| BackgroundRenal fibrosis is a common pathological process of progression from chronic kidney disease to end-stage renal disease caused by hypertension,diabetes and other diseases.Modern pharmacological studies have found that rehmannia rehmannii can lower blood sugar,fight inflammation,regulate immunity,and treat cardiovascular and cerebrovascular diseases,and can treat a variety of basic diseases that induce chronic kidney disease.Modern pharmacological studies have found that catalpol has anti-inflammatory and hypoglycemic effects and can delay myocardial and renal fibrosis caused by diabetes.The research group speculated that rehmanb could inhibit epithelial-mesenchymal transition（EMT）by regulating the Wnt/β-catenin pathway,thus delaying the process of renal fibrosis,and catalpol may be the basis of its pharmacodynamics.This study was supported by the department of nephrology,chongqing hospital of traditional Chinese medicine,a national key clinical specialty of traditional Chinese medicine,and by the national natural science foundation of China（81603611）.PurposeTo observe the effect of Radix rehmanniae on delaying renal fibrosis and explore whether its mechanism is related to the regulation of Wnt/β-catenin signaling pathway and whether its main material basis is catalpol alcohol.Methods1 Quality determination of raw rehmannia glutinosa decoction piecesAccording to the method of rehmannia glutinosa in 2015 edition of Chinese pharmacopoeia,the raw rehmannia glutinosa was identified and its content was determined.2 The rat model of unilateral ureteral obstruction was replicated and administered in groups50 male SD rats were divided into the model group 40（ligation of left ureter）and the sham group 10（no ligation）.The success criterion of the model was that the renal tissue on the obstructed side of the rat was obviously enlarged.HE staining results showed that the renal tubules were obviously dilatated and there were more inflammatory cell infiltration.The results of Masson staining showed a significant increase in the blue staining of Collagen fibers.Rats with successful modeling were randomly divided into model group,raw rehmanb 3.2g·kg-1 group,1.6g·kg-1 group and 0.8g·kg-1 group,with10 rats each.The raw rehmannia group was given decoction once a day by gavage for14 consecutive days.3 Study on the mechanism of renal fibrosis in rats with unilateral ureteral obstruction delayed by water extract of Radix Rehmanniae1 hour after the end of the last gavage,blood was collected under anesthesia and serum creatinine and urea nitrogen were determined by enzyme-linked immunoassay.HE staining was used to observe the pathological changes of kidney tissues.Masson staining was used to observe the renal tubule interstitial fibrosis.Immunohistochemical and western blot methods were used to detect the expression of the marker proteins of renal fibrosis,such asα-SMA,Vimentin,Collagen I,E-cadherin and Wnt/β-catenin signaling pathway proteins,Wnt5a,GSK-3β,β-catenin.Kidney tissue homogenate was taken and the expression of these proteins was observed by western blot.4 Replication and grouping of EMT model of human tubular epithelial cells10 ng·ml-1 TGF-β1 was used to induce the EMT of human renal tubular epithelial cells.The normal group,model group,catalpol 10μg·ml-1 group,catalpol 50μg·ml-1group,and catalpol 100μg·ml-1 group were set,and cultured for 24 hours after administration.5 Study on the mechanism of catalpol on TGF-β1 induced EMT of human tubular epithelial cellsThe morphological changes of cells in each group were observed under the microscope,and the expression of EMT marker proteinsα-SMA was observed by immunofluorescence method.The cells were collected,and the expression of EMT marker proteins Vimentin,α-SMA and Wnt/β-catenin signaling pathway proteins Wnt5a,GSK-3β,β-catenin were detected by western blot.Results1 results of TLC identification and content determination of catalpol in Radix RehmanniaeThe test solution of raw rehmannia glutinosa used in this experiment showed brown spots in the same position as the catalpol control product.The content of catalpol was determined by HPLC to be 1.19%,which was in line with the quality regulation of raw rehmannia glutinosa in the 2015 edition of Chinese pharmacopoeia.2 The success rate and intergroup equilibrium test of the rat model of unilateral ureteral obstruction were replicatedThe obstructed kidney of the model group was larger than that of the sham operation group,and the results of HE and Masson staining showed that the obstructed kidney of the model group had renal tubulointerstitial fibrosis,suggesting that the modeling was successful and the molding rate was 100%.3 The