Study On The Mechanism Of Tangshenfang Improving Diabetic Renal Fibrosis And Oxidative Stress Injury

BackgroundDiabetic kidney disease(DKD)is one of the serious microvascular complication of diabetes(diabetes mellitus,DM)which are the main causes of end stage renal disease(ESRD).In 2019,the global prevalence of diabetes is approximately 9.3%(463 million people).This number is expected to rise to 10.9%(700 million people)in 2045.About 40%of diabetic patients will become diabetic kidney disease patients.DKD has become a serious disease endangering human health.Renal fibrosis is an important pathological process in the development of diabetic kidney disease.However,currently there is a lack of effective treatment options for DKD renal fibrosis.Transforming growth factor ?1(transforming growth factor-?1,TGF?1)plays an important role in the occurrence and development of renal fibrosis.Relevant studies have shown that the TGF?1/Smad3 pathway can regulate kidney fibrosis by regulating long non-coding RNAs(LncRNAs).Related studies have found that the overexpression of LncRNA MEG3(GENE ID:55384)aggravates cell proliferation and induces cell apoptosis.LncRNA MEG3 can regulate the renal fibrosis and inflammation in DKD rats by regulating the miR-181a/Egr-1/TLR4 axis.Oxidative stress plays a vital role in the occurrence and development of diabetic kidney disease.Excessive production of reactive oxygen species can cause early mesangial cell hypertrophy,mesangial expansion,extracellular matrix protein accumulation,glomerular sclerosis,endothelial cell damage,podocyte apoptosis,proteinuria,and renal dysfunction in patients with DKD.Keap1/Nrf2 is a very important endogenous antioxidant pathway.DKD belongs to the categories of diseases such as "edema"," shen-xiao ","xiao-ke" and" obstruction and rejection" in traditional Chinese medicine.DKD renal fibrosis is a typical"long-term disease entering the collaterals" and "kidney collateral stasis".DKD has deficiency of both qi and yin and kidney collaterals,which produce turbid phlegm,blood stasis and other products to block the kidney collaterals.If phlegm and stasis mutually condense for a long time,the collaterals and veins will become blocked.Treatment should replenish qi and nourish yin,dredge collaterals and eliminate evil.Tangshen Formula is a traditional Chinese Formula for the treatment of diabetic kidney disease initiated by Professor PingLi,the head of the research group.It is composed of seven chinese medicines including Astragalus membranaceus,Euonymus alatus,Rehmannia glutinosa,Citrus aurantium,Cornus officinalis,Rheum palmatum L,and Panax Notoginseng.In the early multi-center,randomized,double-blind,and placebo-controlled trials of our research group,Tangshen Formula significantly reduced the proteinuria level of patients with DKD and improved the estimate glomerular filtration rate(eGFR).However,the potential mechanism of Tangshen Formula in the treatment of DKD renal fibrosis and oxidative stress has not been thoroughly explored.Therefore,this study explored the molecular mechanism of Tangshen Formula in treating DKD renal fibrosis and oxidative stress damage by using network pharmacology methods combined with experiments in vivo and in vitroObjective1.To explore the potential mechanism of Tangshen Formula in improving diabetic kidney disease with renal fibrosis by approaching network pharmacology methods;2.To observe the efficacy of different doses of Tangshen Formula in improving DKD rats with right uninephrectomy combined with STZ injection-induced diabetic kidney disease3.To verify the therapeutic effect and related mechanisms of Tangshen Formula on renal fibrosis and oxidative stress in DKD rats with right uninephrectomy combined with STZ injection-induced diabetic nephropathy and HK2 cells stimulated by HGMethods1.Using network pharmacology and bioinformatics methods to explore the main active compounds,targets,activities and pathways of Tangshen Formula in the treatment of DKD and renal fibrosis.Use Cytoscape software to draw visual network structure diagrams,PPI