Inhibition of hepatocyte growth factor on proliferation and transdifferentiation of human Tenon capsule fibroblasts triggered by transforming growth factor-β1in vitro

Objective

To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor -β₁ (TGF-β₁) in vitro.

Methods

Human Tenon capsule fibroblasts were cultured and divided into blank control group, TGF-β₁ treated group and different concentrations HGF+ TGF-β₁ groups.The TGF-β₁ at 10 μg/L was added into culture medium of the TGF-β₁ treated group, and different concentrations of HGF (25, 50, 100, 200 μg/L) and 10 μg/L TGF-β₁ were added into culture medium of the HGF_{25 μg/L}+ TGF-β₁, HGF_{50 μg/L}+ TGF-β₁, HGF_{100 μg/L}+ TGF-β₁, HGF_{200 μg/L}+ TGF-β₁ group respectively, Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm). Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay.

Results

Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025, 0.497±0.101, 0.426±0.062, 0.354±0.040, 0.272±0.084, 0.241±0.011 in the blank control group, TGF-β₁ treated group, HGF_{25 μg/L}+ TGF-β₁ group, HGF_{50 μg/L}+ TGF-β₁ group, HGF_{100 μg/L}+ TGF-β₁group and HGF_{200 μg/L}+ TGF-β₁ group, respectively, showing a significant difference among the groups (F=9.210, P=0.003). Compared with the TGF-β₁ treated group, the proliferation values of the cells were significantly reduced in the blank control group and HGF+ TGF-β₁ group (all at P<0.05). Immunofluorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β₁ treated group and weak red fluorescence in HGF+ TGF-β₁ group, and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)% in the TGF-β₁ treated group and (14.3±3.1)% in the HGF+ TGF-β₁ group, with significant difference between the two groups (t=19.856, P<0.001). The relative expression levels of the α-SMA protein in the cells were 0.642±0.032, 1.330±0.069 and 0.884±0.040 in the blank control group, TGF-β₁ group and HGF_{100 μg/L}+ TGF-β₁ group, respectively, showing a significant difference among the groups (F=13.370, P<0.001), and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank control group and HGF_{100 μg/L}+ TGF-β₁ group than that in the TGF-β₁ treated group (all at P<0.05).

Conclusions

HGF can inhibit the proliferation of human Tenon capsule fibroblasts, down-regulate the expression of α-SMA protein induced by TGF-β₁ and arrest the phenotype transformation of fibroblasts in vitro.

Tenon capsule/cytology; Fibroblasts/metabolism; Hepatocyte growth factor/administration & dosage; Transforming growth factor beta type 1; Fibrosis/prevention & control; α-Smooth muscular actin; Humans; Cells, cultured