process of renal fibrosis in the rat model of unilateral ureteral obstruction was delayed by the water extract of Radix Rehmanniae（1）Compared with the sham operation group,the cortical and medullary mass of ligated kidney tissues in the model group were thinner.Cystic dilatation of renal tubules,thickening of basement membrane,epithelial cells of renal tubules flattened,exfoliated,swollen and vacuolated.Glomerular hyaline degeneration,balloon adhesion,atrophy,interstitial widening,inflammatory cell infiltration;Fibrosis,infiltration of inflammatory cells,cell proliferation and tubulotype of renal medulla interstitium were more obvious than that of cortex.Compared with the model group,the above pathological changes in ligated renal tissues of rats in the groups of 3.2g·kg-1,1.6g·kg-1and 0.8g·kg-1 were significantly improved.（2）Compared with sham operation,the formation of interstitial Collagen fibers in ligated renal tissues of rats in the model group was obvious,and the aizen was increased.Compared with the model group,the formation of mesenchymal Collagen fibers in ligated renal tissues of rats in the 3.2g·kg-1,1.6g·kg-1,and 0.8g·kg-1 groups decreased,and the aizen decreased.（3）Compared with sham surgery,the expression of Collagen I,Vimentin and Collagen ii in the kidney tissues of rats in the model group increased significantly,and the expression of e-cadherin decreased significantly（all P<0.01）.Compared with the model group,the expressions of Collagen I,Vimentin and Collagen I in the kidney tissues of rats in the groups of 3.2g·kg-1,1.6g·kg-1 and 0.8g·kg-1 were decreased,and the expression of e-cadherin was significantly increased（all P<0.01）.（4）Collagen I,Vimentin,α-SMA expressions were significantly increased in the model group compared with the sham group（all P<0.01）.Compared with the model group,the expression of Collagen I,Vimentin,α-SMA were significantly decreased in each dose group（all P<0.01）.4 By regulating the Wnt/β-catenin signaling pathway,the water extract of Radix Rehmanniae delayed the process of renal fiber in the rat model of unilateral ureteral obstruction（1）Compared with sham surgery,the expressions of Wnt5a andβ-catenin in kidney tissues of rats in the model group increased（all P<0.01）.Compared with the model group,the expressions of Wnt5a andβ-catenin in the renal tissues of rats in the groups of 3.2g·kg-1,1.6g·kg-1 and 0.8g·kg-1 were significantly reduced（all P<0.01）.（2）Compared with the sham operation group,the expressions of Wnt5a,GSK-3β,β-catenin in the model group were significantly increased（all P<0.01）.Compared with the model group,the expression of Wnt5a,GSK-3β,β-catenin proteins in raw rehsin group were significantly reduced in 3.2g·kg-1,1.6g·kg-1,and 0.8g·kg-1（all P<0.01）.5 Catalpol inhibits cell EMT of human tubular epithelial cells induced by TGF-β1by regulating the Wnt/β-catenin signaling pathway（1）Compared with the control group,the epithelial cells in the model group differentiated into long fusiform.Compared with the model group,the EMT of human renal tubular epithelial cells was inhibited in catalpol 10μg·ml-1 group,50μg·ml-1 group and 100μg·ml-1 group,maintaining a paving stone shape.（2）Compared with the control group,the expression of fibrosis marker proteinα-SMA in the model group was significantly higher than that in the normal group.Compared with the model group,catalpol 10μg·ml-1 group,50μg·ml-1 group and100μg·ml-1 group showed significantly lower expression ofα-SMA（all P<0.01）.（3）Compared with the normal group,the expression of Vimentin,α-SMA in the model group was significantly increased（all P<0.01）.Compared with the model group,the expression levels of Vimentin,α-SMA were significantly reduced in catalpol 10μg·ml-1group,50μg·ml-1 group and 100μg·ml-1 group（all P<0.01）.6 Catalpol interferes with the cellular EMT of human renal tubular epithelial cells by regulating the Wnt/β-catenin signaling pathwayCompared with the normal group,the expressions of Wnt5a,GSK-3β,β-catenin in the model group were significantly increased（P<0.01）.Compared with the model group,the expression of Wnt5a,GSK-3β,β-catenin in the catalpol 10μg·ml-1 group,50μg·ml-1 group,and 100μg·ml-1 group were significantly reduced（P<0.01）.Conclusions1 The process of renal fibrosis in the rat model of unilateral ureteral obstruction was delayed by the water extract of natural diaquin,the mechanism of which was related to the down-regulation of the expression of related proteins in the Wnt/β-catenin signaling pathway.2 Catalpol is one of the material bases of Radix Rehmanniae to inhibit the epithelial interstitial EMT of renal tubules and delay renal fibrosis. |
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