| The incidence of diabetes(Diabetic Mellitus,DM)is increasing year by year,the statistics of the 10th edition of the International Diabetes Federation(IDF)show that there are currently about 537 million DM patients in the world,and the overall prevalence of type II diabetes mellitus(T2DM)in China has reached 14.92%,which is the country with the most DM patients in the world.Diabetic Kidney Disease(DKD)is one of the common microangiopathy complications of DM,accounting for about 20%～40%of DM.The pathogenesis of DKD is very complex,and recent studies have reported that ferroptosis may be related to DKD,but the mechanism of ferroptosis in DKD has not been elucidated,and the relevant literature is relatively scarce and needs to be studied in depth.Previous studies have shown that Tangshenping,based on the theory of "renal flaccidity ",can reduce oxidative stress and inflammatory damage,and improve the clinical symptoms of DKD patients,but the pharmacodynamic basis and potential targets for the prevention and treatment of DKD have not been elucidated.In this study,the main active pharmacodynamic substances and mechanism of action mechanism of Tangshenping in the prevention and treatment of DKD were analyzed by high performance liquid chromatography tandem quadrupole electrostatic field orbital well mass spectrometry(UHPLC-Q-Exactive-orbitrap/MS)combined with network pharmacology,and it was found that its key signaling pathways were mainly related to AGE/RAGE,Nrf2 pathways,etc.,and were related to oxidative stress and cell death regulation mode.Studies have confirmed that the accumulation of advanced glycation end products(AGEs)in renal tissue is an important factor in DKD kidney damage,and Nrf2/Ho-1 is a key signaling pathway that regulates ferroptosis,and is closely related to the System Xc-/GSH/Gpx4 axis.Tubular epithelial cells are sensitive to ferroptosis because of their rich mitochondria,and at present,there are few reports of AGEs inducing ferroptosis in renal tubular cells,so this study took the regulation of ferroptosis by Nrf2/Ho-1 signaling pathway as the starting point,and explored whether there were characteristic manifestations of ferroptosis by establishing a DKD rat model consistent with human T2DM and an AGEs-induced renal tubular epithelial cell injury model.The regulatory effect of Tangshenping and its core pharmacoactive monomers on Nrf2 signaling pathway and ferroptosis was further studied,and new targets for the prevention and treatment of DKD in traditional Chinese medicine were explored.Objective:The key signaling pathways and core monomer components of Tangshenping in the prevention and treatment of DKD were combined with UHPLC-Q-Exactive-Orbitrap/MS technology combined with network pharmacology predictive analysis,and the literature was combined with the results of the predictive analysis into subsequent experimental studies.Biochemical kits,ELISA,transmission electron microscopy,Perls iron staining,HE staining,Mallory staining,immunohistochemistry,in situ hybridization,immunofluorescence,Western Blot,RT-PCR and Real Time PCR were used to explore whether high-sugar,high-fat+Streptozotocin(STZ)induced DKD rat model and AGEs-induced Human Kidney-2 cell(HK2)damage model have the characteristic manifestations of ferroptosis from the tissue,cellular,molecular and other levels.The regulatory effect of Tangshenping and its core pharmacodynamic monomers on the Nrf2 signaling pathway and cellular ferroptosis was also discussed.Methods:1.High performance liquid chromatography combined with network pharmacology predicts the mechanism of action and core pharmacodynamic components of Tangshenping in the prevention and treatment of DKDThe chemicals of Tangshenping compound were identified by UHPLC-Q-ExactiveOrbitrap/MS technology,the active pharmacoactive monomer was screened by the Swiss ADME principle,and the TCMSP and Swiss Target Prediction databases were searched to collect the targets of the active monomer.DKD disease targets were collected by searching