| Diabetic kidney disease(DKD)is a characteristic microvascular complication of diabetes mellitus(DM).It was estimated that there are 451 million people with diabetes worldwide in 2017.The figures were expected to increase to 693 million by 2045.Approximately 30%of patients with T1DM and 40%of those with T2DM further develop DKD.DKD has become a leading cause of end-stage renal disease and increasing mortality in patients with DM.Proteinuria is the main index of clinical diagnosis and effect evaluation of DKD.The tutor thinks that protein belongs to essential substance of body,which is refined from food and water,and transferred from the spleen to all parts of body.Deficiency of Spleen Qi and Kidney Yin is the key pathogenesis of the proteinuria in DKD.DKD belongs to collateral disease with blood stasis throughout in traditional Chinese medicine.Thus,the tutor takes "ascending lucidity and descending turbidity" as the core theory and tonifying Qi and sending up essential substance as the basic treatment principle,supplements with the methods of nourishing Yin,promoting blood circulation,and dredging collaterals,and formulates Qi-Shen-Sheng-Qing-Jiang-Zhuo-Fang(QSF)to treat DKD,which has shown a clear effect of reducing proteinuria.This study took "the spleen acting as the protector of the body" concerned with immune as the breakthrough point and was aimed to evaluate the protective effects of QSF on the proteinuria,podocyte injury,renal inflammation,and oxidative stress in db/db mice,a spontaneous T2DM mouse model,and to explore the regulatory mechanism involved through the Nrf2/HO-1/NLRP3 inflammasome pathway.Aims:1.To observe the effects of QSF on proteinuria and podocyte injury in db/db mice.2.To explore the protective mechanisms of QSF against renal injury of db/db mice through the Nrf2/HO-1/NLRP3 inflammasome pathway.3.Provide modern scientific evidence for the treatment of QSF for DKD,and further enrich the theoretical connotation of "the spleen acting as the protector of the body" and "spleen being in charge of sending up essential substance".Methods:1.Animals:6-week-old male db/db mice(n=58)and wild-type mice(n=10)were housed.After 2-week adaptation,10 db/db mice were randomly selected as an 8-week-old control group,which were anesthetized with isoflurane inhalation after weighing,collecting urine volume and detecting fasting blood glucose(FBG).Blood samples and kidney were removed quickly.The remaining db/db mice(n=48)were randomly divided into 4 groups(n=2)and were then administered with either vehicle(distilled water,10 mL/kg),losartan(6.5mg/kg),or QSF(20.15g/kg,60.45g/kg)once daily via gavage.The 10 wild-type mice were administered vehicle alone2.Evaluation of renal function and glucose and lipid metabolism:24-hour urine samples were collected every 3 weeks;FBG was detect every 6 weeks.All mice were anesthetized with isoflurane inhalation after 12 weeks of administration.Blood samples for serum assessment were collected after confirming anesthesia.Then,the kidney was removed and weighted quickly.The kidney index was determined as:kidney/body weight(g/kg).Elisa kit was used to measure urine microalbumin concentration,and automatic biochemical analyzer was used to detect blood creatinine(Scr),blood urea nitrogen(BUN),and urinary creatinine.Urine albumin excretion(UAE),protein creatinine ratio(ACR),and creatinine excretion rate(Ccr)were calculated according to the corresponding formula.3.Evaluation of kidney pathological morphology and podocyte injury:HE-staining was performed to assess renal morphology;PAS and Masson staining were performed to assess extracellular matrix(ECM)deposition and renal fibrosis;transmission electron microscopy was performed to observe microscopy of podocytes and glomerular basement membrane(GBM).GBM thickness and foot process density were analyzed quantitatively.immunofluorescence staining and reverse transcription-polymerase chain reaction(RT-PCR)for podocin or synaptopodin were performed to assess podocyte injury.RT-PCR was used to detected FN1 and Col-??3 in renal cortex.4.Evaluation of NLRP3 inflammasome activation and inflammatory response:Western blot was used to detect