Effects of FGF21‐secreting adipose‐derived stem cells in thioacetamide‐induced hepatic fibrosis

Abstract Mesenchymal stem cells (MSCs) have been investigated to treat liver diseases, but the efficiency of MSCs to treat chronic liver diseases is conflicting. FGF21 can reduce inflammation and fibrosis. We established FGF21‐secreting adipose derived stem cells (FGF21_ADSCs) to enhance the effects of ADSCs and transplanted them into thioacetamide (TAA)‐induced liver fibrosis mice via the tail vein. Transplantation of FGF21_ADSCs significantly improved liver fibrosis by decreasing serum hyaluronic acid and reducing the expression of fibrosis‐related factors such as α‐smooth muscle actin (α‐SMA), collagen and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) compared with the Empty_ADSCs by inhibition of p‐JNK, NF‐κB and p‐Smad2/3 signalling. α‐lactoalbumin (LA) and lactotransferrin (LTF), secretory factors produced from FGF21_ADSCs inhibited TGF‐β1‐induced expression of α‐SMA and collagen in LX‐2 cells. These results suggest that transplantation of FGF21_ADSCs inhibited liver fibrosis more effectively than Empty_ADSCs, possibly via secretion of α‐LA and LTF.


| INTRODUCTION
Liver fibrosis is the end stage of most of liver diseases and is a serious health care problem worldwide. 1 In the damaged liver, hepatic stellate cells (HSCs) are activated and produce excessive extracellular matrix components (ECM). 2 Transforming growth factor (TGF)-β is a key mediator of hepatic fibrosis and triggers the activation of HSCs, induces ECM synthesis and increases apoptosis of hepatocytes. 3 Liver transplantation is still considered to be the most effective treatment for end-stage liver disease, but has several limitations.
Recent studies suggest that adipose derived stem cells (ADSCs) show beneficial effects to improve liver fibrosis. 4 In addition, fibroblast growth factor (FGF) 21 improves lipid profiles and hepatic steatosis. 5 To enhance the therapeutic effects of ADSCs on liver fibrosis, we established FGF21-secreting ADSCs (FGF21_ADSCs) and investigated their effects on thioacetamide (TAA)-induced liver fibrosis in mice.

| Histological analysis
Hematoxylin and eosin (H&E) or Masson's trichrome staining was carried out using paraffin-embedded sections. For immunofluorescence staining, liver sections were blocked and incubated with primary antibodies and fluorescence-conjugated secondary antibodies.
DAPI was used for nuclear counterstain. Antibodies used for staining are listed in Table S1.

| Western blot analysis
Total proteins were extracted, separated by 8%-12% SDS-PAGE and transferred to membranes. After blocking, membranes were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Signals were detected and quantified with ChemiDoc XRS+ system using Image Lab software (Bio-Rad, Hercules, CA, USA) with the Immobilon Western Chemiluminescent HRP Substrate (Millipore). Antibodies used for western blotting are listed in Table S1.

| Secretome analysis
Proteins were digested and analysed by Q-Exactive orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) with Easy-Nlc 1000 system as described previously. 6

| Statistical analysis
All data are expressed as mean ± SEM of at least three independent experiments. All statistics were performed using one-way ANOVA (SPSS 19.0 statistical software, IBM, Armonk, NY, USA). P-values less than 0.05 were considered statistically significant.

| Effect of FGF21_ADSCs transplantation on amelioration of liver fibrosis
We constructed the Igκ_FGF21 plasmid to facilitate the secretion of

TGF-β1-induced activation of HSCs
We determined whether factors secreted from FGF21_ADSCs affect HSC activation in LX-2 cells using CM from Empty_ADSCs or  (Figure 2A,B). These results suggest that soluble factors from FGF21_ADSCs, other than FGF21, might contribute to the inhibitory effect on HSC activation.   ; 200×). E, Immunofluorescence staining and (F) quantification of α-SMA (n = 3/group). The percentages of areas for the immunostaining of total image were measured by imageJ and expressed as relative values to CON liver. G, Representative western blot and (H) densitometric analysis for α-SMA, TIMP-1 and Col1a1 (n = 6-13/group). GAPDH was used as control for normalization of results. Data are means ± SEM. **P < 0.01, *P < 0.05. vs untreated mice (CON); ## P < 0.01, # P < 0.05. vs TAA (-); $$ P < 0.01, $ P < 0.05 vs TAA+ Empty_ADSCs CM (data not shown). Among the proteins that were increased over 2-fold ( Figure S4), we chose lactotransferrin (LTF) and α-lactoalbumin (α-LA) for further analysis, because they inhibit liver fibrosis. 10,11 Western blot analysis showed that LTF and α-LA secretion was significantly increased in FGF21_ADSCs CM compared with Emp-ty_ADSCs CM ( Figure 2G). When we treated LX-2 cells with LTF or α-LA in the presence of TGF-β1, the TGF-β1-induced expression of α-SMA and Col1a1 was inhibited ( Figure 2H,I). These results indicate that secretory factors such as α-LA and LTF, which are altered in FGF21_ADSCs, can improve hepatic fibrosis compared to Emp-ty_ADSCs.
The therapeutic effect of MSC therapy is due to a variety of biologically active factors produced by them. 14 Our study showed that CM from FGF21_ADSCs significantly inhibited TGF-β-induced expression of fibrogenic factors compared with CM from Emp-ty_ADSCs. Interestingly, when LX-2 cells, a human HSC line, were incubated with the same amount of FGF21 as found in the CM of FGF21_ADSCs, there was almost no effect on the inhibition of JNK and Smas2/3 activation, suggesting that soluble factors other than FGF21 might contribute to this stronger inhibitory effect. Among the secreted proteins upregulated in the FGF21_ADSCs CM, we noted that α-LA and LTF might be involved in the inhibition of fibrogenic activity. α-LA and LTF have a liver protective and anti-fibrotic effect, respectively. 10,11 We confirmed that α-LA or LTF treatment inhibited TGF-β-induced α-SMA and Col1a1 expression in LX-2 cells.
Therefore, α-LA and LTF secreted by FGF21_ADSCs might contribute in part to the potent inhibitory effect of these cells on liver fibrosis in our model.