GenCards,OMIM,Drugbank,TTD and DisGeNET databases,taking the intersection targets of DKD diseases and drugs,using Cytoscape 3.8.0 software to build the "TangshenpingPharmacodynamic Ingredients-Targets-DKD Diseases" network,and using the Metascape database and GO,Wiki Pathways,KEGG Pathways enrichment analysis.2.Animal experiments were used to study the effects of Tangshenping on ferroptosis and Nrf2/Ho-1 signaling pathways in DKD rats2.1 A high-sugar,high-fat+STZ-induced DKD rat model was constructed to study the intervention effect of Tangshenping on relevant biochemical indexesSixty male rats with SPF-grade SD were randomly divided into 8 normal groups and 52 modules,and after 8 weeks of normal feed,STZ(35 mg/kg)was injected intraperitoneally,and the modeled rats were randomly divided into DKD model group,10 in irbesartan group and 10 in Tangshenping low,medium and high dose groups.Daily gavage deionized water was given to rats in the normal group and model group,and the gavage doses of Tangshenping in the low,medium and high dose groups were 3.5 times(0.525 g/kg),7 times(1.05 g/kg)and 14 times(2.1 g/kg)of the clinical dose,respectively,and irbesartan was 10 times(25 mg/kg)of the clinical dose.Observe general conditions,including weight,mental condition,activity,fur,etc.;Weigh and collect urine for 24 h urine protein quantification.After 16 weeks of intervention,serum was collected to detect renal function UREA,SCR,blood lipid TC,TG,LDL,and the pathological morphology of renal histologies was observed by HE staining,Mallory staining and transmission electron microscopy.The ELISA method was used to detect the content of AGEs and RAGE in kidney tissue.2.2 To investigate the changes of biochemical markers of ferroptosis in DKD rats induced by high sugar and high fat+STZ and the intervention effect of TangshenpingBiochemical analyzers and kits were used to detect the contents of serum ROS,NO,SOD,GSH,LPO and Iron in each group,the contents of GSH,MDA and Iron in renal tissues of each group were detected by the kit,and the iron ion deposition of kidney tissues was observed by Perls iron staining.2.3 To investigate the changes of Nrf2/Ho-1 pathway and ferroptosis marker protein gene in rats with high sugar and high lipid+STZ induced by DKD,as well as the intervention mechanism of TangshenpingThe expression location and expression level of Nrf2,Ho-1,Gpx4,Slc7a11,Fth1,Ascl4 protein and mRNA in rat kidney tissues were detected by immunohistochemistry,in situ hybridization,Western Blot,RT-PCR and Real Time PCR.3.Cell experiments were used to explore the effect of Tangshenping-containing serum on the Nrf2/Ho-1 signaling pathway and ferroptosis of HK-2 cells induced by AGEs3.1 Construction of AGEs-induced HK-2 cell damage model and exploration of drug administration concentrationHK-2 cells were cultured in vitro,and the intervention groups of AGEs(25,50,75,150,300 μg/ml)with different concentration gradients were set up,and the cell activity after exposure of 12 h,24 h,48 h,and 60 h was detected,and the time-dose curve of AGEs induced HK-2 cell damage was drawn.On the basis of selecting the optimal stimulus concentration of AGEs,the ferroptosis inhibitor Fer-1,activator RSL-3 and Tangshenping drug-containing serum intervention groups were set up,and after different time periods of intervention,the cell activity was detected by CCK-8 method,and finally the optimal administration concentration and time of Fer-1,RSL-3 and Tangshenping drug-containing serum were selected.3.2 To investigate the pathological and biochemical changes of ferroptosis in HK-2 cells induced by AGEs and the intervention effect of Tangshenping-containing serumHK-2 cells were cultured in vitro and divided into:Control group,AGEs group,AGEs+Fer-1 group(ferroptosis inhibitors),AGEs+RSL-3 group(ferroptosis inducer group),and AGEs+DFs group(drug-free serum group),AGEs+TSPs group(Tangshenping drugcontaining serum group),AGEs+RSL-3+DFs group(ferroptosis inducer+drug-free serum group),AGEs+RSL-3+TSPs group(ferroptosis