| Objective:To investigate the effect of salvianolic acid B(SalB)on the HPSE,SCD1 and HPSE/SDC1 axis in renal interstitial fibrosis in mice induced by unilateral ureteral obstruction(UUO)and in epithelial-mesenchymal transition(EMT)in human renal tubular epithelial cells(HK-2)induced by angiotensinⅡ(AngⅡ).To explore the effect of SalB on renal interstitial fibrosis and its possible mechanism in vivo and in vitro.Methods:1.C57 mice were randomly divided into 7 days and 14 days two groups,each group was divided into sham group,UUO group and three different doses of SalB groups(6.25mg·kg-1·d-1 of low,12.50mg·kg-1·d-1 of middle and 25.00mg kg-1·d-1 of high).Mice with left ureteral ligation under sterile conditions in UUO group and SalB groups.The sham group was the same operation method as the UUO group except the ligation.After first days of operation,the SalB groups was injected with the corresponding dose of SalB,the mice in sham group and UUO group were injected with the same volume of normal saline.The changes of body weight of mice were observed.Each group of mice were treated with 7d and 14d,respectively.The kidneys,blood and urine were taken on 7th day and 14th day after administration.Then,detect the levels of serum creatinine(Cr),blood urea nitrogen(BUN);transforming growth factor-β1(TGF-β1)fibroblast growth factor-2(FGF-2),syndecan-1(SCD1)and the activity of heparanase(HPSE).Hematoxylin eosin staining(HE staining)and collagen staining(Masson staining)to detect the changes of renal pathology.The expression of a-smooth muscle actin(a-SMA),HPSE and SDC1 were detected by immunohistochemistry(IHC).Westem Blotting(WB)is mainly detect a-SMA,E-cadherin,TGF-β1,FGF-2,HPSE and SDC1 expression of the protein in renal tissue.2.MTT assay different concentrations of AngⅡ(0,10-4,10-3,10-2,10-1,1,101,102μmol·L-1)on the HK-2 cells cultivate for 48h to determine the optimum concentration of Ang Ⅱ.MTT assay different concentrations of SalB(0,10-3,10-2,10-1,1,101,102,103,104μmol·L-1)on the HK-2 cells cultivate induced by AngⅡ(μmol·L-1)for 24h to determine the appropriate concentrations of SalB and observe the cell morphology of HK-2 cells by microscope.ELISA kits were used to detect the levels of TGF-(31,FGF-2,SCD1 and the activity of HPSE in the supernatant.WB and Immunofluorescence(IF)is mainly detect a-SMA,E-cadherin,TGF-β1,FGF-2,HPSE and SDC1 expression of the protein in HK-2 cells.3.To synthesize recombinant plasmid of human HPSE(Homo HPSE)gene andempty vector of control plasmid.After transfection of HK-2 cells with different concentrations of Homo HPSE(0,2,3,4,5μg·mL-1)for 24h,the expression level of HPSE protein in the cells was detected by fluorescence microscopy and WB to determine the appropriate transfection concentration of Homo HPSE.Cultured HK-2 cells were randomly divided into control group(Blank group),negative control group(group NC),Homo HPSE transfected group.Real-time PCR and WB were used to detect the expression of HPSE gene and protein in Homo transfected HK-2 cells and normal HK-2 cells in order to verify whether the HPSE gene was overexpressed in HK-2 cells.The HK-2 cells were randomly divided into NC group,NC+AngⅡ group,HomoHPSE+AngⅡgroup,HomoHPSE+AngⅡ+SalB group,using Real-time PCR and WB to detect the expression levels of HPSE,SCD1 gene and protein in HK-2 cells.ELISA kits were used to detect the levels of TGF-β1,FGF-2,SCD1 and the activity of HPSE in the supernatant.Results:1.After mice were treated with unilateral ureteral ligation for 7 and 14 days,compared with the sham group,the weight of UUO mice was decreased(P<0.05,P<0.001),and then slowly increased;the kidney weight and kidney index increased significantly(P<0.001);the levels of Cr,BUN,TGF-(31,FGF-2 and HPSE activity in serum were significantly increased(P<0.01,P<0.001),but the level of SDC1 was significantly decreased(P<0.01).The pathological results showed that UUO mice left kidney volume was significantly larger than the right,which is full of effusion,thinner renal parenchyma,pyelocaliectasis,dilation of renal tubule degeneration and necrosis,infiltration of inflammatory cells,renal collagen blue staining increased significantly,renal interstitial fibrosis area widened,with the obstruction time prolonged,the pathological changes were more significant.The expression levels of a-SMA,TGF-β1,FGF-2 and HPSE proteins increased significantly in the kidneys of mice(P<0.01,P<0.001),but the expression levels of E-cadherin and SCD1 protein significantly decreased(P<0.001),compared with 7d groups,the changes of proteins expression levels of 14d groups were more significant.After intraperitoneal injection of different doses of SalB in UUO mice,compared with UUO group,the mice weight has increased,but not significantly(P>0.05),kidney weight and kidney index decreased significantly(P<0.05,P<0.01,P<0.001);the serum Cr,BUN,TGF-beta 