| Hepatic fibrosis is a reversible wound healing response caused by acute or chronic cell injury of liver tissue,reflecting the balance between liver repair and scarring.Currently,the overall burden of chronic fibrosis of the liver is increasing,causing about 2 million deaths worldwide each year.However,although substantial progress has been made in the pathophysiological mechanism of fibrosis,there is still a gap in the identification of antifibrosis targets and their transformation into effective treatment.Therefore,studies on the alleviation or reversal of liver fibrosis have clinical significance.Hepatic stellate cells(HSCs),as the main regulator of the pathogenesis of liver fibrosis,could control the synthesis,deposition and degradation of extracellular matrix such as collagen fibers in the process of liver fibrosis by transforming growth factor-?(TGF-?)through paracrine or autocrine.Recent studies have shown that SMADs need to interact with other DNA-binding transcription factors to regulate the TGF-?/SMAD signaling pathways,namely ?-catenin and T cell factor(TCF),Forkhead box O1(FOXO1)and hypoxia inducible factor 1(HIF-1).The relative activity of TGF-?/SMAD signaling depends on the combination of ?-catenin transcription "partners".When ?-catenin binds to TCF,it promotes cell proliferation and fibrosis;however,when it binds to FOXO1,it exacts anti-proliferation,promotes apoptosis and cell survival under oxidative stress.It has been found that FOXO1 and TCF compete to bind ?-catenin in human colon cancer cells.The formation of ?-catenin/FOXO1 transcription complex can promote the expression of foxp3 in TGF-?-induced regulatory T cells(Tregs)and play an anti-inflammatory role.However,the role of different " transcriptional partners " of ?-catenin in HSCs and liver fibrosis remains unclear.Therefore,in this study,the mechanism of ?-catenin/TCF and ?-catenin/FOXO1 regulating the proliferation and activation of HSCs was explored from multiple levels of cells,animals and human body,both forward and reverse.And in vivo experiments we have confirmed that regulating transcription factor complexes ?-catenin/TCF and ?-catenin/ FOXO1 can reduce extracellular matrix deposition,enhance the anti-inflammatory effect of liver tissue,reduce the chronic continuous damage of various pathogenic factors on liver,balance the process of pro-fibrosis and anti-fibrosis,promote tissue repair,and reverse liver fibrosis,so as to find a new target for the treatment of liver fibrosis to provide ideas.Objective: 1.Combined with cell level and animal level experiments,to explore the role of ?-catenin/FOXO1 and ?-catenin/TCF in the inhibition of liver fibrosis in different transcriptional mesons formed by ?-catenin in TGF-? signaling pathway and different "transcriptional partners" FOXO1 and TCF in liver fibrosis.The aim is to elucidate the phenomenon and mechanism of inhibiting liver fibrosis by regulating the ?-catenin transcription factor complex of hepatic stellate cells,which can inhibit the proliferation and activation of HSCs and secretion of extracellular matrix,regulate the levels of proinflammatory factors and anti-inflammatory factors.2.The relationship between ?-catenin/ FOXO1,?-catenin/TCF and the progression of fibrosis was analyzed in human liver fibrosis tissues.It enriches the pathophysiological basis of liver fibrosis disease progression,provides new ideas and strategies for the treatment of liver fibrosis,and provides support for the further exploration of its clinical application in the treatment of liver and other organs fibrosis.Methods: 1.In vitro experiment: LX2 cells were cultured in vitro and divided into control group,TGF-? group,TGF-?+ICG-001 group,and TGF-?+ICG-001+AS1842856 group.The levels of ?-SMA and COL-I of extracellular matrix,cell cycle and oxidative stress function of HSCs in each group were detected by Western Blotting and RT-q PCR.Western Blotting,COIP and PLA were used to analyze the changes of ?-catenin/TCF and ?