Effects Of Crosstalk Between The Notch And TGF-β Pathways In Acetaldehyde-mediated Hepatic Stellate Cells Activation

Abstract:Objectives:The proliferation and transdifferentiation into fibroblasts of activated hepatic stellate cells plays an important role in liver fibrosis.Numerous cell signaling pathways are involved in hepatic stellate cells activation, proliferation and transdifferentiation. Acetaldehyde plays a pivotal role in activating HSC and inducing alcoholic liver fibrosis.To investigate the effects of crosstalk between Notch and TGF-βpathways on acetaldehyde-mediated hepatic stellate cells activation and transdifferentiation, and then provide further evidence for cellular mechanism of hepatic fibrosis.Methods:①HSC culture and treatments:Rat hepatic stellate cell line HSC-T6 was maintained at 37℃in a humidified incubator containing 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin.Acetaldehyde was used to stimulate HSC-T6 activation and transdifferentiation.To study the potential cross-talk between the Notch and TGF-βpathways under acetaldehyde treatment in HSC-T6,the effect of Notch inhibition on the TGF-B pathway activation and the effect of TGF-βinhibition on the Notch pathway activation were investigated.SB-431542 and DAPT were used to block the TGF-βand Notch pathway respectively. HSC-T6 were synchronised by placing in 0.4% FBS DMEM medium for 12h when the confluence of cells was approximately 70%-80% before acetaldehyde (final concentration 200μmol·L-1) treatment for the indicated time period.②RT-PCR:Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions.The reverse transcription reactions (RT-PCR) were carried out using the M-MLV Reverse Transcriptase (Promega) according to the manufacturer's protocol.PCR products were resolved by electrophoresis in agarose gel,stained with ethidium bromide. The gel images were digitally captured with Quantity One analysis software.③Western blot:Cells were lysed in Triton lysis buffer and centrifuged at 12,000 x g for 30 min.Protein concentrations were measured using Coomassie BrilliantBlue G-250.Proteins were applied to SDS-PAGE. After electrophoresis, proteins were blotted to polyvinylidene fluoride (PVDF) membranes and then blocked with 5% skim milk powder with 0.1%Tween-20.The blots were then probed at room temperature for 2h with relevant antibodies,washed by TBST (TBS containing 0.1% Tween-20) three times and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 2h.Immunoreactive material was detected using the ECL kit according to manufacturer's instruction.The bands were digitally captured with Quantity One analysis software.④Detection of targets: detecting HSC proliferation by MTT and VG staining; detecting the expression of TIMPI and FN by ELISA;detecting the mRNA levels of RBP-JK, Hesland Smad3 by RT-PCR;detecting the protein levels of RBP-JK, Hesl,Smad3,phospho-Smad3 (S425) and a-SMA by Western blotting.Results:①Acetaldehyde treatment promoted HSC-T6 adherent increased the level of collagen fibers,indicating that acetaldehyde stimulated the proliferation of HSC-T6.Acetaldehyde stimulation induced HSC-T6 activation.Importantly, both SB-431542 and DAPT pre-incubation inhibited acetaldehyde treatment resulted in HSC-T6 activating.②Acetaldehyde treatment up-regulated the expression of TIMPI and FN, and both SB-431542 and DAPT pre-incubation inhibited acetaldehyde treatment resulted in TIMP I and FN up-regulating.③Acetaldehyde treatment had no appreciable effects on RBP-JK expression regulating.However, both SB-431542 and DAPT pre-incubation inhibited RBP-JK expression under acetaldehyde treatment in HSC-T6, and double-blocking reduced RBP-JK expression more significantly. Either SB-431542 or DAPT pre-incubation inhibited Hesl up-regulation mediated by acetaldehyde treatment, and double-blocking reduced Hes1 expression more significantly. Acetaldehyde treatment increased Smad3 mRNA level, but this effect was inhibited by both SB-431542 and DAPT pre-incubation.However, acetaldehyde treatment had effect on Smad3 protein level, further studies are needed to clarify the mechanisms. Moreover, acetaldehyde treatment resulted in p-Smad3-S425 level up-regulation, and either SB-431542 or DAPT pre-incubation blocked p-Smad3-S425 up-regulation, and double-blocking reduced Hes1 expression more significantly.④Acetaldehyde treatment up-regulated a-SMA expression, and which was inhibited by TGF-βor Notch pre-incubation.Double-blocking reduced acetaldehyde-mediated a-SMA up-regulation more significantly. Conclusion:①Acetaldehyde treatment significantly activate HSC, and promote HSC proliferation and fibrosis;②Both SB-431542 and DAPT can antagonize acetaldehyde-mediated HSC activation and proliferation through TIMP I,FN and a-SMA regulation;③SB-431542 can inhibite the Notch pathway nodes RBP-JK and Hesl expression.Notch signaling pathway stimulated by acetaldehyde in HSC activation with a clear regulatory role. It may exist "cross-talk" between the TGF-βand Notch signaling pathway, and CSL (RBP-JK) may be "cross point" of this two signaling pathways, and Hes1 is a direct target of signaling pathways.