Use of fh535 as a drug for promoting differentiation of adscs into hepatocytes

Abstract

The application of FH535 as a drug for promoting the differentiation of ADSCs into hepatocytes. FH535 inhibits the expression levels of β-catenin, p-β-catenin, GSK-3β and p-GSK-3β of ADSCs; thereby promoting the differentiation of ADSCs into hepatocytes.

Description

Technical Field

[0001] This invention relates to the application of FH535, and particularly to the application of FH535 as a drug to promote the differentiation of ADSCs into hepatocytes. Background Technology

[0002] The liver is a vital organ regulating physiological processes such as glycogen storage, lipid metabolism, plasma protein secretion, and biological detoxification. End-stage liver diseases, including hepatic metabolic disorders, cirrhosis, and fulminant hepatic failure, are leading causes of death worldwide. Liver transplantation is currently the most ideal treatment. However, its clinical application is limited by factors such as donor scarcity and immune rejection. In recent years, primary hepatocyte (PH) transplantation and artificial liver support devices have been considered alternative or adjuvant therapies to liver transplantation, aiming to promote liver regeneration and recovery from acute liver failure, offering hope for the clinical treatment of end-stage liver diseases. However, both face the problem of severe cell shortage; therefore, how to generate a large number of replaceable functional hepatocytes is a crucial issue that urgently needs to be addressed.

[0003] Mesenchymal stem cells (MSCs) possess the ability to differentiate into endoderm, mesoderm, and ectoderm cells. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into functional hepatocytes in vitro, applicable to various end-stage liver diseases. ADSCs, after being cultured in liver differentiation media containing HGF, bFGF, nicotinamide, and hemostatin M, can differentiate into hepatocyte-like cells (ihep). ihep transplantation can restore liver function and prolong survival in mice with acute liver failure and metabolic liver disease. The Wnt signaling pathway is an evolutionarily conserved pathway that plays a crucial role in the fate and differentiation of mesenchymal stem cells. Xu H et al. reported that Lnc13728 may promote adipogenic differentiation of hADSCs by upregulating ZBED3 and inhibiting the Wnt / β-catenin pathway. It has been reported that inhibition of Wnt signaling can promote hepatocyte differentiation from bone marrow-derived stem cells (BMSCs). Ji Yangdan et al. found that miR-26b can promote the differentiation of BMSCs into hepatocyte-like cells and their chemotactic migration ability by regulating the Wnt / β-catenin signaling pathway. Their study also found that the traditional Chinese medicine Yi Guan Jian could promote the differentiation of BMSCs into hepatocytes. Ping Jian et al. found that Yi Guan Jian could induce the differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells, and the mechanism was related to the downregulation of the Wnt / β-catenin signaling pathway. Mechanistically, downregulation of the Wnt signaling pathway inhibits stem cell proliferation and promotes differentiation into functional hepatocytes. Whether inhibition of the Wnt signaling pathway in ADSCs promotes their differentiation into hepatocytes is unknown.

[0004] FH535 is a small molecule inhibitor of the Wnt signaling pathway. It targets and inhibits the recruitment of β-catenin and β-catenin / Tcf-mediated transcription, and also inhibits the aggregation of PPARδ and PPARγ by the co-activators GRIP1 and β-catenin. This invention aims to intervene in the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into hepatocytes by utilizing FH535. By detecting the number, function, and gene expression levels of hepatocytes at different differentiation stages, the study seeks to clarify the role of the Wnt pathway inhibitor FH535 in the differentiation of adipose-derived mesenchymal stem cells into hepatocytes, thereby laying a cell biology foundation for cell therapy of end-stage liver disease. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide FH535 as a drug for promoting the differentiation of ADSCs into hepatocytes.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] The application of FH535 as a drug to promote the differentiation of ADSCs into hepatocytes.

[0008] FH535 inhibited the expression levels of β-catenin, p-β-catenin, GSK-3β, and p-GSK-3β in ADSCs, thereby promoting the differentiation of ADSCs into hepatocytes.

