Study On The Mechanism Of Tangshen Decoction Against Oxidative Damage And Improving Renal Interstitial Fibrosis

Renal interstitial fibrosis(RIF)is mainly characterized by the substantial proliferation of fibroblasts and myofibroblasts(MFs)and accumulation of extracellular matrix(ECM).RIF is the common pathological basis for nearly all the chronic kidney diseases(CKDs)to progress into end-stage renal diseases(ESRD).The pathogenesis of RIF is complicated,where oxidative stress response is one of the key factors.Traditional Chinese medicine has huge potential in the anti-oxidation and anti-fibrosis treatment.Tangshen Formula(TSF)is comprised of seven Chinese herbal medicines,i.e.Astragali Radix,Radix Rehmanniae,Corni Fructus,Panax notoginseng,Euonymus alatus,Radix et Rhizoma Rhei,and Citrus aurantium,based on the principles of reinforcing Qi and nourishing Yin,promoting blood circulation to remove meridian obstruction and purging turbidity for detoxication.The preliminary studies showed that TSF had a clear protective effect on the kidneys of db/db mice in the model of type 2 diabetes mellitus,where reduction of oxidative stress was one of the possible mechanisms.In the model of uninephrectomized rats induced by streptozotocin(STZ)and the model of mice with unilateral ureteral obstruction(UUO),TSF intervention showed a good effect of RIF improvement.In this study,the animal model of mice with UUO was used to discuss the mechanism of kidney protective effect of TSF through down-regulation of expression of NADPH oxidase and improvement of oxidative damage to kidneys.On this basis,it was validated in the experiment in vitro that the TSF in the drug-containing serum had a protective effect on the oxidative damage to the epithelial cell line(HK-2)in proximal convoluted tubules of human kidneys induced by hydrogen peroxide,providing an experimental basis for the anti-oxidation effect of TSF in treatment of RIF.ObjectiveTo study the protective effect of TSF in RIF,and to discuss whether its mechanism is related to the down-regulation of expression of NADPH oxidase and reduction of oxidative stress in the kidneys.Methods1.Study on protective effect and mechanism of TSF on the kidneys of mice with UUO56 male C57BL/6N mice at the age of 7 weeks were randomly divided into sham operation group(only the left ureter was dissociated,but not ligated or cut off),model group(UUO group),TSF group(UUO+TSF therapy)and fosinopril group(UUO+fosinopril THERAPY),with adaptive feeding for 3 days and pre-administration for 5 days before the operation with left ureteral obstruction,with post-operative continued intragastric administration.TSF group was given TSF at 3.6g/kg/d,fosinopril group was given fosinopril at 1.5mg/kg/d,and the sham operation group and model group were given the same volume of normal saline.The mice of the model group,TSF group and fosinopril group were sampled twice respectively on postoperative days 7 and 14,and the mice of the sham operation group were sacrificed on postoperative day 14.Sera were collected to detect the renal function(serum creatinine and urea nitrogen),MDA content and SOD activity.The obstructed kidney was removed to detect the MDA content in the renal tissue homogenate by the TBA method,and to detect the SOD activity by the WST-1 method.The kidney paraffin section was made to observe the cell infiltration by HE staining,to observe the extent of RIF by MASSON and semi-quantitative analysis,and to observe the type of renal collagen fiber hyperplasia by Sirius red staining.Immunohistochemistry,Western blotting,and real-time fluorescent quantitative real-time PCR methods were used to detect the expression of RIF related indicators(Collagen Type Ⅰ,Collagen Type Ⅲ,transforming growth factor(TGF)-β1 and connective tissue growth factor(CTGF))and oxidative stress-related indicators(3-nitrotyrosine,NADPH oxidase subunit,and Nrf2 pathway)in renal tissues.2.Study on the intervention of TSF in the drug-containing serum for the oxidative damage to HK-2 cellsMale SD rats(weight:200±10g)were randomly divided into the blank group and TSF group,10 rats in each group.TSF group was given TSF at 2.4g/kg/d,and the blank group was given the same volume of normal saline intragastrically for 7 consecutive days.2h after administration on an empty stomach on the morning of day 7,the rats were narcotized for blood sampling from the abdominal aorta.The blood samples were allowed to stand for 2h,and then refrigerated and centrifuged for 20 min at 3000r/min.The supernatant was separated,and then the sera from the same group were combined,inactivated at 56℃ for 30min and stored at-20℃.HK-2 cells cultured in vitro were intervened with hydrogen peroxide at different concentrations at different times,and the CCK8 method was used to detect the cell survival rate to screen the hydrogen peroxide concentration and time required for establishing the HK-2 cell oxidative damage model.CCK8 method was then further applied to detect the effect of drug-containing serum at different concentrations on the hydrogen peroxide-induced HK-2 cell survival rate to screen the concentration of drug-containing serum with the strongest effect of recovering the damaged cell proliferation.HK-2 cells were divided into blank group(culture medium+blank serum),model group(culture medium+hydrogen peroxide + blank serum)and TSF group(culture medium+hydrogen peroxide+drug-containing serum).TBA method was used to detect the MDA content of cell culture supernatant in each group,WST-1 method was used to detect the SOD activity,and quantitative real-time PCR techniques were used to detect the expression of TGF-β1 in the cells.Results1.TSF could improve the renal tissue damage to the mice with UUO,the mechanism of which was related to the response to oxidative stress.