-catenin/ FOXO1 transcription complex levels in each group after HSCS activation.2.In vivo experiment: According to different etiology of liver fibrosis,two kinds of liver fibrosis mouse models,carbon tetrachloride(CCl4)and common bile duct ligation(BDL),were established to observe the appearance of mice liver,and rate method was used to detect alanine aminotransferase and aspartic aminotransferase.At the same time,liver tissue sections and staining were performed in different stages of liver fibrosis.Furthermore,RNA was extracted from liver tissue,and RT-q PCR was used to analyze the levels of fibrosis and inflammatory factors in different stages of liver fibrosis mouse model,and to judge the intervention time of different models.The proximity ligation assay(PLA)was used to analyze the changes of ?-catenin transcription complex in different stages of liver fibrosis.3.On the basis of the above experiments,?-catenin /TCF inhibitor ICG-001 was used to intervene in two liver fibrosis mouse models.The mice were divided into control group,model group and ICG-001 intervention group.To evaluate the inhibition of liver fibrosis in mice after ICG-001 intervention,the serum level of liver enzymes,histological staining,immunohistochemistry,?-SMA and Col-1 RNA expression were detected.The levels of inflammatory cytokines IL-10,IL-17,TNF-? and IL-6 in liver tissues of BDL groups were determined by ELISA.4.To further explore the mechanism of TGF-? signaling pathway transcription factor complex in inhibiting liver fibrosis from both positive and negative perspectives,TGF-? group and FOXO1 inhibitor intervention group were added to liver fibrosis animal model of BDL mice.Immunohistochemistry,Western blotting,RT-q PCR,Co IP and PLA were used to analyze the conversion relationship between ?-catenin/TCF and ?-catenin/FOXO1 transcriptional complex.The effect of TGF-? signaling pathway transcription factor complex on HSCs cells in vivo was analyzed by tissue immunofluorescence co-localization and tissue HSCs specific cell PLA assay.The expression levels of foxp3,TNF-? and p27Kip1 were detected by RT-q PCR.5.Finally,immunohistochemistry,RT-q PCR and PLA tests were carried out in the liver tissues of children with biliary atresia at different hepatic fibrosis stages to analyze whether TGF-? levels,cell proliferation levels,IL-10,?-catenin/FOXO1,?-catenin/TCF in human liver tissues were correlated with hepatic fibrosis stages.To provide the basis for the clinical strategy of redirecting TGF-? signaling pathway transcription factor complex ?-catenin/FOXO1 and ?-catenin/TCF in the treatment of liver fibrosis.Results: 1.In vitro experiment: ?-catenin/TCF and ?-catenin/ FOXO1 were involved in the proliferation and activation of hepatic stellate cells.1.1 ICG-001 interferes with activated LX2 cells.Western blotting results showed that the expression level of ?-SMA protein in LX2 cells in ICG-001 group was significantly decreased compared with TGF-? group.Compared with ICG-001 group,the expression of ?-SMA protein in ICG-001+AS1842856 group was significantly higher than that in ICG-001 group.There was no significant difference in ?-SMA protein expression between ICG-001 group and control group,and there was no significant difference in ?-SMA protein expression between ICG-001+AS1842856 group and TGF-? group.These results indicated that ICG-001 could inhibit the transdifferentiation of activated HSCs into myofibroblasts,and the expression levels of ?-SMA and COL-I were significantly decreased.1.2 RT-q PCR results of Mn SOD and p27Kip1 showed that the expression of Mn SOD and p27Kip1 in LX2 cells of ICG-001 group was opposite to that of ?-SMA and Col-I Western blotting.These results indicated that ICG-001 intervention of activated HSCs could arrest the cell cycle of HSCs and enhance the level of oxidative stress.1.3 The detection results of ?-catenin,TCF and FOXO1 Western blotting were significantly higher than those of the control group,but ?-catenin only in the ICG-001 group was significantly lower than that in the ICG-001+AS1842856 group,and there was no statistically significant difference between the other groups.1.4 The results of immunoprecipitation with ?-catenin as Co IP antibody showed that the level of ?-catenin binding TCF was significantly decreased and the FOXO1 level was significantly increased in ICG-001 group compared with TGF-? group.?-catenin,TCF,FOXO1 PLA results showed that the ?