[0009] The concentration of FH535 is 20 μmol / L.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0011] This invention utilizes FH535 as a drug to promote the differentiation of adipocyte-derived mesenchymal stem cells (ADSCs) into hepatocytes. Primary ADSCs were extracted using the collagenase method. ADSCs were identified using flow cytometry, immunofluorescence (IF), and adipogenic (Oil Red O staining) and osteogenic (Alizarin Red staining) differentiation experiments. The expression of key proteins in the Wnt pathway was detected by Western blotting (WB) to determine the optimal inhibitory concentration of FH535 on ADSCs. The ADSCs-to-hepatocyte induction experiment was divided into a hepatocyte induction control group (HDM group) and an HDM+FH535 intervention group (HDM+FH535 group). At 3, 7, 14, and 21 days after induction, the percentage and number of hepatocyte-like cells (ihep), glycogen area, and protein and mRNA expression levels of liver-specific markers were detected in both groups using phase-contrast microscopy, glycogen staining (PAS), immunofluorescence (IF), and qRT-PCR. The t-test was used for intergroup comparisons. Flow cytometry results showed that ADSCs expressed stem cell markers CD29 (positive rate 97.6%), CD44 (positive rate 76.8%), and CD90 (positive rate 99.1%), but did not express hematopoietic and endothelial cell markers CD31 (negative, 0.016%), CD34 (0.28%), and CD45 (negative, 0.016%). A 20 μmol / L concentration of FH535 significantly inhibited the expression levels of β-catenin, p-β-catenin, GSK-3β, and p-GSK-3β in ADSCs, with statistically significant differences between groups (t = 20.03, 65.89, 24.48, 50.65, P < 0.05). The percentage of iHEP cells in the FH535-treated group was significantly higher than that in the HDM group (Day 3: 13.18 ± 0.82 vs). 8.68±0.27, t=11.63, P<0.05)(Day7: 62.42±8.01 vs 20.74±4.79, t=9.988, P<0.05)(Day14: 75.16±12.12 vs 25.13±2.24, t=9.079, P<0.05)(Day21: 81.16±4.52 vs 51.62±8.36, t=6.950, P<0.05); The number of iHEP cells in the FH535 treatment group was significantly higher than that in the HDM group (Day7: 98.6±7.44 vs 62.2±14.57, t=4.977, P<0.05)(Day14: 149.6±16.68 vs 8.68±0.27, t=11.63, P<0.05)(Day14: 75.16±12.12 vs 25.13±2.24, t=9.079, P<0.05)(Day21: 81.16±4.52 vs 51.62±8.36, t=6.950, P<0.05); 56.2±4.21, t=12.14, P<0.05)(Day21: 201.6±27.5vs 137±25, t=3.887, P<0.05); The glycogen staining area of ​​cells in the FH535 treatment group was significantly higher than that in the HDM group (Day14: 4655.9±2079.8μm). 2vs 1360.5±360.8μm 2 , t=3.491, P<0.05) (Day21: 6244.2±1827.9μm 2 vs 3799.6±1395.1μm 2 The fluorescence intensity of ALB and CK18 staining in the FH535-treated group was higher than that in the HDM group on days 14 and 21 after hepatocyte differentiation induction. The mRNA expression levels of liver genes (ALB: albumin; Transferrin: ferritin; HNF-1B: hepatocyte nuclear factor 1B; ASGPR1: desialized glycoprotein receptor 1; TAT: thrombin-anthiopin complex; CK8: cytokeratin 8) in the FH535-treated group were significantly higher than those in the HDM group on day 14 after induction (ALB: 44.53±7.80 vs 19.25±9.50, t=3.563, P<0.05) (Transferrin: 37.91±13.20 vs 19.25±9.50, t=3.563, P<0.05) (Transferrin: 37.91±13.20 vs 19.25±9.50, t=2.377, P<0.05). The expression levels of liver gene mRNA in the FH535-treated group were significantly higher than those in the HDM group on day 21 after induction (8.16±2.76, t=3.820, P<0.05)(HNF-1B: 7.23±1.90vs3.14±0.23, t=3.695, P<0.05)(ASGPR1: 9.47±1.00vs 5.26±2.20, t=3.018, P<0.05)(TAT: 6.92±2.53vs 2.09±0.41, t=3.269, P<0.05)(CK8: 7.71±3.05vs 0.81±0.14, t=3.916, P<0.05); The expression levels of liver gene mRNA in the FH535-treated group were significantly higher than those in the HDM group on day 21 after induction (ALB: 26.27±10.52vs). (6.60±1.85, t=3.192, P<0.05)(Transferrin: 35.82±6.60vs23.39±2.68, t=3.024, P<0.05)(CK8: 19.99±2.30vs3.62±1.55, t=10.20, P<0.05). 20 μmol / L FH535 significantly promoted the differentiation of ADSCs into hepatocytes. In this study, we demonstrated that FH535 promotes the hepatic-derived differentiation of ADSCs and enhances the expression of hepatocyte-specific marker proteins. This effect improves the differentiation efficiency of ADSCs into hepatocyte-like cells. Therefore, based on the results of this study, FH535 may be a potential target for the treatment of various acute and chronic liver diseases, providing new insights for clinical stem cell therapy of end-stage liver disease. Attached Figure Description

