3.4 | Effect of Hedgehog-Gli1 signaling on fibrogenesis
in vitro
We next elucidated the mechanism by which activated Hedgehog-Gli1
signaling promoted the fibrogenesis
in vitro. It has been well demonstrated that the hedgehog pathway
modulates several important aspects of function, including cell
proliferation, activation and differentiation. Targeting the hedgehog
pathway can be a promising direction in fibrosis treatment (Lim et al.,
2018). To further confirm that the activation of Hedgehog-Gli1 signaling
on the effect of LF cell viability, we analyzed the effect of Gli1
expression on the proliferation and apoptosis of the LF fibroblasts. As
shown in the immunofluorescence staining (Fig. 4 A), most of the cells
were stained with collagen I or collagen Ⅲ (markers of LF fibroblasts),
indicating a high purity of LF fibroblasts obtained. Subsequently, we
analyzed the cell viability with or without Gli1 overexpression or
knockdown (Fig. 4B) using the MTS assay and flow cytometry. The data
from Figures 4C showed that over-expressing Gli1 in LF fibroblasts
markedly promoted cell proliferation as compared with the control group,
whereas this effect was alleviated by the knockdown of Gli1. Moreover,
flow cytometry suggested that LF cells over-expressing Gli1 exhibited a
much lower percentage of cell apoptosis as compared to the control group
(Fig. 4D). By contrast, The rate of apoptosis cell of the Gli1 knockdown
LF cells was higher than that in the control group (Fig. 4D). Further
study results revealed that the mechanism underlying anti-apoptosis in
Gli1-overexpressing LF cells involved the inhibition of the Bax and the
activation of Bcl-2 (Fig. 4E). Together, these above data suggest that
activated Hedgehog-Gli1 signaling enhanced fibroblast viability by
promoting cell proliferation and inhibiting cell apoptosis.
To further determine the relationship between the activation of
Hedgehog-Gli1 signaling and fibrogenesis in fibroblasts, we analyzed the
effect of Gli1 over-expression or knockdown on the expression of
fibrosis-related proteins such as collagen I and III in LF fibroblasts.
As illustrated in Figure 4E, over-expression of Gli1 significantly
increased the mRNA and protein expressions of collagen I and III in LF
cells (Fig. 4F). By contrast, the expression levels of collagen I and
III in the Gli1 knockdown LF cells were lower than those in the control
group (Fig. 4F). Furthermore, immunohistochemistry assay also
demonstrated that over-expression or knockdown Gli1 increased or
abrogated the expression of fibrosis-related proteins (Fig. 4G) in LF
cells, suggesting that the activated Hedgehog-Gli1 signaling in LF cells
promoted fibrogenesis in vitro.