Effect Of Clearing Turbid And Invigorating Collaterals Based On The Canonical Wnt/?-catenin Pathway And Transformation And Differentiation Of TGF-?1-Indured Renal Tubular Epithelial Cells

1 ObjectiveTo investigate the role of Wnt/?-catenin signaling pathway in transforming growth factor-?₁-induced renal tubular epithelial cell transdifferentiation and the effect of Qingshen granule on clearing heat and turbidity.2 MethodsRat renal tubular epithelial cell line?NRK-52E?with good growth status was inoculated into 12-well culture plates.When NRK-52E grown to 50%confluency,it was replaced with serum-free medium for 24 h.The cells were divided into 5 groups:blank group?normal rat serum?,model group?TGF-?₁ was added to the serum of normal rats?,and Dkk-1 group?TGF-?₁,Dkk-1 was added to the serum of normal rats?.Qingqi granule group?TGF-?₁ was added to the serum of Qingshen granule rats?and Dkk-1 group of Qingshen granules?TGF-?1 and Dkk-1 were added to the serum of Qingshen granule rats?.The morphological changes of renal tubular epithelial cells were observed.The viability of each group was determined by MTT assay.The expression of E-cadherin and?-SMA in renal tubular epithelial cells was detected by immunofluorescence.Western-blot assay was used to observe the expression of E-cadherin,?-SMA,Wnt4 and?-catenin proteins in the group.3 Results3.1.Cell morphology changesNormal NRK-52E cells are epithelial cells in the form of paving stones,which were round or elliptical.After NRK-52E cells induced by TGF-?₁,the morphology of NRK-52E cells became longer and larger,and the gap between cells and cells was broadened,and the number of cells in the same field of view decreased.Under the action of Qingshen granules,the"fusiform deformation"of the cells was significantly reduced,the cell gap was reduced,and the number was increased,but there were still significant differences with the morphology,gap and number of normal cells.3.2.Cell viability was determined by MTT assayAfter 24 hours and 48 hours of MTT treatment,the cell viability of TGF-?₁ group decreased with time,and decreased to 50%at 24 hours.Therefore,it is more suitable to select 24 hours for drug action time.3.3.Immunofluorescence assay for E-cadherin and?-SMA protein expressionE-cadherin showed bright red fluorescence in the blank group cells and the strongest fluorescence in each group,and the cell red fluorescence appeared weaker in the model group.Compared with the model group,the red fluorescence of Dkk-1 group,Qingshen granule group and Qingshen granule Dkk-1 group increased gradually,but the fluorescence intensity of the cells in the blank group was still significantly different.Alpha-SMA showed almost no red fluorescence in the blank group,bright red fluorescence in the model group cells and the strongest fluorescence in each group.Compared with the model group,the red fluorescence of Dkk-1 group,Qingshen granule group and Qingshen granule Dkk-1 group decreased gradually,but the fluorescence intensity of the cells in the blank group was still different significantly.3.4.Western Blot was used to detect the expression of Wnt4,?-catenin,E-cadherin and?-SMA protein in each groupWnt4,?-catenin and?-SMA showed almost no protein expression in the blank group,and the expression of histone was the most obvious in the model.Compared with the model group,the protein expression of Dkk-1 group,Qingshen granule group and Qingshen granule Dkk-1 group gradually decreased,but there was still significant difference compared with the blank group.E-cadherin showed significant protein expression in the blank group and was the strongest in each group,with less protein expression in the model group.Compared with the model group,the protein expression of Dkk-1 group,Qingshen granule group and Qingshen granule Dkk-1 group gradually increased,but there were still significant differences compared with the blank group.4 Conclusion1.During the transduction of NGF-?E cells induced by TGF-?₁,the Wnt/?-catenin signaling pathway is activated,and the activation of Wnt/?-catenin signaling pathway is involved in the EMT process of NRK-52E cells;2.Qingqi granules with clearing heat and turbidity can partially inhibit the activated Wnt/?-catenin signal and inhibit the TGF-?₁-induced EMT process in NRK-52E cells.