[0012] Figure 1 ADSCs identification results

[0013] A. Morphological characteristics of primary (P0) and third-generation (P3) ADSCs under phase-contrast microscopy (200×); B. Oil Red O staining and Alizarin Red staining of ADSCs (P3) after induction with adipogenic and osteogenic differentiation media (right side), and Oil Red O staining and Alizarin Red staining of uninduced ADSCs (left side) (400×); C. Flow cytometry identification results of ADSCs (P3) (CD29, CD44, CD90, CD31, CD34, CD45).

[0014] D. Immunofluorescence staining of stem cell markers CD29, CD44, CD90, and CD105 (400×)

[0015] Figure 2 Inhibitory effects of different concentrations of FH535 on the ADSCs-Wnt signaling pathway: A. Western blot results of expression and phosphorylation of key proteins β-catenin and GSK-3β in the Wnt signaling pathway, detected by Western blot after 24 hours of intervention with different concentrations of FH535 in ADSCs; B. Statistical results of relative expression and phosphorylation levels of key proteins β-catenin and GSK-3β in the Wnt signaling pathway, detected by Western blot after 24 hours of intervention with different concentrations of FH535 in ADSCs.

[0016] Figure 3 Effects of FH535 on the differentiation of ADSCs into hepatocytes: A. Morphological changes of hepatocyte-like cells (ihep) under phase contrast microscopy (200×) at different time points (3, 7, 14, 21 days) after hepatocyte induction in the HDM group and HDM+FH535 group; B. Morphological characteristics of uninduced ADSCs and normal human hepatocyte cell line (HL-7702) under phase contrast microscopy (200×); C. Statistical results of the percentage and number of hepatocyte-like cells (ihep) at different time points (3, 7, 14, 21 days) after hepatocyte induction in the HDM group and HDM+FH535 group.

[0017] Figure 4 The effect of FH535 on the glycogen storage function of hADSCs after hepatocyte induction.

[0018] A. Shows PAS staining results (200×) at different time points after hADSCs hepatocyte induction in the HDM group and FH535 intervention group. The staining results show that purplish-red granules were deposited in the cells on day 7 after hepatocyte differentiation in both groups. With the increase of induction time, the glycogen staining area also increased. On days 14 and 21 after induction, the number of purplish-red glycogen granules in the cells gradually increased. B. Shows the statistical graph of PAS staining area at different time points after hADSCs hepatocyte induction in the HDM group and FH535 intervention group.

[0019] Figure 5Immunofluorescence staining of hADSCs after hepatocyte induction to hepatocytes to express hepatocyte-specific proteins

[0020] A. Immunofluorescence staining results of ALB at different time points after hADSCs were induced to become hepatocytes in the HDM group and the FH535 intervention group (200×). The results showed that ALB was expressed in the cells of both groups starting on day 7 after hepatocyte differentiation, and green fluorescence was visible in the cytoplasm. The intensity of green fluorescence increased with the increase of induction time. At 21 days after induction, the ALB fluorescence intensity of the cells in the FH535 treatment group was significantly higher than that in the HDM group. B. Immunofluorescence staining results of CK18 at different time points after hADSCs were induced to become hepatocytes in the HDM group and the FH535 intervention group (200×). The results showed that CK18 was expressed in small amounts in the cells of both groups starting on day 7 after hepatocyte differentiation, and weak green fluorescence was visible in the cytoplasm. At 21 days after induction, the fluorescence intensity of CK18 expression in the cells of both groups increased.