1,FGF-2 content and HPSE activity were significantly decreased(P<0.05,P<0.01,P<0.001),but the level of SDC1 was increased significantly(P<0.05,P<0.001),and these changes were positively correlated with the administration time and dose of SalB.The pathological results showed that SalB had a significant improvement in the renal injury of the UUO mice,which was dose and time dependent.After intraperitoneal injection of different doses of SalB in UUO mice,the expression levels of α-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE in renal tissue were decreased(P<0.05,P<0.01,P<0.001),but the expression levels of E-cadherin and SCD1 were increased(P<0.05,P<0.01,P<0.001),with the prolongation of SalB administration time and the increase of dose,the effect of UUO on the expression of these proteins in kidney was significantly improved.2.The results of MTT showed that,compared with DMEM group,the treatment of HK-2 cells with 1μmol·L-1AngⅡ for 48h could significantly inhibit the proliferation of HK-2 cells(P<0.05);then HK-2 cells were treated with different concentrations of SalB for 24h,the low concentration of SalB(0.1μmol·L-1)could promote its proliferation(P<0.05),but the high concentration of SalB(102μmol·L-1)showed inhibitory effect.The morphology of HK-2 cells in each group did not change significantly.Compared with DMEM group,the levels of TGF-β1,FGF-2 and HPSE activity were significantly increased(P<0.01,P<0.001),but the level of SCD1 was significantly decreased in the supernatant of AngⅡ group(P<0.01).The expression levels of α-SMA,E-cadherin,TGF-β1,FGF-2 and HPSE protein in cells were significantly increased(P<0.001),but the expression levels of E-cadherin and SCD1 protein were significantly decreased(P<0.001).After cells were treated with different doses of SalB for 24h,the levels of TGF-β1,FGF-2 and HPSE activity were significantly decreased(P<0.05,P<0.01,P<0.001),but the level of SCD1 was significantly increased in the supernatant(P<0.05,P<0.01)in a dose-dependent manner of SalB;The expression levels of a-SMA,E-cadherin,TGF-β1,FGF-2 and HPSE protein in cells were decreased but the expression levels of E-cadherin and SCD1 protein were increased(P<0.05,P<0.01)in a dose-dependent manner of SalB.3.Fluorescence microscopy and WB results showed that the appropriate concentration of Homo HPSE plasmid transfected HK-2 cells was 3μg·ML-1L Compared with blank group,the expression of mRNA and protein of HPSE increased significantly after transfection with Homo HPSE in HK-2 cells(P<0.001).After the overexpression of HPSE gene in HK-2 cells were induced by AngⅡ for 48h,treated with SalB for 24h,compared with the NC+AngⅡ group,the expression levels of HPSE mRNA,α-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE protein in HK-2 cells of NC group were lower(P<0.05,P<0.01,P<0.001),the expression levels of E-cadherin and SCD1 protein were higher(P<0.001);The levels of TGF-β1 FGF-2 and HPSE activity in supernatant were lower(P<0.05,P<0.01),the level of SCD1 was higher(P<0.05).The expression levels of HPSE mRNA,a-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE protein in HK-2 cells of HomoHPSE+AngⅡ group were significantly increased(P<0.05,P<0.01,P<0.001),the expression levels of E-cadherin and SCD1 protein were significantly decreased(P<0.01);The levels of TGF-β1,FGF-2 and HPSE activity in supernatant were significantly increased(P<0.05),but the level of SCD1 was significantly decreased(P<0.05).After the treatment of HK-2 cells with concentration of 1μmol L-1 SalB for 24h,the expression levels of HPSE mRNA,a-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE protein were significantly decreased(P<0.05,P<0.01),the expression levels of E-cadherin and SCD1 protein increased significantly(P<0.01,P<0.001),but the expression level of SCD1 mRNA in each group did not change significantly;The levels of TGF-β1,FGF-2 and HPSE activity in supernatant were decreased(P<0.05,P<0.01),but the level of SCD1 was increased(P<0.01).Conclusion:1.SalB may reduce the HS side chain cleavage of SCD1 and reduce the release of FGF-2 and TGF-β1 by decreasing the activity and the protein expression level of HPSE,in order to delay the progression of renal interstitial fibrosis caused by unilateral ureteral obstruction,in a time-and dose-dependent manner.2.SalB may reduce the HS side chain cleavage of SCD1 and reduce the release of FGF-2 and TGF-β1 by decreasing the activity and the protein expression level of HPSE,in order to inhibit the occurrence of EMT induced by AngⅡ and the deposition of ECM in HK-2 cells,in dose-dependent manner.3.SalB may regulate the HPSE/SCD1 axis by directly regulating the expression level of HPSE,decrease the release of cytokines such as FGF-2 and TGF-1,in order toimprove the EMT induced by AngⅡ and the deposition of ECM in HK-2 cells. |
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