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.