[0021] Figure 6 The effect of FH535 on the expression levels of liver-specific genes at different induction stages of hADSCs hepatocytes. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0023] Experimental Example: Application of FH535 as a drug to promote the differentiation of ADSCs into hepatocytes

[0024] like Figure 1-6 As shown,

[0025] 1. Obtaining adipose tissue specimens

[0026] Adipose tissue samples were obtained from the Plastic Surgery Clinic of the First Affiliated Hospital of Xinjiang Medical University. Patients aged 25-45 years were selected after liposuction or subcutaneous excision. Waste adipose tissue was collected and stored in sterile centrifuge tubes. This invention was approved by the hospital's ethics committee and informed consent was obtained from the patients.

[0027] 2. Reagents, consumables and instruments

[0028] DMEM medium, penicillin antibodies, 0.25% trypsin, and fetal bovine serum (FBS) were purchased from Gibco (USA); type I collagenase was purchased from Worthington Biotech (USA); adipogenic and osteogenic induction media were purchased from Weitong Biotechnology (USA); flow cytometry antibodies CD31, CD34, and CD45 were purchased from BD Biosciences (USA), and CD29, CD44, and CD90 were purchased from eBioscience (USA); FH535 was purchased from Selleck Biotech (USA); hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor-4 (FGF-4), and oncostatin M were also purchased. M, OSM) were purchased from PeproTech (USA); dimethyl sulfoxide (DMSO) and dexamethasone were purchased from Sigma (USA); ALB chicken anti-human antibody and CK18 rabbit anti-human antibody; FITC-labeled goat anti-chicken and goat anti-rabbit secondary antibodies were purchased from Abcam (USA); Dapi was purchased from Roche (Switzerland); PAS kit was purchased from Solarbio (China); β-catenin, p-β-catenin, GSK-3β, p-GSK-3β, and GAPDH antibodies were purchased from CST (USA); RNA extraction kit was purchased from Fujitsu (China); cDNA reverse transcription kit and SYBR Premix RT-qPCR kit were purchased from Takara (Japan); Western chemiluminescence kit was purchased from Millipore (Germany); four-well glass-bottom confocal microplates, 6-well plates, and 6cm / 10cm cell culture dishes were purchased from Corning (USA); laser confocal microscope and inverted fluorescence microscope were purchased from Leica (Germany); and cell culture incubator was purchased from Thermo Fisher Scientific (USA).

[0029] 3. Methods

[0030] 3.1 Isolation, culture, and stemness identification of human adipose-derived mesenchymal stem cells

[0031] Sterile human adipose tissue was washed three times with PBS containing 1% penicillin and streptomycin for 5 minutes each time. The adipose tissue was then minced into a homogenate using sterile surgical scissors. PBS containing 1 mg / ml type I collagenase was added and mixed well. The mixture was then placed in a 37°C shaker and shaken for 30 minutes, manually inverting the container twice during the process. Digestion was terminated with 15 ml of DMEM low-glucose medium containing 10% FBS. The mixture was centrifuged at 1500 rpm for 10 minutes, and the adipose supernatant was discarded. The cell pellet was seeded into 10 cm cell culture dishes and placed in a 5% CO2 incubator. The medium was changed every other day. When the cells reached 80% confluence, they were digested with 0.25% trypsin and passaged at a 1:3 ratio. The first passage was marked as P1.

[0032] P3 generation cells were used for adipogenic and osteogenic differentiation and flow cytometry identification. Adipogenic and osteogenic induction identification: Cells were induced to differentiate according to the kit instructions. The induction medium was changed every 2-3 days. Oil Red O staining was performed 10 days after adipogenic differentiation, and Alizarin Red staining was performed 18 days after osteogenic differentiation. Cell morphological changes were recorded using a phase-contrast microscope. Flow cytometry identification: After passage of P3 generation cells, cells were collected and divided into 3 tubes. The cells were resuspended in 100 μL of pre-chilled PBS. 2.5 μL of CD31-FITC and CD34-PE, CD90-PE and CD45-FITC, and CD29-PE and CD44-FITC antibodies were added to each tube according to the fluorescent labeling. The cells were then incubated at 4°C in the dark for 30 min. 400 μL of PBS was added to each tube to stop the reaction before flow cytometry analysis.