inducer+Tangshenping drug-containing serum group).After the intervention,HK-2 cell morphology was observed under light microscopy,mitochondrial morphology changes were observed by transmission electron microscopy,HK-2 cell activity was detected by CCK-8 method,Fe2+/total iron ion,GSH and MDA content were detected by kit,and ROS fluorescence level in HK-2 cells was observed by DCFH-DA fluorescence probe method.3.3 To investigate the changes of Nrf2/Ho-1 signaling pathway and ferroptosis marker protein gene induced by AGEs,and the intervention mechanism of Tangshenpingcontaining serumHK-2 cells were cultured in vitro,grouped with 3.2,and stimulatory drugs were given to each group of HK-2 cells,and the expression of ferroptosis marker proteins Gpx4 and Acsl4 was detected by immunofluorescence and Nrf2,Ho-1,Gpx4,Slc7a11,Fth1,Acsl4 proteins and mRNA were detected by Western Blot and RT-PCR.4.In vitro experiments were used to preliminarily explore the effect of Tangshenping core monomer on AGEs-induced ferroptosis and Nrf2 signaling pathways in HK-2 cellsThe structure of ligand proteins and monomer components was queried from Pubchem and PDB databases,and Auto Dock Tools software was used to virtually verify the molecular docking of key drug monomer components and ligand proteins,and PyMOL and LigPlot software were used for image visualization.HK-2 cells were cultured in vitro,and the effect of Tangshenping core monomers(kaempferol,quercetin,and formononetin)on the activity of HK-2 cells induced by AGEs was detected by CCK-8 method,and the appropriate cell experimental concentration was screened.HK-2 cells were divided into:Control group,AGEs intervention group,AGEs+TBHQ(Nrf2 activator group),AGEs+kaempferol group,AGEs+quercetin group,AGEs+Formononetin group,DCFH-DA probe was used to detect ROS content,kit to detect GSH and MDA content,and Nrf2,Ho-1,Gpx4,Slc7a11 and Acsl4 protein expression were detected by Western Blot.Results:1.Ultra-high performance liquid chromatography combined with network pharmacology was used to predict the mechanism of action of Tangshenping in the prevention and treatment of DKD1.1 UHPLC-Q-Exactive-Orbitrap/MS technology for the analysis of Tangshenping chemical compositionA total of 96 compounds of Tangshenping compound were identified by UHPLC-QExactive-Orbitrap/MS technology,including glycosides,organic acids,flavonoids,purines,isoflavones,pyranoids,benzoic acids and amino acids,of which hedysarum multijugum maxim had the most components,followed by rehmanniae radix praeparata,hedyotis diffusae herba.1.2 The mechanism of action of Tangshenping in the prevention and treatment of DKD was predicted by network pharmacologyBased on the SWISS ADME database,32 potential pharmacodynamic components in 96 compounds of Tangshenping were screened out,and a "drug-target-disease" network was constructed,and the analysis showed that the core compounds of Tangshenping for the prevention and treatment of DKD included flavonoid monomers such as quercetin,kaempferol and formononetin,and the key pathways predicted by enrichment analysis mainly involved AGE/RAGE pathway,NRF2 pathway,reactive oxygen reaction,inflammatory response,apoptosis,and insulin resistance.endoplasmic reticulum stress,etc.2.Effects of Tangshengping on ferroptosis and Nrf2/Ho-1 signaling pathways in DKDinduced rats with high sugar and high lipid+STZ2.1 Establishment of a rat model of DKD induced by high glucose and high fat+ STZ and intervention effect of TangshenpingCompared with the normal group,the general state of rats in the DKD model group was poor,the body weight decreased to varying degrees,the quantitative increase of urine protein in 24 h(P<0.01),the ratio of renal weight to body weight(P<0.01),the serum SCR,UREA,TC,TG,LDL increased significantly(P<0.05,P<0.01),protein casts,renal interstitial inflammatory infiltrates,glomerulosclerosis and renal interstitial fibrosis appeared in the kidney tissue of rats in the model group,and the degree of foot process fusion and adhesion decreased