-catenin/TCF interaction of LX2 cells in ICG-001 group was significantly decreased compared with that in TGF-? group,and the ?-catenin/ FOXO1 result was contrary to that of ?-catenin/TCF.TF ratio(?-catenin/TCF versus ?-catenin/FOXO1)was used to analyze the difference in transcription factor levels between the groups.The results showed that the change trend of TF ratio was consistent with the expression of ?-SMA,a specific marker of LX2 cell activation,and the activation of LX2 cells was closely related to the increase of TF ratio.The above results suggested that ?-catenin/TCF can promote the proliferation and activation of TGF-?-induced HSCs.2.In vivo experiment: animal model of liver fibrosis in mice and the relationship between ?-catenin/TCF,?-catenin/ FOXO1 and the course of liver fibrosis 2.1 ALT and AST levels of the CCl4 model reached the highest level at 4w,and decreased at 6w and 8w with the extension of modeling time.Mice liver HE staining, reticular fiber staining results showed that portal area increased neutrophils and lymphocytes infiltration,disorganized hepatocyte and fibrous tissue hyperplasia gradually significant,enlargement and breaking of the reticular fibers,fiber interval has been formed at 4w after injection of CCl4;Immunohistochemical results of ?-SMA in mouse liver showed that the expression level of ?-SMA increased significantly compared with the control group at 4w.The m RNA expression levels of IL-6 and TNF-? in mouse liver tissues reached the peak at 4w,and the expression level of foxp3 gradually increased at the early stage of the model,reached the peak at 6w,and decreased at 8w.2.2 ALT and AST of BDL model were significantly higher than those of Sham at 1w after BDL operation,and reached the peak at 2w,and showed a downward trend at 3w and 4w.HE staining,reticular fiber staining,Masson staining and bright green sirius red staining of mouse liver tissue were detected from different dimensions of inflammatory cell infiltration,reticular fiber staining and collagen fiber hyperplasia,respectively.As it turns out,1w after BDL operation,there was hyperplasia of bile duct,enlargement of portal area,slight edema and degeneration of liver cells,a small amount of neutrophils and lymphocytes infiltration.With the extension of postoperative time,focal necrosis was observed,the number of bile duct increased,and the vacuolation of liver cells was aggravated.Immunohistochemical results of ?-SMA showed that ?-SMA in portal area was significantly increased at 1w after BDL compared with the control group.The expression level of ?-SMA continued to increase after BDL.The expression level of TNF-? m RNA in mouse liver gradually increased at 1w,2w and 3w,and the expression level of foxp3 gradually increased at the early stage of model,and significantly increased at 2w and 3w.The above results indicated that inflammatory cell infiltration and fibrous tissue hyperplasia appeared in the mouse liver fibrosis models induced by two different mechanisms of CCl4 and BDL after 4W and 1W respectively.2.3 In situ PLA assay was performed on paraffin liver tissue sections at different stages of CCl4 and BDL models to detect the changes of ?-catenin/TCF and ?-catenin/FOXO1 transcription complex at different stages of the disease course.The results showed that ?-catenin/TCF level was significantly increased at 4w in CCl4 models,while ?-catenin/FOXO1 level was the highest in the control group,but significantly decreased at 4w.The level of ?-catenin/TCF was significantly increased at 1w after BDL operation,while the level of ?-catenin/FOXO1 was the highest in the control group and decreased at 1w. These results indicated that ?-catenin/FOXO1 and ?-catenin/TCF transcription complex levels were correlated with the course of liver fibrosis in the two models.3.Inhibiton of ?-catenin/TCF reversed liver fibrosis in CCl4 and BDL mouse models.3.1 The liver appearance,liver weight/body weight ratio,liver enzymes,histology,?