[0033] 3.2 Experiment on differentiation of adipose-derived mesenchymal stem cells into hepatocytes

[0034] 3.2.1 Hepatocyte Differentiation Induction Experiment

[0035] Hepatocyte differentiation was induced using third-generation ADSCs, with the induction method following the research of Yin, L, et al. During induction, serum-free culture with added cytokines was used. Hepatocyte differentiation medium (HDM) was supplemented with various cytokines in the order of induction time. 1-3 days after induction (pre-induction stage), 20 ng / ml FGF-4 and EGF were added; 4-7 days after induction (differentiation induction stage), 20 ng / ml FGF-4 and HGF were added; 7-21 days after induction (maturation induction stage), 20 ng / ml FGF-4 and HGF, 40 ng / ml OSM, 1 μmol / L dexamethasone, and 1‰ DMSO were added.

[0036] 3.2.2 Experimental Grouping

[0037] This experiment was divided into two groups: a hepatocyte differentiation-inducing control group (HDM group, where ADSCs were induced to differentiate using conventional hepatocyte differentiation-inducing medium) and an FH535 treatment group (HDM+FH535 group, where ADSCs were co-cultured with FH535 at a concentration of 20 μmol / L in addition to the HDM group).

[0038] 3.4 Periodic acid-Schiff (PAS) staining

[0039] After ADSCs were induced to become hepatocytes, at 7, 14, and 21 days, the cells in both groups were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. Staining was performed according to the periodic acid-Schiff (PAS) staining kit instructions. Glycogen staining results of each group of cells were photographed using an inverted microscope, and the staining area of ​​the images was analyzed using Image Pro Plus 6 software.

[0040] 3.5 Cell Immunofluorescence Staining Detection

[0041] After ADSCs were induced to become hepatocytes, at 7, 14, and 21 days, the cells in both groups were washed twice with cold PBS, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 at room temperature for 15 minutes, washed three times with PBS, blocked with 5% goat serum for 30 minutes, and then incubated overnight at 4°C with the corresponding antibodies (chicken anti-human ALB, 1:500; rabbit anti-human CK18, 1:1000). After washing with PBS, the cells were incubated with fluorescent secondary antibodies (FITC-labeled goat anti-chicken secondary antibody and FITC-labeled goat anti-rabbit secondary antibody, 1:250) for 30 minutes, counterstained with Dapi, and photographed under a fluorescence inverted microscope.

[0042] 3.6 Real-time quantitative PCR detection

[0043] Two groups of cells were collected at 7, 14, and 21 days after ADSCs were induced to become hepatocytes. Total RNA was extracted from the cells, and 1 μg of total RNA was used for reverse transcription to synthesize first-strand cDNA. The expression of liver genes was detected using the SYBR Premix RT-qPCR kit, with GAPDH used as an internal control gene. -ΔΔCT The relative expression level of mRNA was calculated using a method, and the primers were synthesized by Shanghai Sangon Biotech.

[0044]

[0045]

[0046] Table 1 Primer sequence information used in this invention

[0047] 3.7 Western Blot Experiment

[0048] After 24 hours of treatment with different concentrations of FH535, ADSCs were collected, and total protein was extracted and quantified using cell lysis. 20 μg of protein samples were loaded and subjected to SDS-PAGE electrophoresis. Protein transfer was performed using a PVDF membrane, blocked with 5% skim milk powder for 1 hour, and then incubated at 4°C with primary antibodies (rabbit anti-human β-catenin, p-β-catenin, GSK-3β, p-GSK-3β, and GAPDH, diluted 1:2000, 1:2000, 1:2000, 1:2000, and 1:5000, respectively). After washing four times with PBST, a secondary antibody (goat anti-rabbit-HRP antibody, concentration: 1:5000) was added and incubated at room temperature for 1 hour. The mixture was then developed using ECL chemiluminescence reagent in the dark, and gel imaging was performed using a gel imaging system. Protein grayscale analysis was performed using ImageJ software.

[0049] 3.8 Statistical Analysis

[0050] SPSS 20.0 software was used for statistical analysis. Data are expressed as mean ± standard deviation (x ± s). The t-test was used to compare the two groups, and P < 0.05 was considered statistically significant.