the protein expression of NLRP3,ASC,pro-caspase-1,and caspase-1 in renal cortex;caspase-1 were localized with synaptopodin by double immunofluorescence staining in renal cortex;Liquid suspension chip(Luminex)and Elisa kit were performed to detect the levels of IL-1?,IL-18,IL-6,TNF-?,and MCP-1 in serum;RT-PCR was performed to detect the gene expression of NLRP3,ASC,caspase-1,GSDMD,IL-1?,and IL-18 in renal cortex;Immunohistochemistry was performed to determine the F4/80 level and assess the degree of macrophage infiltration in renal tissues.5.Detection of anti-oxidative stress factors and Nrf2/HO-1 pathway in renal cortex:Colorimetric method was performed to detect the levels of SOD and GSH in renal cortex;RT-PCR was used to detect the gene expression of Nrf2 and HO-1 in renal cortex.Results:1.Effects of QSF on renal function in db/db mice:(1)Kidney weight and KI:The kidney weight of 8-week-old and vehicle-treated db/db mice was higher than that of WT mice(P<0.05,P<0.01,respectively).The kidney weight of vehicle-treated db/db mice was 1.5 folds of that in WT mice(P<0.01),and the KI increased significantly in vehicle-treated db/db mice(P<0.01).Losartan and QSF-H significantly reduced KI(P<0.05,P<0.01,respectively).(2)Renal function(Scr,BUN,Ccr):Scr was lower(P<0.01)and Ccr was higher in 8-week-old db/db mice(P<0.01),while no changes in Scr and Ccr levels were observed in vehicle-treated db/db mice compared with those of WT mice(P>0.05).Scr increased by 66.6%(P<0.01)and Ccr decreased to 59.8%(P<0.05)compared with those of 8-week-old db/db mice.Losartan and QSF administration did not reduce the levels of Scr and Ccr(P>0.05).No changes in BUN levels were observed among different groups(P>0.05).(3)24-hour urine volume and urinary protein excretion:The levels of 24-hour urine volume,UAE,and ACR of db/db mice at each time point were significantly higher than those of WT mice(P<0.01)Losartan administration reduced 24-hour urine volume at each time point but without statistical difference(P>0.05).There was no significant decrease in 24-hour urine volume in QSF-treated mice(P>0.05).At 12th week,losartan and QSF-H significantly reduced the levels of UAE and ACR(P<0.05 or P<0.01),and QSF-L only reduced UAE level(P<0.05).2.Effect of QSF on glucose and lipid metabolism in db/db mice:Compared with WT mice,8-week-old db/db mice showed higher FBG,CHOL,TG,and LDL-C levels.(P<0.05 or P<0.01),the levels of LDL-C and HDL-C were also higher than those in vehicle-treated db/db mice(P<0.05 or P<0.01);neither losartan nor QSF reduced the levels of FBG,CHOL,TG,and LDL-C(P>0.05).3.Effect of QSF on pathological morphology and podocyte injury in db/db mice:(1)Pathological morphological changes:Compared with WT mice,vehicle-treated db/db mice displayed glomerular hypertrophy(P<0.01),obvious mesangial proliferation,ECM deposition,and severe renal fibrosis.QSF and losartan reduced the mean glomerular volume(P<0.01)and improved mesangial cell proliferation,ECM deposition and renal fibrosis.(2)Observation of ultrastructure:The GBM thickness is uniform,and the shape of foot processes is regular and closely arranged in WT mice.8-week-old db/db mice displayed obvious swelling and irregular shape of the foot process.No significant fusion of foot processes and podocytes detachment were observed in 8-week-old db/db mice,but the density of the foot process was reduced(P<0.01).Vehicle-treated db/db mice displayed significant GBM thickening(P<0.01),extensive fusion and detachment of the foot processes,and a significant decrease in foot process density(P<0.01).The GBM thickness in vehicle-treated db/db mice increased significantly compared with that of 8-week-old db/db mice(P<0.01),and there was no significant difference in foot process density(P>0.05).After 12 weeks of administration,losartan and QSF reduced GBM thickness(P<0.01),fusion and detachment of foot processes,and increased the density of the foot processes(P<0.01).