diagrams,and perform data analysis.2.Using right uninephrectomy combined with a single strep tozo tocin injection induced DKD rat model to observe the efficacy of Tangshen Formula low-dose,Tangshen Formula medium-dose,Tangshen Formula high-dose,and Fosinopril Sodium.8-week-old Wistar rats were adaptively fed for 3 days and measured for basic information,including body weight,blood glucose,24-hour urine output,and urine protein quantitative.Then randomly divided into groups according to blood glucose and weight,Sham group and DKD group.DKD group underwent right uninephrectomy and a single intraperitoneal injection of STZ;after the DKD rat model was successfully done,they were randomly divided into 5 groups:DKD group,DKD+TSF low-dose group(1.2g/kg/d),DKD+TSF medium-dose group(2.4g/kg/d),DKD+TSF high-dose group(4.8g/kg/d),Fosinopril sodium group(1.33mg/kg/d).The experimental animals were given intragastric administration at the age of 10 weeks.During the experiment,the body weight was measured once a week,and the blood glucose was measured once every 4 weeks.After 20 weeks of continuous intragastric administration,the experiment ended.Sample materials were collected,including blood serum,kidney,liver and others.Serum biochemical testing items included renal function,liver function,and blood lipid-related indicators.Elisa kit was used to detect urine protein.Pathological tissue sections were stained with PAS and MASSON to evaluate the pathological damage of kidney.3.Using uninephrectomy combined with a single streptozotocin injection induced DKD rat model and HG-stimulated HK2 cell fibrosis model to study the efficacy and mechanism of Tangshen Formula in treating DKD with renal fibrosis.Basing on the results of the pharmacodynamics experiment,the low-dose group of TSF was selected for the following experiment in this chapter.Using immunohistochemistry,Western Blotting,immunofluorescence,and Real-time PCR to evaluate the effects of Tangshen Formula on fibrotic indicators in the renal cortex of DKD rats,including:Collagen I,Collagen ?,Fibronectin,TGF?1,Smad 3,p-Smad 3,p-Smad 2/3,LncRNA MEG3.After the intervention of Tangshen Formula treatment on HG-stimulated HK2 cells,the expression of collagen I,fibronectin,p-Smad 2/3,LncRNA MEG3 in HK2 cells were measured.4.Using uninephrectomy combined with a single streptozotocin injection induced DKD rat model and HG-stimulated HK2 cell oxidative stress injury model to explore the underlying mechanism of Tangshen Formula in ameliorating oxidative stress damage of DKD.Basing on the results of the pharmacodynamics experiment,the experiment group is the same as method 3.The expression of Keap1,Nrf2,HO1,3-NT in the kidney of DKD rats were detected by immunohistochemistry,Western Blotting,immunofluorescence,and Real-time PCR.The DHE staining method was used to detect the quantification of ROS in the kidney cortex of DKD rats.The expression of Keap1,Nrf2,HO1 in HK2 cell stimulated by high glucose were detected after Tangshen Formula treatment.Results1.Network pharmacology found that the main active ingredients of Tangshen Formula are quercetin,beta-sitosterol,kaempferol,Diincarvilone A,Ferulic acid methyl ester,rehmapicrogenin,4-Hydroxy-3-methoxycinnamaldehyde,Stigmasterol,Methyl 4-hydroxycinnamate,7-O-methylisomucronulatol.The core targets of TSF for the DKD renal fibrosis were as follows:vascular endothelial growth factor(VEGFA),epidermal growth factor receptor(EGFR),fibronectin 1(FN1),transforming growth factor ?1(TGF ? 1),signaling protein 3(SMAD3),signaling protein 2(SMAD2),etc.GO enrichment analysis found that TSF was involved in the biological process of anti-oxidant stress.Therefore,we found the oxidative stress-related factors of TSF,superoxide dismutase 1(SOD1),heme oxygenase 1(HOMX1),Kelch-like epichlorohydrin-related protein-1(KEAP1),nuclear factor E2-related factor 2(NFE2L2).TSF might ameliorate DKD by regulating the TGF?