by electron microscopy,and the thickness of the glomerular basement membrane increased(P<0.01),The levels of AGEs and RAGE in renal tissue were elevated(P<0.01,P<0.05).After Tangshenping intervention,the above general conditions,biochemical indexes,and renal histopathology of DKD rats were improved to varying degrees.2.2 Changes of biochemical markers of ferroptosis in DKD rats induced by high-sugar and high-fat diet+STZ and the intervention effect of TangshenpingPerls iron staining showed that there was a large amount of iron ion deposition in the renal tubular epithelial cells of high-sugar high-fat diet+STZ-induced DKD model rats.Biochemical analysis and kit detection showed that the serum ROS and LPO contents of DKD rats were increased,the serum NO,SOD,GSH and total Iron contents were decreased(P<0.01,P<0.05),the total Iron and MDA contents of renal tissue were increased,and the GSH content was decreased(P<0.05,P<0.01).After each dose of Tangshenping intervention,the serum ROS and LPO contents of DKD rats decreased,the serum NO,SOD,GSH and total Iron contents increased(P<0.01,P<0.05),the total Iron and MDA contents of renal tissue decreased,and the GSH content increased(P<0.05,P<0.01).2.3 Changes in protein genes associated with ferroptosis and Nrf2 signaling pathway in renal tissues of rats with high glucose and high lipid +STZ and intervention mechanism of Tangshenping2.3.1 The immunohistochemical results showed that compared with the glomerulus,ferroptosis marker proteins Gpx4,Slc7a11 and Fth1 were expressed significantly in the tubular epithelial cells of DKD-induced rats with high glucose and high lipid+STZ.Semi-quantitative statistical analysis showed that compared with the normal group,the expression of Gpx4,Slc7a11 and Fthl proteins decreased in the kidney tissue of the DKD model group,and the expression of Acsl4,Nrf2 and Ho-1 proteins increased(P<0.01,P<0.05).Compared with the DKD model group,the expression of Gpx4s Slc7a11,Fthl,Nrf2 and Ho-1 proteins in the low,medium and high dose groups of Tangshenping was increased,while the expression of Acsl4 protein decreased(P<0.01,P<0.05).2.3.2 The results of Western Blot protein quantification showed that compared with the normal group,the expression of Gpx4,Slc7a11 and Fthl proteins decreased in the kidney tissue of rats in the DKD model group,the expression of Acsl4 protein increased(P<0.05,P<0.01),while the expression of Nrf2 and Ho-1 proteins increased slightly.Compared with the DKD model group,the expression of Gpx4,Slc7a11,Fth1,Nrf2 and Ho-1 proteins in Tangshenping was increased,while the expression of Acsl4 proteins decreased(P<0.01,P<0.05).2.3.3 The results of in situ hybridization experiments showed that ferroptosis marker proteins Gpx4 and Fthl were significantly expressed differently in renal tubules.Semiquantitative statistical analysis showed that compared with the normal group,the expression of Gpx4 and Fthl mRNA in kidney tissue of rats in the DKD model group decreased significantly(P<0.01),the expression of Ho-1 mRNA increased(P<0.01),and the expression of Nrf2 mRNA tended to increase.Compared with the DKD model group,the expression of Gpx4,Fth1,Nrf2 and Ho-1 mRNA in the renal tissues of rats in each dose group of Tangshenping was significantly increased(P<0.01).2.3.4 The results of RT-PCR and Real Time PCR showed that compared with the normal group,the expression of Gpx4,Slc7a11 and Fthl mRNA decreased in the kidney tissue of rats in the DKD model group,and the expression of Acsl4 mRNA increased(P<0.05,P<0.01),while the expression of Nrf2 and Ho-1 mRNA tended to increase.Compared with the DKD model group,the expression of Gpx4,Slc7a11,Fth1,Nrf2 and Ho-1 mRNA in each dose group of Tangshenping increased,while the expression of Acsl4 mRNA decreased(P<0.05,P<0.01).3.Effects of Tangshenping-containing serum on the Nrf2/Ho-1 signaling pathway and ferroptosis of HK-2 cells induced by AGEs3.1 The results of the construction of an AGEs-induced model of HK-2 cell damage and the exploration of drug