3.1 PIC Ⅱ significantly alleviates hepatic fibrosis and inflammation in Mdr2−/− mice
To broadly analyze the potential pharmacological effects of PIC Ⅱ on hepatic fibrosis, we attempted to use the Mdr2-/- mice that mimics the immunopathogenesis and symptoms of patients with liver fibrosis in the clinic (Fig. 1A , left panel ). As shown in Fig. 1A , right panel and Fig. S1A , there was little difference in the weight loss, ratio of liver or spleen to body weight between different groups. We further applied H & E and Masson’ s trichrome staining to evaluate the pathological change before or after PIC Ⅱ administration and found that the obvious inflammatory infiltration, ECM deposition, and fibrous scar were observed in the liver of Mdr2−/− mice but were markedly alleviated by different doses of PIC Ⅱ to varying degrees (Fig. 1B ). Consistently, although it had less effects on γ-glutamyl transpeptidase (γ-GGT) (a cell surface enzyme for indicating hepatobiliary injury, Fig. S1B ), PIC Ⅱ at different concentrations also significantly downregulated the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT, two typical markers for hepatic injury), total biliary acid (TBA), total bilirubin (TBIL) and alkaline phosphatase (AKP, three markers for reflecting bile acid accumulation and liver fibrosis degree) induced by the deficiency of Mdr2 (Fig. 1C and Fig. S1B ). To better figure out the protective effects and underlying mechanisms of PIC Ⅱ, we further performed RNA-sequencing analysis of fibrotic livers in Mdr2−/− mice and plotted DEGs implicated in fibrosis-related genes as a heatmap (Fig. 1D ). A total of 8274 DEGs with P -value < 0.05 was identified between three different groups. In cluster 1, the expression of genes was downregulated in the Mdr2−/−mice and further decreased in Mdr2−/− + PIC Ⅱ group, such as Stat3 , ribosomal protein S6 (Rps6 ) and small mother against decapentaplegic family member 3 (Smad3 ), which often early increased and sustained in liver injury. The gene expression in cluster 2 was upregulated in the Mdr2−/− group and further increased in Mdr2−/− + PIC Ⅱ group, such as the key genes involved in inhibiting HSC proliferation and activation like peroxisome proliferator activated receptor gamma (Pparg ) and Smad7 as well as collagen degradation related gene including matrix metallopeptidase 13 (Mmp13 ). The expression of DEGs in cluster 3 including cellular communication network factor 2 (Cnn2 ) as well as collagen formation genes such as tissue inhibitor of metalloproteinase 1 (Timp1 ) and collagen type XXIII alpha 1 chain (Col23a1 ) in cluster 4 were remarkably downregulated in the Mdr2-/- + PIC Ⅱ group when compared to the Mdr2-/- group (Fig. 1D ). Consistently, the qPCR results validated the mRNA changes of representative fibrosis-related genes in the heatmap and showed that PIC Ⅱ mainly inhibited the expression of the collagen-synthesis related genes (Fn1 , Acta2 , H19 and Col1a1 ) and positively supported the collagen-degradation related ways (increased Mmp13and Timp1 ) (Fig. 1E ). Furthermore, the immunofluorescence staining also confirmed the anti-fibrotic effects of PIC Ⅱ on the Mdr2-/- mouse model, as illustrated by the lower distribution of Fibronectin, α-SMA and Collagen1 (Fig. 1F and Fig. S1C ). Of noted, the direct inhibition of PIC Ⅱ on the protein level of a-SMA and fibronectin in aHSC had not been observed clearly (Fig. S2A and S2B ), which suggested that PIC Ⅱ might possess a suppressive effect on aHSCs through another indirect way.