[0051] 1. ADSCs identification

[0052] After primary ADSCs adhered to the culture vessel, the number of cells gradually increased and formed colonies, exhibiting a typical elongated shape. It took about 10 days for the primary cells to fuse to 80%. Then, the ADSCs were passaged and purified to P3. Under the microscope, the ADSCs showed long spindle-shaped whorl growth, which is consistent with the morphological characteristics of mesenchymal stem cells. Figure 1 A). Seven days after adipogenic induction, Oil Red O staining of P3 generation ADSCs showed bright red lipid droplets of varying sizes within the cells. Fourteen days after osteogenic induction, Alizarin Red staining showed dense cell growth with large areas of orange-red calcium nodules. Figure 1 B). Flow cytometry was used to identify the stem cell phenotype of hADSCs. The results showed that the isolated hADSCs expressed stem cell markers CD29 (positive rate 97.6%), CD44 (positive rate 76.8%), and CD90 (positive rate 99.1%), but did not express hematopoietic and endothelial cell markers CD31 (negative, 0.016%), CD34 (0.28%), and CD45 (negative, 0.016%). Figure 1 C). Furthermore, immunofluorescence staining morphologically validated the flow cytometry results; red fluorescence showed that spindle-shaped ADSCs expressed stem cell surface markers such as CD90, CD105, CD29, and CD44. Figure 1 D). These results suggest that we have isolated high-quality hADSCs, which can be used for subsequent cell differentiation experiments.

[0053] Figure 1ADSCs identification results

[0054] B. Morphological characteristics of primary (P0) and third-generation (P3) ADSCs under phase-contrast microscopy (200×); B. Oil Red O staining and Alizarin Red staining of ADSCs (P3) after induction with adipogenic and osteogenic differentiation media (right side), and Oil Red O staining and Alizarin Red staining of uninduced ADSCs (left side) (400×); C. Flow cytometry identification results of ADSCs (P3) (CD29, CD44, CD90, CD31, CD34, CD45).

[0055] D. Immunofluorescence staining of stem cell markers CD29, CD44, CD90, and CD105 (400×) 2. Inhibitory effect of different concentrations of FH535 on the ADSCs-Wnt signaling pathway

[0056] To clarify the effective concentration of FH535 on ADSCs, we treated ADSCs with different concentrations of FH535 (20, 40, and 80 μmol / L). After 24 hours, total protein from ADSCs was collected, and the expression and phosphorylation levels of key Wnt signaling pathway proteins β-catenin and GSK-3β were detected. Western blot results showed that, compared with the DMSO group, 20 μmol / L FH535 significantly inhibited the expression levels of β-catenin, p-β-catenin, GSK-3β, and p-GSK-3β in ADSCs, with statistically significant differences between groups (t = 20.03, 65.89, 24.48, and 50.65, P < 0.05). Intervention with 80 μmol / L FH535 in ADSCs only inhibited the expression levels of GSK-3β and p-GSK-3β, with statistically significant differences between groups (t = 34.20, 33.30, P < 0.05), but did not inhibit the expression and phosphorylation level of β-catenin (t = 0.27, P > 0.05). Intervention with 40 μmol / L FH535 in ADSCs did not inhibit the phosphorylation of the corresponding proteins, and there was no statistically significant difference compared with the DMSO group (t = 0.17, P > 0.05). These results indicate that 20 μmol / L FH535 has a significant inhibitory effect on the Wnt signaling pathway in ADSCs and can be considered the optimal inhibitory concentration for subsequent hepatocyte differentiation assays.

[0057] Figure 2B. Inhibitory effect of different concentrations of FH535 on ADSCs-Wnt signaling pathway. 24 hours after intervention with different concentrations of FH535 in ADSCs, Western blot results of expression and phosphorylation of key proteins β-catenin and GSK-3β in the Wnt signaling pathway were detected. 24 hours after intervention with different concentrations of FH535 in ADSCs, Western blot results of relative expression and phosphorylation levels of key proteins β-catenin and GSK-3β in the Wnt signaling pathway were also analyzed.