administration concentrationThe results of CCK-8 experiments showed that AGEs inhibited HK-2 cell activity in a time-dose-dependent manner,and 150μg/ml AGEs stimulation was the best model of HK-2 cell damage induced by 48 h.Fer-1 can increase the activity of HK-2 cells exposed to AGEs and the optimal administration concentration is 2.5μmol/L;RSL-3 can further inhibit the activity of HK-2 cells exposed to AGEs,and the optimal administration concentration is 1μmol/L.4%of Tangshenping drug-containing serum could significantly improve the activity of HK-2 cells exposed to AGEs,and excessive concentration of drug-containing serum was not conducive to cell activity.3.2 AGEs-induced ferroptosis-related biochemical and pathological changes in HK-2 cells,and the intervention effect of Tangshenping-containing serumTransmission electron microscopy showed that the volume of mitochondria in HK-2 cells induced by AGEs decreased,the membrane density increased,and the crest fracture,reduction or even disappearance were visible,and some membrane rupture occurred.The mitochondrial structure of HK-2 cells in the AGEs+RSL-3 group was more obviously damaged,and it could be seen that most mitochondrial crest disappeared,the mitochondrial volume decreased,the membrane density increased,and even became vacuoles.Fer-1 and Tangshenping drugcontaining serum can significantly improve the degree of pathological damage of mitochondria.The results of biochemical kit and DCFH-DA fluorescent probe showed that compared with the Control group,the contents of Fe2+/total iron,ROS and MDA in HK-2 cells in the AGEs group increased,and the contents of cell activity and GSH decreased(P<0.05,P<0.01).Compared with the AGEs group,the contents of Fe2+/total iron,ROS and MDA in the AGEs+Fer-1 group decreased,and the contents of cell activity and GSH increased(P<0.05,P<0.01).Compared with the AGEs+DFs group and the AGEs+RSL-3+DFs group,the contents of Fe2+/total iron,ROS and MDA in the AGEs+TSPs group and the AGE+RSL-3+TSPs group decreased and the contents of cell activity and GSH increased(P<0.05,P<0.01)under the intervention of Tangshenping-containing serum.3.3 AGEs-induced changes in ferroptosis and Nrf2 signaling pathway-related protein genes in HK-2 cells and the intervention mechanism of Tangshenping3.3.1 The results of immunofluorescence experiments showed that compared with the Control group,the expression of Gpx4 protein in HK-2 cells in the AGEs group was significantly reduced,and the expression of Acsl4 protein was significantly increased(P<0.01).Compared with the AGEs group,the expression of Gpx4 protein in the AGEs+Fer-1 group was significantly increased,and the expression of Acsl4 protein was significantly reduced(P<0.01).Compared with the AGEs+DFs group and the AGEs+RSL-3+DFs group,the expression of Gpx4 protein in HK-2 cells in the AGEs+TSPs group and the AGEs+RSL-3+TSPs group was increased,while the expression of Acsl4 protein was reduced(P<0.05,P<0.01).3.3.2 The quantitative analysis results of Western Blot showed that compared with the Control group,the expression of Gpx4,Slc7a11 and Fthl proteins in HK-2 cells in the AGEs group decreased(P<0.05),and the expression of Nrf2,Ho-1 and Acsl4 proteins increased or tended to increase(P<0.05).Compared with the AGEs group,the expression of Nrf2,Ho-1,Gpx4,Slc7a11 and Fthl proteins in HK-2 cells in the AGE+Fer-1 group increased(P<0.05,P<0.01),while the expression of Acsl4 protein tended to decrease.Compared with the AGEs+DFs group and the AGEs+RSL-3+DFs group,the expression of Nrf2,Ho-1,Gpx4,Slc7a11 and Fthl proteins in HK-2 cells in the AGEs+DFs group and the AGEs+RSL-3+DFs group was increased,while the expression of Acsl4 protein decreased(P<0.05,P<0.01).3.3.3 The results of RT-PCR experiments showed that compared with the Control group,the expression of Gpx4,Slc7a11 and Fthl mRNA in HK-2 cells in the AGEs group decreased(P<0.05),the expression of Acsl4 mRNA increased significantly(P<0.01),and there were no significant differences in the expression of Nrf2,Ho-1 