3.2 PB ameliorated CCl4-induced liver injury and hepatic fibrosis in mice
In order to evaluate whether large doses of PB has any hepatotoxicity, mice were intraperitoneal injection of PB 40mg/kg. HE staining, Sirius red staining and Masson staining showed that PB (40mg/kg) did not cause any pathological or fibrous damage (Supplementary Fig. 1B). Mice were intraperitoneal injection with 2% DMSO as the vehicle group. Similarly, HE staining, Sirius red staining and Masson staining showed that 2% DMSO did not cause any pathological or fibrous damage (Supplementary Fig. 1C). Based on these results, we next evaluated the anti-hepatic fibrosis effect of PB in mouse fibrosis models in vivo .
Carbon tetrachloride (CCl4) is a hepatotoxin that has been used extensively to induce liver injury. It induces liver fibrosis by producing various reactive metabolites and damaging hepatocytes. To assess the pharmaceutical activities of PB in vivo , we performed CCl4 challenge in mice. After 4 weeks of CCl4 injections, the mice exhibited marked increases in hepatic inflammatory cell infiltration, mild thickening of the central venous wall and fibrous hyperplasia. However, PB treatment ameliorated these pathological changes and protected against CCl4‐induced fibrosis (Figure 2A). Serum ALT and AST levels were significantly elevated in the CCl4-treated mice, while decreased in PB treated group indicating improved liver injury (Figure 2B). The severity of liver fibrosis was also biochemically assessed by measuring the hepatic hydroxyproline content. PB markedly decreased the CCl4-induced increase of hydroxyproline content (Figure 2C). Sirius red and Masson’s trichrome staining visualizes collagen, which is used to evaluate the degree and characteristics of fibrosis. Sirius Red, Masson’s trichrome stained liver sections indicated that fibrous collagen deposition, fibroplasia, and bridging fibrosis significantly decreased in PB treated mice (Figure 2D). The expression of hepatic fibrogenic markers, including αsma , Col1a1 ,Tgfb1 and tissue inhibitor of metalloproteinase 1(Timp1 ) were detected to assess the antifibrotic effects of PB in vivo . PB treatment significantly decreased the mRNA expression of these fibrogenic genes. Western blot assays also validated that PB treatment decreased α‐SMA and COL1A1 expression in the liver tissues (Figure 2E, F). Accordingly, PB has the potential to inhibit the progression of CCl4-induced hepatic fibrosis in mice.