Effects Of Tanshinone ⅡA Combined With DAPT On TGF-β1 Induced Notch1/Jagged1 Signaling Pathway In Human Renal Tubular Epithelial Cell Transdifferentiation

Objective: To study the effects of Tanshinone IIA combined with DAPT on the expression of transdifferentiation markers E-cadherin,Snail,N-cadherin and FN,and Notch1 / Jagged1 signaling pathway in human renal tubular epithelial cells(HK-2)induced by TGF-β1,and determine whether the synergistic effect of Tanshinone IIA combined with DAPT on anti-renal interstitial fibrosis,and it may provide a new research model and theoretical basis for the research and treatment of renal interstitial fibrosis with the compatibility of Chinese and Western medicine molecules.Methods:(1)The optimal drug concentration was selected by cell proliferation rate treatment with Tanshinone IIA and its combination with DAPT which detected by CCK-8 method;(2)HK-2 cells were randomly divided into five groups: Normal group(no treatment),Model group(TGF-β1 10 ng / ml),Tanshinone IIA group(TGF-β1 10 ng /ml + Tanshinone IIA 5 μmol / L),DAPT group(TGF-β1 10 ng / ml + DAPT 10 μmol /L),Tanshinone IIA + DAPT group(TGF-β1 10 ng / ml + Tanshinone IIA 5 μmol / L +DAPT 10 μmol / L),the morphological changes of each group were observed though microscope;(3)The localization and expression of EMT-related indicators E-cadherin,N-cadherin,FN,and channel proteins Notch1 and Jagged1 in five groups of cells detected by Immunofluorescence;(4)The m RNA and protein expressions of E-cadherin,Snail,N-cadherin,FN,and channel proteins Notch1 and Jagged1 in the five groups of cells detected by RT-q PCR and Western bolt techniques,respectively.Results:(1)The optimal drug concentration of Tanshinone IIA was 5 μmol / L in the Tanshinone IIA group and the Tanshinone IIA + DAPT group screened by CCK-8 method;(2)Observation under a microscope: HK-2 cells in the normal group showed cobblestone-like adhesion and growth,with typical epithelial-like cell morphology.The cell shape of the model group is usually a long spindle shape,the cells of model group with different sizes,similar to fibroblasts,with a large number and close arrangement.Long spindle cells in Tanshinone IIA group,DAPT group,and Tanshinone IIA + DAPT group were significantly reduced,morphologically close to the normal group,and uniform in density;(3)Cell immunofluorescence results showed that E-cadherin,N-cadherin,Notch1 and Jagged1 were expressed on HK-2 cell membrane,and FN was expressed in HK-2 cell cytoplasm;compared with the normal group,the model group E-cadherin expression decreased(P<0.05),FN,N-cadherin,Notch1 and Jagged1 expression increased(P<0.05);compared with the model group,E-cadherin expression increased in various intervention groups(P<0.05),N-cadherin,FN,Notch1,and Jagged1 expression decreased(P<0.05);(4)The results of RT-q PCR and Western bolt showed that the relative expressions of Snail,N-cadherin,FN m RNA and protein in HK-2 model group induced by TGF-β1 were higher than those in the normal group(P<0.05);The relative expressions of E-cadherin m RNA and protein of HK-2 in each intervention group were higher than that in the model group(P<0.05).Compared between the intervention groups,the relative expressions of E-cadherin m RNA and protein in the Tanshinone IIA + DAPT group were significantly higher than in the Tanshinone IIA group(P<0.05),but the difference was not statistically significant between the DAPT group and the Tanshinone IIA + DAPT group(P>0.05);(5)The results of RT-q PCR and Western bolt showed that compared with the normal group with low expression of Notch1,Jagged1 m RNA and protein,the relative expressions of Snail,N-cadherin,FN m RNA and protein in the model group HK-2 cells were increased(P<0.05);Compared with the model group,the relative expressions of Snail,N-cadherin,FN m RNA and protein in each intervention group HK-2 cells were decreased(P<0.05);Compared between the intervention groups,the relative expressions of Snail,N-cadherin,FN m RNA and protein in the Tanshinone IIA + DAPT group were significantly lower than the Tanshinone IIA group or the DAPT group(P<0.05).(6)The results of RT-q PCR and Western bolt showed that the expression of Notch signaling pathway indicators Notch1,Jagged1 m RNA and protein in the normal group was lower than the model group,and the relative expressions of Notch1,Jagged1 m RNA and protein in HK-2 cells in the model group were higher than that in the normal group(P<0.05);The relative expressions of Notch1,Jagged1 m RNA and protein in the intervention group were lower than those in the model group(P<0.05);the relative expressions of Notch1 m RNA and protein in the Tanshinone IIA + DAPT group were lower than those in the Tanshinone IIA group(P<0.05),although there was no significant difference between the DAPT group and the Tanshinone IIA + DAPT group(P>0.05).The relative expressions of Jagged1 m RNA and protein in the Tanshinone IIA+ DAPT group were lower than in the Tanshinone IIA or the DAPT group(P<0.05).Conclusions: Tanshinone IIA combined with DAPT can inhibit the expression of EMT-related proteins such as Snail,N-cadherin,and FN secreted by HK-2 cells induced by TGF-β1,and reduce the expression of its channel ligand Jagged1 m RNA and protein,which superior to the single drug.Tanshinone IIA combined with DAPT also up-regulated E-cadherin secretion and decreased Notch1 m RNA and protein expression induced by TGF-β1 in HK-2 cells,and the effect was better than that of Tanshinone IIA group.These results indicated that that Tanshinone IIA combined with DAPT can inhibit epithelial cell transdifferentiation and matrix deposition during the TGF-β1-induced interstitial fibrosis of HK-2 cells,and its mechanism may be associated with regulating the expression of Notch1 and Jagged1,inhibiting EMT changes and interfering with the transmission of Notch1 / Jagged1 signal pathway.