/Smad pathway and Keap1/Nrf2 pathway2.Pharmacodynamic results confirmed that TSF could ameliorate kidney damage in DKD rats.The body weight and condition of the DKD rats were worse than those in the Sham group from the beginning of the experiment.As the experiment progressed,the weight difference continued to increase.After 20 weeks of drug intervention,compared with the DKD group,the state of the rats in each group was improved.The body weight of TSF low-dose group and TSF medium-dose group were higher than that of DKD group from the 14th week to the 20th week of the experiment.The blood glucose level of rats in the DKD group was significantly higher than that in the Sham group during the whole experiment.The blood glucose levels of the TSF low-dose,middle-dose,high-dose group and fosinopril sodium group decreased from the 16th week to the 20th week of the experiment,which was significantly lower than that of the DKD group.The organ index,24h urine output,UACR,Scr,UREA,ALT,AST,and blood lipid levels of DKD rats were significantly higher than those of the Sham group.After treating DKD rats with TSF low-dose,medium-dose,high-dose and fosinopril sodium for 20 weeks,they can significantly reduce 24h urine output,reduce urine protein excretion,improve kidney function,liver function,and correct dyslipidemia.Kidney PAS staining and Masson's trichrome staining showed that there were significant mesangial matrix proliferation,renal tubular damage,and renal interstitial fibrosis in DKD rats.The treatment of TSF low-dose.TSF middle-dose,TSF high-dose and fosinopril sodium could significantly ameliorate the glomerular mesangial matrix proliferation and tubulointnterstitial injury.3.TSF could regulate the TGF?1/Smad 3 pathway and the expression of LncRNA MEG3 to improve the renal fibrosis of DKD.Compared with the Sham group,the protein and gene expression levels of Collagen I,Collagen ?,and Fibronectin in the kidney cortex of DKD rats were significantly higher elevated.The protein expression levels of TGF?1,p-Smad 2/3,Smad3,and p-Smad3 in the renal cortex of DKD rats were significantly higher than those in the Sham group.The gene expression level of LncRNA MEG3 in the kidney cortex of DKD rats was significantly higher than that in the Sham group.After 20 weeks of interventional treatment with TSF,the expression of Collagen I,Collagen ?,Fibronectin,TGF?1,p-Smad 2/3,Smad3,p-Smad3,LncRNA MEG3 were significantly reduced.TSF could improve the deposition of Collagen I,Fibronectin protein and the phosphorylation of Smad 2/3 protein in HK2 cells stimulated by high glucose.What's more,down-regulating the expression levels of TGF?1 and Lnc RNAMEG3 mRNA.4.TSF could improve the oxidative stress damage of DKD by regulating the Keapl/Nrf 2 signaling pathway.MDA content in DKD rats was significantly elevated compared with sham group,while the SOD content in DKD rats was lower than that in Sham group.The protein and gene expression levels of 3-NT,Keapl in the kidney cortex of DKD rats were significantly increased,and the expression levels of Nrf2,HO1 protein were significantly decreased.Immunofluorescence results showed that the content of ROS in the kidney cortex of DKD rats increased.After 20 weeks of intervention,TSF could significantly reduce the protein and gene expression levels of 3-NT,Keapl in DKD rats,and up-regulate the protein expression levels of Nrf2 and HO1 in the kidney cortex of DKD rats.In addition,TSF could reduce the expression of NOX4 gene level and increase the expression of TXNRD1 and GCLC gene levelTSF could reduce the content of ROS in HK2 cells stimulated by high glucose,upregulate the expression of Nrf2 in protein and gene level,and reduce the expression of Keap1 in protein and gene level.ConclusionThis is the first study to predict the potential targets of TSF in the treatment of DKD based on the network pharmacology.And the results of network pharmacology were verified in DKD rat animal experiments and HG-induced HK2 cells,which confirmed that TSF could regulate the TGF?1/Smad 3 pathway.and the expression of LncRNAMEG3 to improve the renal fibrosis of DKD.What's more,TSF could improve the oxidative stress damage of DKD by regulating the Keap1/Nrf2 signaling pathway.