The Study Of Crosstalk Between Wnt And TGF-β In Activated Hepatic Stellate Cells

Objective:Wnt signal transduction pathway is a hot topic in molecular biology, cell biology and cancer research recent years, involved in embryonic development and adult cell proliferation, differentiation and apoptosis regulatory process. The abnormal activation of Wnt signaling pathway is closely related with the occurrence and progress of fibrosis at kidney, lung, liver, heart and skin tissues.In this study, we use detection methods such as hepatic stellate cells in vitro, MTT, VG staining, ELLSA, Western blotting, to show Wnt and TGF-βsignaling pathway in the activation of hepatic stellate cells stimulated by acetaldehyde in the crosstalk of the possible mechanisms, and then from the perspective of stellate cell activation, Observe the functions of the two pathways cross talk effects. First, use acetaldehyde to stimulate HSC cell, by VG staining, observed from the morphological point of HSC activation; MTT assay the proliferation level of activated HSC; use ELISA method to detect the content of TIMP I and fibronectin (FN). Prove that acetaldehyde can stimulate the activation of hepatic stellate cells. Then use SB-431542 to specific blocking TGF-β, westernblotting detect the phosphorylation ofβ-catenin which is the downstream products of the Wnt signaling pathway; use TDZD-8 specific blocking GSK-3β, further activate Wnt signaling pathway, westernblotting detect the phosphorylation status of Smad3, which is a key factor of TGF-β; using the SB-431542 and TDZD-8 at the same time, Westernblotting detect P-catenin and Smad3 phosphorylation, to analyze the crosstalk which may exist between the two pathways. Methods:(1) HSC-T6 cells were divided into untreated group (negative control), activated acetaldehyde group (positive control group), TDZD-8 treatment group (Wnt way activation), acetaldehyde activation+TDZD-8 treatment group (Wnt way to further activation), acetaldehyde activation+SB-431542 treatment group (TGF-βpathway blocked, wnt way activated), acetaldehyde activation+SB-431542+TDZD-8 processing (Wnt way to further activation, TGF-βpathway blocked).After the cells cultured to confluence 70%-80%,0.4% FBS DMEM medium to synchronize cells for 12h, replace the medium to 10% FBS DMEM complete medium. Use inhibitors of the corresponding training to treat the control group, stimulate group use the corresponding aldehyde inhibitor pretreatment for 1h after stimulation (final concentration of 200μmol/L) stimulation of additional time per 12h, stimulated 24h.Then extract of total cellular protein, G250 determination of protein content, SDS-PAGE electrophoresis separation, Western blotting to detect the protein content, test targets including:Wnt way downstream product:the total (3-catenin, phosphorylation ofβ-catenin, TGF-βway: total smad3, phosphorylation smad3-S425, phosphorylation smad3-S204. According to westernblotting protein band intensity,using Quantity One analysis software for processing, analysis different role of receptor antagonist or activator protein phosphorylation subsequent. (2) Group as (1), all cells are inoculated in six well board climbing film, cell treatment program with (1), then use VG staining and observed under the microscope. ELISA detect fibronectin and collagen I prime content of cell culture supernatant. (3) 96-well plates inoculated HSC-T6 cells in beads, the same group as (1), each group with 3 holes,and the cell treatment as (1), light microscope observe the growth conditions of cells, MTT detect activity of cells. Results:(1) After being activated by acetaldehyde, MTT assay the activation of HSC cells were significantly increased, ELISA detect fibronectin FN,collagen I and smooth muscle actin a-SMA content increase in different degrees, VG staining showed collagen content increased, the alcohol metabolite acetaldehyde on the activation of hepatic stellate cells stimulated significantly. (2) After using the wnt activator TDZD-8, Wnt way downstream products of P-catenin expression was not significantly changed, but the p-β-catenin levels decrease; in the other group using SB431542 blocked TGF-βpathway, the downstream products ofβ-catenin was no significant change, p-β-catenin levels decrease; use TDZD-8 and SB431542 also blocked TGF-βpathway, activation of Wnt way, P-catenin expression was no significant change, p-β-catenin levels were significantly decreased. (3) After using the Wnt activator TDZD-8(GSK-3(3 specific inhibitor), TGF-P pathway critical factor Smad3 phosphorylation of S425 and S204 were significantly increased; after using SB431542 blocked TGF-βway, S425 and S204 are significantly reduced; simultaneously TDZD-8 and SB431542, S425 and S204 are increased, but the level of expression is not significant than TDZD-8 or SB431542 alone; Conclusion:(1) Acetaldehyde activated HSC cells, HSC cells can be further activation, the secretory product of collagen I and fibronectin significantly increased, cells proliferation and transdifferentiation.(2) Acetaldehyde-activated hepatic stellate cells, TGF-βsignaling pathway can cause inhibition of Wnt signaling pathway, so TGF-P signaling pathway can inhibit the effects of Wnt signaling pathway. (3) Acetaldehyde can lead to activation of TGF-βP signaling pathway in hepatic stellate cells, and Wnt and TGF-βsignaling pathway exists "crosstalk", Wnt signaling pathway has a antagonistic effects on the Smad3-S425 and Smad3-S204 phosphorylation.