and mRNA.Compared with the AGEs group,the expression of Nrf2,Ho-1,Gpx4,Slc7a11 and Fthl mRNA in HK-2 cells in the AGE+Fer-1 group increased or tended to increase(P<0.05),while the expression of Acsl4 mRNA decreased.Compared with the AGEs+DFs group and the AGEs+RSL-3+DFs group,the expression of Nrf2,Ho-1,Gpx4,Slc7a11 and Fth1 mRNA in HK-2 cells in the AGEs+TSPs group and the AGEs+RSL-3+TSPs group was increased,while the expression of Acsl4 mRNA decreased(P<0.05,P<0.01).4.Effects of Tangshenping core monomers on AGEs-induced ferroptosis and Nrf2 signaling pathways in HK-2 cellsComputer molecular docking prediction showed that Tangshenping core monomers quercetin,kaempferol and formononetin could interact with key target proteins Nrf2,Ho-1,Gpx4,Slc7a11 and Fthl in van der Waals forces and hydrogen bonds,and the binding free energy was less than-5.0 kcal/mol,which had good affinity.Kit detection,cell proliferation toxicity experiments and DCFH-DA fluorescent probe experiments showed that quercetin,kaempferol and mangananthin could increase the activity of HK-2 cells induced by AGEs,reduce the content of intracellular ROS and MDA,and increase the content of GSH(P<0.05,P<0.01).Compared with the Control group,the expression of Gpx4 and Slc7a11 proteins in HK-2 cells in the AGEs group decreased,and the expression of Nrf2,Ho-1 and Acsl4 proteins increased(P<0.05,P<0.01).Compared with the AGEs group,the expression of Nrf2,Ho-1,Gpx4 and Slc7a11 proteins in HK-2 cells in the quercetin group,kaempferol group and formononetin group increased(P<0.05,P<0.01),while the expression of Acsl4 protein tended to decrease.Conclusion:1.UHPLC-Q-Exactive-Orbitrap/MS technology combined with network pharmacological analysis showed that Tangshenping mainly plays the role of preventing and treating DKD through antioxidant,anti-inflammatory,regulating cell death and other ways,involving Nrf2,AGE/RAGE and other signaling pathways,and its main active pharmacodynamic components include quercetin,kaempferol,formononetin and other flavonoids.2.Renal function was impaired in DKD rats induced by high sugar and high lipid+STZ,the content of AGEs and RAGE in renal tissue cells increased,iron ion deposition appeared in renal tubular epithelial cells,the content of iron ions,lipid peroxide and ROS in kidney tissue increased,while antioxidants such as GSH decreased,and the biochemical characterization of ferroptosis appeared.Tangshenping treatment reversed this process,thereby protecting kidney function in DKD rats.3.High-sugar,high-fat+STZ-induced DKD rat renal tissue cells exhibited ferroptosis based on System Xc-/GSH/Gpx4 axis,and mainly in renal tubular epithelial cells,accompanied by the inhibition of Nrf2/Ho-1 signaling pathway,Tangshengping can activate Nrf2/Ho-1 signaling pathway,upregulate the expression of Nrf2,Ho-1,Gpx4,Slc7a11,Fth1,and reduce the expression of Acsl4.Thus resisting the occurrence of ferroptosis in kidney tissue cells of DKD rats.4.HK-2 cells exposed to AGEs exhibited characteristic manifestations of ferroptosis,such as mitochondrial crest cleavage or decrease,increased membrane density,increased Fe2+/total iron ions,and increased lipid peroxidation products,while Fer-1 and Tangshenping-containing serums could improve the characterization.5.AGEs-induced HK-2 cells exhibited ferroptosis based on the System Xc-/GSH/Gpx4 axis,accompanied by the inhibition of the Nrf2 signaling pathway,and Tangshenping-containing serum could significantly activate the Nrf2/Ho-1 pathway,upregulate the expression of Nrf2,Ho-1,Gpx4,Slc7a11,and Fth1,and reduce the expression of Acsl4.Thus resisting the occurrence of AGEs and RSL-3-induced HK-2 cell ferroptosis.6.In vitro experiments have preliminarily shown that the core monomeric compounds of Tangshenping,quercetin,kaempferol and formononetin,regulate ferroptosis based on the System Xc-/GSH/Gpx4 axis by relying on the Nrf2 pathway,increasing the resistance of HK2 cells exposed to AGEs to ferroptosis. |
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