(1)Renal protection:Based on the changes in the renal pathology,TSF could significantly reduce the degeneration and necrosis of proximal tubular epithelial cells,reduce the renal interstitial inflammatory cell infiltration and reduce the renal interstitial collagen fibroplasia.The results of semi-quantitative analysis by Masson staining also showed that TSF could significantly reduce the collagen fiber area percentage on both days 7 and 14 of modeling(P<0.001,P<0.001).The results of immunohistochemistry showed that intervention with TSF could significantly reduce the expression of Collagen Type Ⅰ(P<0.001)on day 7 of modeling,and could significantly reduce the expression of Collagen Type Ⅰ and Collagen Type Ⅲ(P<0.001,P<0.001)on day 14 of modeling.The results of immunohistochemistry and real-time fluorescent quantitative real-time PCR showed that on day 7 of modeling,TSF could significantly reduce the TGF-β1 protein and mRNA relative expression(P<0.05,P<0.001)and could significantly reduce the CTGF mRNA relative expression(P<0.001);on day 14 of modeling,TSF could also significantly reduce the TGF-β1 protein and mRNA relative expression(P<0.01,P<0.001)and could significantly reduce the CTGF protein and mRNA relative expression(P<0.05,P<0.001).Based on the renal function,on day 14 of modeling,TSF could significantly reduce the serum creatinine level in the mice with UUO(P<0.01),without significant effect on the urea nitrogen.(2)Mechanism of response to oxidative stress:On both days 7 and 14 of modeling,the MDA content was significantly increased in the renal tissue(P<0.001,P<0.001)and serum(P<0.001,P<0.001)of the mice with UUO,while the SOD activity was significantly reduced in the renal tissue(P<0.001,P<0.001)and serum(P<0.05,P<0.05)of the mice with UUO;on day 7 of modeling,TSF could significantly reduce the MDA content in the renal tissue and serum(P<0.001,P<0.001),and significantly increase the SOD activity in the renal tissue and serum(P<0.001,P<0.05);on day 14 of modeling,TSF could significantly reduce the MDA content in the renal tissue and serum(P<0.001,P<0.001),and significantly increase the SOD activity in the renal tissue(P<0.001).The results of immunohistochemistry showed that TSF could significantly reduce the 3-nitrotyrosine protein relative expression(P<0.01)on day 14 of modeling.For the effect of NADPH oxidase subunit,the results of Western blotting and real-time fluorescent quantitative real-time PCR showed that compared to the model group,on both days 7 and 14 of modeling,TSF could significantly reduce the Nox protein(P<0.001,P<0.01)and mRNA(P<0.001,P<0.001)relative expression,and could also significantly reduce the phosphorylated p47phox protein(P<0.001,P<0.001)and mRNA(P<0.001,P<0.001)relative expression.For the effect of Nrf2 pathway,the results of Western blotting and real-time fluorescent quantitative real-time PCR showed that compared to the model group,on both days 7 and 14 of modeling,TSF could significantly reduce the Nrf2 protein(P<0.001,P<0.001)and mRNA(P<0.001,P<0.001)relative expression,and could also significantly reduce the NQO1 protein(P<0.01,P<0.05)and mRNA(P<0.001,P<0.001)relative expression.2.TSF in the drug-containing serum could reduce the hydrogen dioxide induced HK-2 cell oxidative damage.(1)The effect of hydrogen dioxide at the concentration of 200μmol/L was screened for 48h to establish the HK-2 cell oxidative damage model,and the concentration of drug-containing serum with the strongest effect of recovering the damaged cell proliferation was screened as 10%.(2)The MDA content in the hydrogen dioxide induced HK-2 cell culture supernatant was significantly increased(P<0.01),and the SOD activity was significantly reduced(P<0.01).Intervention with 10%drug-containing serum could significantly reduce the MDA content(P<0.01)and significantly increase the SOD activity(P<0.01).The result of real-time fluorescent quantitative real-time PCR showed that compared to the model group,intervention with 10%drug-containing serum could significantly reduce the TGF-β1 mRNA relative expression(P<0.01).Conclusions1.TSF could improve the renal tissue damage to the mice with UUO and reduce the expression of Collagen Type Ⅰ and Collagen Type Ⅲ.RIF improvement by TSF was possibly related to the down-regulation of expression of NADPH oxidase and the significant reduction of oxidative stress levels in the serum and renal tissue of the mice with UUO.2.The TSF in the drug-containing serum could reduce the oxidative stress level in the hydrogen peroxide-induced HK-2 cell damage model,and down-regulate the expression of TGF-β1.