[0058] 3. Effects of FH535 on the differentiation of ADSCs into hepatocytes

[0059] To assess the effect of FH535 on the differentiation of hADSCs into hepatocytes and to observe morphological changes, we counted and statistically analyzed the number and percentage of hepatocyte-like cells (ihep) at different time points (3, 7, 14, and 21 days) after induction in both groups of cells. Morphologically, before induction, ADSCs were elongated spindle-shaped. Compared with the normal human hepatocyte line HL-7702, ADSCs gradually transformed into hepatocyte-like cells after induction with differentiation medium, changing their morphology from elongated spindle-shaped to polygonal and roundish (like epithelial cells of normal hepatocytes). Figure 3 (A&B). The results showed that with the increase of induction time, the number and proportion of ihep cells (round and polygonal) gradually increased in both groups. Starting from day 7 of ADSCs-induced differentiation into hepatocytes, the percentage and number of ihep cells in the FH535 treatment group were as follows: Day 3: 13.18±0.82 vs 8.68±0.27; Day 7: 62.42±8.01 vs 20.74±4.79; Day 14: 75.16±12.12 vs 25.13±2.24; Day 21: 81.16±4.52 vs 51.62±8.36) and number (Day 7: 98.6±7.44 vs 62.2±14.57; Day 14: 149.6±16.68 vs 56.2±4.21; Day 21: 201.6±27.5 vs 8.68±0.82). 137±25) was significantly higher than that of the HDM group ( Figure 3 (A&C) The differences between the two groups were statistically significant (percentage of cells, Day 3: t = 11.63, Day 7: t = 9.988, Day 14: t = 9.079, Day 21: t = 6.950; number of cells, Day 7: t = 4.977, Day 14: t = 12.14, Day 21: t = 3.887; P < 0.05 for all groups).

[0060] Figure 3 Effect of FH535 on the differentiation of ADSCs into hepatocytes

[0061] A. Morphological changes of hepatocyte-like cells (ihep) in the HDM group and HDM+FH535 group at different time points (3, 7, 14, 21 days) after hepatocyte induction under phase contrast microscopy (200×); B. Morphological characteristics of uninduced ADSCs and normal human hepatocyte cell line (HL-7702) under phase contrast microscopy (200×); C. Statistical results of the percentage and number of hepatocyte-like cells (ihep) in the HDM group and HDM+FH535 group at different time points (3, 7, 14, 21 days) after hepatocyte induction.

[0062] 4. Effects of FH535 on hepatocyte function after ADSCs induction

[0063] To clarify whether FH535 can promote the differentiation of hADSCs into hepatocytes, thereby enabling them to store glycogen and express mature hepatocyte-specific proteins, we used periodic acid-Schiff (PAS) staining and immunofluorescence staining with albumin (ALB) and cytokeratin 18 to stain hepatocyte-like cells at different stages (days 7, 14, and 21) after induction in both groups of cells. PAS staining results showed that purplish-red granules were deposited within the cells 7 days after hepatocyte differentiation in both groups. With increasing induction time, the number of purplish-red glycogen granules gradually increased. Figure 4 A). PAS staining area statistics showed that on days 14 and 21 after ADSCs were induced to differentiate into hepatocytes, the glycogen staining area in the FH535 treatment group was significantly higher than that in the control group, and there was a statistically significant difference between the two groups (Day 14: 4655.9±2079.8μm). 2 vs 1360.5±360.8μm 2 , t=3.491, P<0.05) (Day21: 6244.2±1827.9μm 2 vs 3799.6±1395.1μm 2 (t = 2.377, P < 0.05) Figure 4 B). ALB immunofluorescence results showed that both groups of cells began to express a small amount of ALB (green fluorescence) 7 days after induction of hepatocyte differentiation, and the fluorescence intensity increased with increasing induction time. The ALB fluorescence intensity of ADSCs in the FH535 treatment group was higher than that in the control group on days 14 and 21 after induction of hepatocyte differentiation. Figure 5 A). Immunofluorescence results for CK18 showed that the HDM group began to express a small amount of CK18 (green fluorescence) on day 14 after ADSCs were induced to differentiate into hepatocytes, and no significant difference in CK18 expression was observed up to day 21. In contrast, the FH535 treatment group showed increased green fluorescence intensity of CK18 on days 14 and 21 after ADSCs were induced to differentiate into hepatocytes compared to the HDM control group. Figure 5 B).

[0064] Figure 4 The effect of FH535 on the glycogen storage function of hADSCs after hepatocyte induction.

