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.