-SMA,COL-I m RNA and protein levels were compared in CCL4 and BDL liver fibrosis mice models with ICG-001 for 1w and 2w,respectively.The results showed that in the CCL4 and BDL models,there was no significant difference after 1w of ICG-001 intervention,but decreased significantly after 2w ALT and AST of mice in 1w and 2w intervention groups were significantly decreased compared with model group.The results of HE staining and mesh staining of CCL4 model liver tissue showed that the infiltration of inflammatory cells and the number of hepatocyte clusters surrounded by fibrous septa in ICG-001 group were decreased compared with the positive control group.The results of HE,Masson,reticular fiber and sirius red staining of liver tissue of BDL model showed that the proliferation and fibrosis of bile duct and inflammatory cell infiltration were reduced in ICG-001 group compared with the same period BDL model group.The semiquantitative results of sirius red staining in BDL model showed that collagen deposition in ICG-001 group was significantly lower than that in the positive control group.RT-q PCR results showed that the positive proportion of COL-I and ?-SMA in ICG-001 group of the two models decreased significantly compared with the model group at intervention for 2w.Immunohistochemical results showed that COL-I level in ICG-001 group of CCL4 model was significantly lower than that in the positive control group at intervention for 2w.The level of ?-SMA in ICG-001 group of BDL model was significantly lower than that in the positive control group at intervention for 2w.These results suggest that inhibition of ?-catenin/TCF for 2w can improve hepatic fibrosis significantly.3.2 The expression of foxp3 and p27Kip1 level was significantly increased compared with the positive control group after ICG-001 intervention.3.3 In BDL liver fibrosis mouse model,IL-6 level decreased significantly compared with the positive control group after ICG-001 intervention for 2w.The change trend of TNF-? and IL-17 was consistent with that of IL-6,while the increase of IL-10 was significant.4.BDL model was used to study the molecular mechanism of ?-catenin/TCF and ?- catenin/ FOXO1 in regulating liver fibrosis.4.1 In BDL mice,after intraperitoneal injection of AS1842856 to inhibit FOXO1,immunohistochemistry and Western blotting were used to detect the level of ?-SMA in the liver tissue of mice.The results showed that the level of ?-SMA was significantly increased and liver fibrosis was aggravated after the intervention of AS1842856.When AS1842856 was applied on the basis of ICG-001,the number of ?-SMA positive cells was significantly increased compared with ICG-001 group.The results of Western blotting and semiquantitative analysis of ?-SMA and COL-I showed that the protein expression levels of ?-SMA and COL-I in ICG-001 group were significantly decreased compared with those in positive control group.4.2 IP test was performed on nucleoprotein extracts from liver tissues of mice in each group.The results showed that ?-catenin/TCF and ?-catenin/FOXO1 in BDL group were increased and decreased compared with Sham group.After ICG-001 intervention,?-catenin/TCF and ?-catenin/FOXO1 were consistent with Sham group.The results of PLA test showed that the interaction trend of TCF,FOXO1 and ?-catenin in each group was basically consistent with the results of liver nuclear protein COIP test.After BDL operation and under the action of TGF-?,the interaction trend of ?-catenin and TCF was increased,while the interaction of ?-catenin and FOXO1 was decreased,which was related to the progress of liver fibrosis.After ICG-001 intervention,the binding of ?-catenin and TCF was weakened,while the binding of ?-catenin and FOXO1 was enhanced.However,in ICG-001 combined AS1842856 group,the interaction between ?-catenin and TCF and FOXO1 was weakened,which was consistent with the results of liver nuclear protein Co IP test.4.3 Tissue immunofluorescence results showed that the proliferation and activation level of HSCs in the BDL model group was significantly higher than that in the Sham group,the ICG-001 group was significantly lower than that in the positive control group,the number and activation of HSCs in the TGF-? group were increased compared with that in the BDL model group,and the proliferation and activation level of HSCs in the TGF-? group was significantly decreased after ICG-001 intervention.The ?