(3)Protein and mRNA expression of podocin and sypaptopodin:Immunofluorescence analysis showed intense and linear staining of both podocin and synaptopodin in WT mice.This expression pattern was no significant difference in 8-week-old db/db mice but was weak and discontinuous in vehicle-treated db/db mice.Meanwhile,the relative expression of both podocin and synaptopodin decreased noticeably in vehicle-treated db/db mice compared with that of WT mice(P<0.01);this effect was partly reversed by losartan and QSF administration(P<0.05 or P<0.01).The mRNA levels of both podocin and synaptopodin in 8-week-old and vehicle-treated db/db mice were higher than in WT mice(P<0.05,P<0.01,respectively).Losartan and QSF administration increased the gene expression of podocin and synaptopodin but with no statistical significance(P>0.05).4.Results of QSF on NLRP3 inflammasome and inflammatory responses in renal tissues of db/db mice:The protein and mRNA expression of NLRP3,ASC,and caspase-1 in renal cortex of 8-week-old and vehicle-treated db/db mice significantly increased compared with those of WT mice(P<0.05 or P<0.01).Meanwhile,colocalization of caspase-1 with synaptopodin in the glomeruli,mRNA expression of IL-1? and GSDMD in the renal cortex,and the levels of IL-1? and IL-18 in serum also increased in 8-week-old and vehicle-treated db/db mice compared with those in WT mice(P<0.05 or P<0.01).In addition,serum IL-6 increased in 8-week-old db/db mice and the levels of TNF-? and MCP-1 in serum increased in vehicle-treated db/db mice compared with those in WT mice(P<0.05 or P<0.01).Losartan administration significantly reduced the protein and mRNA expression of NLRP3,caspase-1,colocalization of caspase-1 with synaptopodin,and serum IL-18 levels(P<0.05 or P<0.01).QSF significantly reduced the protein and mRNA expression of NLRP3,ASC,and caspase-1 in renal cortex,colocalization of caspase-1 with synaptopodin in the glomeruli,mRNA expression of IL-1? and GSDMD in renal cortex as well as the levels of IL-1?,IL-18 and MCP-1 in serum(P<0.05 or P<0.01).In addition,QSF also reduced the expression of F4/80 and improved macrophage infiltration in the renal cortex of db/db mice(P<0.05).5.Effects of QSF on oxidative stress factors and Nrf2/HO-1 pathway in renal cortex of db/db mice:No changes were observed in the levels of SOD and GSH and the mRNA expression of Nrf2,HO-1 in renal cortex of 8-week-old db/db mice compared with WT mice;these parameters decreased in vehicle-treated db/db mice than those in WT mice(P<0.05 or P<0.01)except the mRNA expression of Nrf2.Both losartan and QSF increased the levels of SOD and GSH(P<0.05 or P<0.01)and up-regulated the mRNA expression of HO-1(P<0.05)in renal cortex of db/db mice.Conclusions:1.8-week-old db/db mice display significant glucose and lipid metabolism disturbances,increased urinary protein levels,and hypertrophy of glomeruli and podocytes.NLRP3 inflammasome is activated in renal tissue and serum inflammatory cytokines levels increase,but the anti-oxidative stress factors are not significantly changed in renal tissues.Inflammation predates oxidative stress in kidney of db/db mice.2.QSF reduces the levels of UAE and ACR and improves weight loss in db/db mice at the end of the experiment.3.QSF inhibits glomerular hypertrophy,GBM thickening and ECM deposition.QSF improve podocyte injury and renal fibrosis with increased protein and gene expression of both podocin and synaptopodin and decreased gene expression of both FN1 and Col-??3 in db/db mice.4.QSF inhibits the activation of NLRP3 inflammasome and some downstream inflammatory cytokines,improves the degree of renal macrophage infiltration,suppress the immune response and thus improves the inflammatory cascade response in db/db mice.5.QSF increases the levels of SOD and GSH as well as the gene expression of Nrf2 and OH-1 and increase the anti-oxidative stress ability of db/db mice.6.QSF is formulated according to the theory of "ascending lucidity and descending turbidity".It is possible to regulate Nrf2/HO-1/NLRP3 inflammasome pathway and improves oxidative stress and immune inflammatory response by regulating the functions of "the spleen acting as the protector of the body" and "the spleen being in charge of sending up essential substance",thereby improving podocyte injury and reducing urinary protein excretion in db/db mice. |
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