-2 cells in Dapa group(P>0.05);compared with PA group,ROS production in HK-2 cells in PA+Dapa group was reduced,and the difference was statistically significant(P<0.05).(3)JC-1 fluorescence detection of the changes of mitochondrial membrane potential of HK-2 cells treated with palmitic acid by dapagliflozin and transmission electron microscopy observation of mitochondrial morphology:compared with the CON group,the ratio of red fluorescence to green fluorescence intensity of HK-2 cells in the PA group Decrease,suggesting that the mitochondrial membrane potential decreased(P<0.05).There was no significant change in the mitochondrial membrane potential of HK-2 cells in the Dapa group(P>0.05);compared with the PA group,the red fluorescence and green fluorescence of HK-2 cells in the PA+Dapa group The increase in intensity ratio indicates an increase in mitochondrial membrane potential(P<0.05).Transmission electron microscopy showed that compared with the control group,the mitochondria of the PA group were swollen and deformed,and the mitochondria appeared ridge breaks and vacuoles;compared with the PA group,the swelling of the mitochondria in the PA+Dapa group was reduced,and the mitochondria were basically normal.(4)SOD and MDA kits detect the effect of dapagliflozin on oxidative stress in HK-2 cells treated with palmitic acid:The results show that compared with the CON group,the content of SOD in HK-2 cells in the PA group decreased(P<0.05),and MDA The level increased(P<0.05);the SOD and MDA in the HK-2 cells of the Dapa group did not change significantly(P>0.05);compared with the PA group,the level of SOD in the HK-2 cells of the PA+Dapa group increased.The level of MDA decreased,and the difference was statistically significant(P<0.05).(5)Western blotting was used to detect the changes in the levels of apoptotic proteins Bax,Bcl-2,Cleaved caspase-3 and cytoplasmic Cyto C of HK-2 cells treated with palmitic acid by dapagliflozin:The results suggest that compared with the CON group,the PA group HK-2 The apoptotic proteins Bax and Cleaved caspase-3 tended to increase,the anti-apoptotic protein Bcl-2 tended to decrease,and the cytoplasmic Cyto C protein expression increased.The difference was statistically significant(P<0.05),the above mentioned in the Dapa group There was no significant change in the index(P>0.05).Compared with the PA group,the HK-2 cell protein Bax and Cleaved caspase-3 in the PA+Dapa group decreased,the Bcl-2 increased,and the cytoplasmic Cyto C decreased.The difference was statistically significant(P<0.05).(6)Hoechst33258 apoptosis staining and Annexin V-FITC flow cytometry to detect the apoptosis of HK-2 cells treated with palmitic acid by dapagliflozin The results showed:compared with the CON group,the fluorescence intensity of the HK-2 cell apoptosis staining in the PA group Flow cytometry showed that the rate of apoptosis was increased(P<0.05),and there was no significant change in the rate of HK-2 cell apoptosis in the Dapa group(P>0.05);compared with the PA group,HK-2 cells in the PA+Dapa group The fluorescence intensity of apoptosis staining was reduced,and flow cytometry showed that the rate of apoptosis was reduced(P<0.05).(7)The effect of dapagliflozin on the expression of autophagy-related proteins LC3,P62 and Beclinl in HK-2 cells treated with palmitic acid was detected by western blotting:The results showed that compared with the CON group,the trend of the HK-2 cell protein LC3II and Beclinl in the PA group Increased,the difference was statistically significant(P<0.05);the expression of autophagy protein in HK-2 cells in the Dapa group did not change significantly(P>0.05);compared with the PA group,the Dapa+PA group HK-2 Cell proteins LC3Ⅱand Beclinl tended to decreased(P<0.05).ConclusionsDapagliflozin protects the renal tubular epithelial cells treated with palmitic acid.The possible mechanism is related to the modification of mitochondrial function,oxidative stress and the regulation of partial autophagy.