-catenin/TCF ligation of HSCs cells in the BDL model group was significantly increased compared with that in the Sham group,and was significantly decreased after ICG-001 intervention compared with that in the positive control group.TGF-? group promoted TGF-? signaling pathway transcription factor activity.ICG-001 intervention TGF-? group,?-catenin/TCF ligation decreased significantly compared with TGF-? group.?-catenin/FOXO1 ligation in positive control group was significantly lower than that in Sham group.It was significantly higher than that in positive control group after ICG-001 intervention.TGF-? group promoted that ?-catenin/FOXO1 increased significantly compared with positive control group,and after ICG-001 intervention,the ligation between FOXO1 and ?-catenin increased significantly.As the ratio of ?-catenin/TCF and ?-catenin/ Fox O1,TF ratio can be used to comprehensively evaluate the dynamic changes between TCF and FOXO1 in the competition binding with ?-catenin.The TF Ratio of HSCs cells in the BDL model group was significantly increased compared with that in the Sham group,and was significantly decreased after ICG-001 intervention compared with that in the BDL group.There was no significant difference between the TGF-? and BDL groups.The TF Ratio in TGF-? group after ICG-001 intervention was significantly lower than that in TGF-? group,and had no difference with that in ICG-001 group.Combined with the results of Section 2.1 in this section,TF ratio decreased and the degree of liver fibrosis decreased;otherwise,it was aggravated.4.4 The results of RT-q PCR showed that the expression levels of TNF-? and Foxp3 in the liver of mice in BDL model group were significantly higher than those in Sham group.After ICG-001 intervention,TNF-? decreased significantly and foxp3 increased.On the basis of ICG-001,inhibition of FOXO1 by AS1842856 significantly increased the level of TNF-? and decreased the expression of foxp3.4.5 After ICG-001 intervention,p27Kip1 levels were significantly higher than those in the BDL model group.The level of p27kip1 decreased significantly after inhibition of FOXO1 by AS1842856 on the basis of ICG-001.5.Relationship between ?-catenin/TCF,?-catenin/ FOXO1 transcription complex and fibrosis progression in human liver tissue 5.1 Sixteen patients with biliary atresia and other patients diagnosed by laparoscopic cholecystectomy with initial diagnosis by Doppler ultrasound were graded by Ishak score according to HE,Masson and Sirius red staining results.Among them,there were 3 patients with no fibrosis(NC),4 patients with stage 1,3 patients with stage 2,stage 3 and 4 respectively.5.2 Masson staining,IL-10,Ki67 semi-quantitative analysis of liver tissue of patients with different stages showed that the degree of fibrosis,anti-inflammatory factors,and cell proliferation were all correlated with the increasing expression level of TGF-?.5.3 The PLA results of paraffin section of liver tissue from patients with different stages of liver fibrosis showed that ?-catenin/TCF ligation increased gradually in S0,S1,S2 and S3 stages,and tended to be stable in S3 and S4 stages.?-catenin/FOXO1 was the highest at S0 stage and gradually decreased at S0-S2 stage.TF ratio was consistent with the changing trend of liver fibrosis degree.Conclusions: 1.In vitro,TCF and FOXO1 competitively bind to ?-catenin to form different transcription factor complexes that regulate LX2 transdifferentiation,cell cycle and oxidative stress levels.2.In vivo animal experiments,TCF and FOXO1,the downstream transcription factors of TGF-? pathway,competitively bind ?-catenin.?-catenin/TCF and ?-catenin/ FOXO1 act as switches for different biological effects of the TGF-? pathway leading to pro-fibrosis and anti-inflammatory,cellular inhibition--different biological event outcomes.By regulating the TF ratio of HSCs,the proliferation and activation of HSCs cells can be inhibited and the deposition of extracellular matrix can be reduced.Immune response of liver tissue was regulated,chronic tissue injury was alleviated,and liver fibrosis was reversed.3.TF Ratio,an indicator reflecting the relative levels of ?-catenin/TCF and ?-catenin/ FOXO1,were positively correlated with the early progression of liver fibrosis in children with BA. |
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