Study On The Mechanism Of Glycyrrhizic Acid Inhibiting The Transdifferentiation Of Renal Tubular Epithelial Cells Induced By IS

BackgroundRenal interstitial fibrosis(RIF)is the necessary pathway for various chronic kidney diseases to progress to End stage renal disease(ESRD).Epithelial-mesenchymal transition(EMT)plays an important role in the occurrence and development of RIF.Indoxyl sulfate(IS)is a protein-bound uremic toxin.After being accumulated in the body of CKD patients,it can cause excessive production of reactive oxygen species(ROS)in the kidneys,which in turn causes EMT in renal tubular epithelial cells.Reduced nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase,NOX)is the main source of renal ROS,and NOX4 is most abundantly expressed in kidneys.The research group found that glycyrrhizic acid(Glycyrrhizic acid,GA)has anti-fibrosis potential through high-throughput screening in the early stage.With the help of the "drug-target" molecular docking method in virtual screening technology,it was found that glycyrrhizic acid and NOX4 have a higher molecular docking score.Literature studies have also shown that the antioxidant function of GA may be related to the AMPK signaling pathway.Based on this,this study further explored the antioxidant effect of GA and its molecular mechanism by observing the effect of GA on the ROS level and EMT of HK-2 cells induced by IS.ObjectiveConstruct an IS-induced oxidative stress damage model of HK-2 cells,observe the effects of GA on HK-2 cells oxidative stress damage and EMT,and explore the molecular mechanism of GA's anti-fibrosis effect.Methods1.Obtain the "glycyrrhizic acid-target" network in CNKI,and filter the target name.KEGG pathway analysis and GO enrichment analysis were performed on the obtained collection to explore the mechanism of GA.The molecular docking technique was used to analyze the correlation between GA and AMPK protein.2.After stimulating HK-2 cells with 250 ?M IS for different times,the DCFH-DA method was used to determine the ROS level evaluation model.Western Blotting was used to observe the expression of interstitial cell marker proteins ?-SMA and Vimentin after HK-2 cells stimulated by IS for 24 h,and to preliminarily evaluate the effect of IS on the EMT of HK-2 cells.After pre-incubating HK-2 cells with NOX4 inhibitor DPI(10?M)for 1h,IS was added to stimulate them for 3 h,and the intracellular ROS levels in each group were detected by the DCFH-DA method.Western Blotting detects the expression of P22phox and NOX4 protein.3.After different concentrations of GA acted on the cells for 48 h.the CCK-8 method was used to detect its effect on cell viability.The cell wound healing experiment was used to observe the effect of different concentrations of GA on the migration of HK-2 cells.This was used to set the concentration of GA used in subsequent experiments.HK-2 cells were preincubated with GA for 24 h or HK-2 cells were pre-treated with DPI(10 ?M)for 24 h,and then IS stimulated for 24 h.Western Blotting was used to detect the effect of interstitial cell marker proteins.Next,observe the effect of GA on the increase of ROS in HK-2 cells induced by IS.DCFH-DA method was used to detect the ROS level of each group after GA preincubation for 24 h.Western Blotting detected the expression levels of P22phox and NOX4 proteins in each group after GA pre-incubation for 24 h.4.After pre-incubating HK-2 cells with GA 200 ?M for 24 h,the cells were stimulated with IS for 24 h.Western Blotting was used to detect the expression of AMPK protein in each group.The AMPK specific inhibitor Compound C(10 ?M)pretreated HK-2 cells for 1 h,and then added GA 200 ?M to treat the cells for 24 h,and then added IS and incubated them for 24 h.Western Blotting detects the expression levels of P22phox,NOX4 and mesenchymal cell marker proteins.Results1.A total of 109 specific targets were obtained.KEGG and GO analysis showed that GA is significantly enriched in signaling pathways such as interleukin signaling,response to toxic substances,cytokine production,MAPK cascade,cell proliferation,and reactive oxygen metabolism regulation.The results of molecular docking showed that the amino acid residues THR21,THR26,VAL24,VLY25,GLU143,and LYS141 of GA and AMPK can form a good bonding force.2.With the change of induction time,the amount of ROS production showed an inverted U-shaped change.In the range of 1-4 h,the level of ROS increased with time,and in the range of 4-72 h,the level of ROS decreased with the increase of induction time.Based on this,250?M IS was used to stimulate the HK-2 cell oxidative stress model for 3 h to complete the follow-up experiment.After IS treated for 24 h,compared with the normal group,the expressions of a-SMA and Vimentin were significantly increased,suggesting that HK-2.cells had EMT.After adding the NOX4 inhibitor DPI,the ROS level and Western Blotting results showed that compared with the model group,the ROS level in the DPI group decreased significantly,and the difference was statistically significant(P<0.0l).This suggests that DPI can inhibit the increase of ROS levels in HK-2 cells induced by IS.The protein expression of P22phox and NOX4 was significantly lower than that of the model group(P<0.0l).indicating that DPI can inhibit the expression of P22phox and NOX4.3.Different concentrations of GA(6.75-216 ?M)treatment group had no significant effect on HK-2 cell viability.GA 100 ?M,GA 150 ?M,and GA 200 ?M can improve the ISinduced decline in HK-2 cell viability and increase in cell migration,and are dose-dependent.The 200 ?M group has the most obvious effect,so GA 200 ?M is the best choice Intervention concentration.GA 200 ?M intervention can inhibit the decrease in E-cadherin expression(P<0.05)and the increase in expression of Vimentin and ?-SMA(P<0.05)in HK-2 cells caused by IS,and this is similar to the effect of the inhibitor DPI.Compared with the model group,the GA intervention group showed a significant decrease in ROS expression.Western Blotting results showed that GA intervention can inhibit the increase of P22phox and NOX4 protein expression levels caused by IS.4.Compared with the normal group,the expression of AMPK in the model group was significantly reduced,and the expression of AMPK protein could be up-regulated after GA pre-incubation.Pretreatment with AMPK specific inhibitor Compound C(10 ?M)can block the inhibition of IS-induced increase in P22phox and NOX4 expression by GA,and GA can inhibit IS-induced increase in expression of ?-SMA and Vimentin in HK-2 cells And the expression of epithelial cell marker E-cadherin is reduced,and the addition of AMPK inhibitor Compound C(10 ?M)can significantly inhibit the above-mentioned effects of GA.Conclusion1.GA is closely related to oxidative stress signal targets and pathways,and GA and AMPK have potentially better binding activity.2.250 ?M IS stimulation for 3 h is the best condition for constructing HK-2 cell oxidative stress model.IS induced EMT in HK-2 cells by promoting the generation of ROS derived from NOX4.3.GA can significantly increase the viability of HK-2 cells when exposed to IS,and at the same time inhibit the increase in ROS induced by IS.GA reverses the EMT of HK-2 cells induced by IS by inhibiting ROS derived from NOX4.4.GA can up-regulate AMPK protein expression in HK-2 cells inhibited by IS.GA can inhibit IS-induced EMT in HK-2 cells by regulating AMPK/NOX4/ROS signaling pathway.