| ObjectiveTo study the inhibitory effects of Compound Shelong Capsules(CSC)on renal tubular EMT and the effects on Wnt4/β-catenin signaling pathway in an in vivo renal fibrosis model and an in vitro epithelial-mesenchymal transition(EMT)model of renal tubular epithelial cells to provide an experimental basis for the treatment of renal fibrosis in chronic kidney disease(CKD).Methods1.In vivo experiments(1)Modeling method and drug administration: The unilateral ureteral obstruction(UUO)method was used to establish the rat model of renal fibrosis.SD rats were randomly divided into blank group(Control group),sham operation group(Sham group),and modeling group.After UUO surgery in the modeling group,the rats were randomly divided into model group(UUO group),positive drug control group(LP group,7.5mg/kg·d),Compound Shelong Capsules high dose group(CSC-H group,1.5g/kg·d),Compound Shelong Capsules medium dose group(CSC-M group,0.75g/kg·d),Compound Shelong Capsules low dose group(CSC-L group,0.375g/kg·d).The drug was administered 24 h after surgery and was given for 14 consecutive days.(2)Renal function tests: Cystatin C(Cys-C)in serum was measured by enzyme-linked immunosorbent assay;creatinine(Cr)was measured by sarcosine oxidase;urease was measured by urea nitrogen(BUN).(3)Hematoxylin-eosin staining,Sirius red staining and transmission electron microscopy: pathomorphology,degree of fibrosis and ultrastructure of the affected kidney in different groups.(4)Immunohistochemistry(IHC)assay: protein expression and distribution of α-SMA,E-cadherin,Wnt4,β-catenin and TGF-β1 in rat kidney.(5)Western blot assay: protein phosphorylation levels of rat kidney α-SMA,Ecadherin,Wnt4 and β-catenin.(6)Fluorescence quantitative PCR assay: m RNA expression levels of α-SMA,Ecadherin,Wnt4 and β-catenin in rat kidney.2.In vitro experiments(1)Modeling method and drug administration: TGF-β1(10ng/ml)was used to induce human renal tubular epithelial cells(HK-2)for 48 h to construct EMT model.The Blank group was cultured using basal medium,the TGF-β1 group was cultured with TGF-β110ng/ml,the Dkk-1 group was cultured with TGF-β1 10ng/ml and Dkk-1 100ng/ml,the CSC group was cultured with TGF-β1 10ng/ml and CSC 3mg/ml,and the CSC+Dkk-1group was cultured with TGF-β1 10ng/ml,Dkk-1 100ng/ml and CSC 3mg/ml were incubated.(2)Western blot assay: protein phosphorylation levels of α-SMA,E-cadherin,Wnt4 and β-catenin in each group of cells.Results1.In vivo experiments(1)Results of rat renal function indexes: compared with Sham group,the levels of Cr,BUN and Cys-C were increased in UUO group(P<0.01);compared with UUO group,the levels of Cr were decreased in LP group,CSC-H group and CSC-M group(P<0.05,P<0.01);compared with UUO group,the levels of BUN were decreased in CSC-H group and CSC-L group(P<0.05,P<0.01);Cys-C levels were significantly lower in each administration group compared with the UUO group(P<0.01).(2)HE staining results of rat kidney: compared with Sham group,the kidney pathological damage was obvious in UUO group;compared with UUO group,the degree of lesion in each administration group was improved,and the degree of renal tubular dilatation or atrophy,renal tubular epithelial cell edema or degenerative necrosis,and renal interstitial lymphocyte infiltration were all reduced,and the CSC-H and CSC-M groups were better.(3)The results of Sirius red staining in rat kidney: under ordinary light microscopy,compared with Sham group,orange-red collagen fiber deposition was seen at the edge of renal tubules and between renal tubules and renal interstitium in UUO group;compared with UUO group,the extent of red staining was reduced in LP group and each dose group of CSC,and the collagen deposition in renal interstitium was reduced,and the dilatation of renal tubules was reduced.The results under polarized light microscopy were generally consistent with those under ordinary light microscopy.(4)The results of transmission electron microscopy of rat kidney: compared with the UUO group,the edema and disorder of microvilli arrangement of renal tubular epithelial cells were reduced in each dosing group,and the number of mitochondria and rough endoplasmic reticulum was increased.(5)Results of rat kidney IHC assay: α-SMA,Wnt4,β-catenin and TGF-β1 protein expressions were compared in different groups.Compared with the Sham group,protein expression was enhanced in the UUO group,and renal tubular dilation was obvious;compared with the UUO group,protein expression was reduced in each administration group,and a small amount of expression was seen in the interstitium.Compared with the Sham group,E-cadherin protein expression was decreased in the UUO group,and a small amount was expressed in the cytoplasm of renal tubular epithelial cells,and renal tubular dilation was obvious;compared with the UUO group,E-cadherin protein expression was increased in each administration group.(6)Western blot test results of rat kidney: compared with the Sham group,the expression levels of α-SMA,Wnt4 and β-catenin in the UUO group increased(P<0.01,P<0.05)and the expression levels of E-cadherin decreased significantly(P<0.01);compared with the UUO group,the expression levels of α-SMA,Wnt4 and β-catenin in the LP group,CSC-M group and Compared with the UUO group,the α-SMA protein expression level was significantly decreased in the LP,CSC-M and CSC-L groups(P<0.01);the E-cadherin protein expression level was increased in the CSC-M group(P<0.05);the Wnt4 protein expression level was decreased in the CSC-M group(P<0.05);and the β-catenin protein expression level was significantly decreased in the LP and CSC-H groups(P<0.01).(7)The results of fluorescence quantitative PCR in rat kidney: compared with Sham group,the expression levels of α-SMA,Wnt4 and β-catenin m RNA in the affected kidney of UUO group were significantly increased(P<0.01)and E-cadherin m RNA expression levels were significantly decreased(P<0.01);compared with UUO group,α-SMA m RNA expression levels in CSC-H group were significantly decreased(P<0.01).expression level decreased in the CSC-H group(P<0.05);E-cadherin m RNA expression level increased significantly in the CSC-H,CSC-M and CSC-L groups(P<0.01);Wnt4 m RNA expression level decreased in each administration group(P<0.05,P<0.01);β-catenin m RNA expression levels decreased in LP,CSC-H and CSC-M groups(P<0.05,P<0.01).2.In vitro experimentWestern blot assay results of HK-2 cells: compared with Blank group,α-SMA,Wnt4,β-catenin protein expression levels were significantly increased(P<0.01)and E-cadherin protein expression levels were significantly decreased(P<0.01)in TGF-β1 group;compared with TGF-β1 group,α-SMA protein expression levels were decreased(P<0.05,P<0.01)in Dkk-1 group,CSC group,and CSC+Dkk-1 groups compared with TGF-β1 group(P<0.05,P<0.01);E-cadherin protein expression level was significantly increased in CSC group and CSC+Dkk-1 group(P<0.01);Wnt4 protein expression level was decreased in Dkk-1 group,CSC group and CSC+Dkk-1 group(P<0.05,P<0.01);β-catenin protein expression level decreased in CSC group,CSC+Dkk-1 group(P<0.05,P<0.01).Conclusions1.CSC may inhibit the process of tubular EMT in renal fibrosis by inhibiting the activation of Wnt4/β-catenin signaling pathway,which has a significant ameliorating effect on renal fibrosis.2.CSC may inhibit the process of EMT in HK-2 cells by suppressing the activation of Wnt4/β-catenin signaling pathway induced by TGF-β1(10ng/ ml)in HK-2 cells. |
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