[0065] A. Shows PAS staining results (200×) at different time points after hADSCs hepatocyte induction in the HDM group and FH535 intervention group. The staining results show that purplish-red granules were deposited in the cells on day 7 after hepatocyte differentiation in both groups. With the increase of induction time, the glycogen staining area also increased. On days 14 and 21 after induction, the number of purplish-red glycogen granules in the cells gradually increased. B. Shows the statistical graph of PAS staining area at different time points after hADSCs hepatocyte induction in the HDM group and FH535 intervention group.

[0066] Figure 5 Immunofluorescence staining of hADSCs after hepatocyte induction to hepatocytes to express hepatocyte-specific proteins

[0067] Immunofluorescence staining results of ALB (200×) at different time points after hADSCs were induced to become hepatocytes in the B.HDM group and the FH535 intervention group showed that ALB was expressed in both groups of cells starting on day 7 after hepatocyte differentiation, with visible green fluorescence in the cytoplasm. The intensity of the green fluorescence increased with the increase of induction time. At 21 days after induction, the ALB fluorescence intensity in the FH535 treatment group was significantly higher than that in the HDM group. Immunofluorescence staining results of CK18 (200×) at different time points after hADSCs were induced to become hepatocytes in the B.HDM group and the FH535 intervention group showed that CK18 was expressed in small amounts in both groups of cells starting on day 7 after hepatocyte differentiation, with visible weak green fluorescence in the cytoplasm. At 21 days after induction, the fluorescence intensity of CK18 expression in both groups of cells increased.

[0068] 5. Effects of FH535 on hepatocyte-specific gene expression induced by hADSCs

[0069] To clarify whether FH535 promotes the expression levels of hepatocyte-specific genes after induction of hADSCs into hepatocytes, we used quantitative real-time PCR to detect the expression levels of liver-specific genes (albumin, ALB; transferrin; hepatocyte nuclear factor-1B, HNF-1B; Asialoglycoprotein receptor 1, ASGPR1; thrombin-antithrombin complex, TAT; cytokeratin, CK8) at different induction stages. qPCR results showed that on day 14 after ADSCs were induced to differentiate into hepatocytes, the mRNA expression levels of six liver genes in the FH535-treated group were significantly higher than those in the HDM group (ALB: 44.53±7.80 vs 19.25±9.50, t=3.563, P<0.05)(Transferrin: 37.91±13.20 vs 8.16±2.76, t=3.820, P<0.05)(HNF-1B: 7.23±1.90 vs 3.14±0.23, t=3.695, P<0.05)(ASGPR1: 9.47±1.00 vs 5.26±2.20, t=3.018, P<0.05)(TAT: 6.92±2.53 vs 19.25±9.50, t=3.563, P<0.05)(ASL ...TAT: 9.92±2.53 vs 19.25±9.50, t=3.563, P<0.05)(TAT: 9.92±2.53 vs 19.25±9.50, t=3. 2.09±0.41, t=3.269, P<0.05)(CK8: 7.71±3.05vs 0.81±0.14, t=3.916, P<0.05); On day 21 after ADSCs were induced to differentiate into hepatocytes, the mRNA expression levels of three liver genes in the FH535-treated group were significantly higher than those in the HDM group, and the differences between the groups were statistically significant (ALB: 26.27±10.52vs 6.60±1.85, t=3.192, P<0.05)(Transferrin: 35.82±6.60vs 23.39±2.68, t=3.024, P<0.05)(CK8: 19.99±2.30vs 3.62±1.55, t=10.20, P<0.05)( Figure 6 FH535 antagonizes β-catenin / Tcf-mediated transcription and inhibits the aggregation of PPARδ and PPARγ by the coactivators GRIP1 and β-catenin. FH535 exhibits selective antiproliferative activity in certain cancer cells expressing high levels or with active Wnt / β-catenin pathways.

[0070] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. The application of FH535 in the preparation of an inducing agent for the in vitro differentiation of adipose-derived mesenchymal stem cells into hepatocytes, characterized in that, The concentration of FH535 in the inducing agent is 20 μmol / L. The inducing agent is added to the hepatocyte differentiation culture medium to co-culture and induce adipose-derived mesenchymal stem cells to differentiate into hepatocyte-like cells.

2. The application according to claim 1, characterized in that, FH535 inhibited the expression levels of β-catenin, p-β-catenin, GSK-3β, and p-GSK-3β in ADSCs, thereby promoting the differentiation of ADSCs into hepatocytes.

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