3 Results
3.1 PB inhibited
the proliferation and activation of HSCs
HSC proliferation and transformation play vital roles in the process of
fibrosis (Tan, Liu, Ke, Jiang & Wu, 2020; Wu et al., 2016). Thus,
intervention of HSC proliferation, apoptosis and inhibition of the
fibrogenic function, are proposed therapeutic approaches to reverse
liver fibrosis. To evaluate the anti-hepatic fibrosis effect of drug
candidates, we employed a luciferase screening cell model based on the
COL1A1 promoter activity. Results of dual-luciferase reporter assay
showed that COL1A1 promoter activity was significantly increased with
TGFβ1 treatment in LX-2 cells. PB significantly inhibited COL1A1 promoter
luciferase activity in a concentration-dependent manner (Figure 1B).
To
determine whether PB has an effect on LX-2 cell proliferation and
apoptosis, LX-2 cells were treated with 0.25μM, 0.5μM or 1μM PB, and
the
cell proliferation and apoptosis were measured by CCK-8 assay and
Annexin V-propidium iodide (PI) staining, respectively. The results
showed that PB could inhibit TGFβ1
induced
HSC proliferation, however,
scarcely influenced the apoptosis
of LX-2 cells (Figure 1C and Supplementary Fig. 1A). These results
indicated that PB is a potential anti-liver fibrosis drug.
Next, we assessed the effect of PB on HSC activation. In the context of
FBS-free starvation and TGFβ1 stimulation, LX-2 cells display an
activated phenotype in vitro . PB inhibited the mRNA expression of
various fibrogenic genes, including TGFβ1 , α-SMA , andCOL1A1 in LX-2cells (Figure 1D).
The protein expression also showed
a corresponding decrease after treatment with PB in LX-2 cells (Figure
1E). Meanwhile, the expression of α-SMA was assessed by
immunofluorescence, which showed that α-SMA expression was increased by
the TGFβ1 treatment while significantly attenuated by PB in LX-2 cells
(Figure
1F).
We
then confirmed the effect of PB on cultured mouse pHSC. As mouse pHSCs
exhibit self-activation, they do not require starvation and stimulation
during in vitro culture. Similarly, RT-qPCR and western blot
results showed that PB reduced the mRNA and protein expression of α-SMA,
immunofluorescence analysis further confirmed that PB could decrease
a-SMA expression during differentiation (Figure 1G-I). These results
suggest that PB is a potent antifibrogenic agent in HSC activation.