To investigate the therapeutic effect and potential molecular mechanisms of cyclin-dependent kinase inhibitor-73 (CDKI-73), the Rab11 inhibitor, on liver fibrosis.

Human LX2 cells were divided into four groups: negative control group, transforming growth factor-β (TGF-β) group, CDKI-73 group and TGF-β+ CDKI-73 group. Fifteen 5-week-old female C57 mice with body weight of (18.04±0.62) g were divided into 3 groups with 5 mice in each group: control group (intraperitoneal injection of olive oil + vehicle gavage), carbon tetrachloride (CCl₄) group (intraperitoneal injection of CCl₄ + vehicle gavage) and CCl₄+ CDKI-73 group (intraperitoneal injection of CCl₄+ CDKI-73 gavage). Another 15 5-week-old female C57 mice with body weight of (18.06±0.34) g were divided into 3 groups with 5 mice in each group: sham operation group (Sham), bile duct ligation (BDL) group + vehicle group (BDL+ vehicle gavage) and bile duct ligation+ CDKI-73 group (BDL+ CDKI-73 gavage). The expression of α-smooth muscle actin (α-SMA) and fibronectin(FN)in LX2 cells were analyzed by Western blot. Masson and Sirius red were used to examine the liver fibrosis after CDKI-73 treatment in vivo. Immunohistochemistry (IHC) was utilized to examine the expression of α-SMA in mice liver.

Collagen content assessed by Sirius red and Masson staining and α-SMA expression evaluated by IHC were all increased in CCl₄ group compared with control group (q=38.47, 24.99, 36.79). Moreover, the collagen content and α-SMA expression in CCl₄ + CDKI-73 treatment group were obviously decreased compared with CCl₄ group (q=24.72, 14.87, 27.50), and the differences were statistically significant (all P<0.001). Compared with Sham group, collagen content and α-SMA expression in bile duct ligation group were increased (q=28.23, 41.01, 44.16). Furthermore, in BDL group, after treatment with CDKI-73, the collagen content and α-SMA expression were notably decreased (q=22.88, 34.31 and 33.97, all P<0.001). Consistent with in vivo results, the relative expression levels of α-SMA and FN protein in TGF-β group were higher than those in TGF-β+ CDKI-73 group (α-SMA: 3.71±0.34 vs. 1.28±0.31; FN: 3.21±0.39 vs. 0.83±0.06, all P<0.001). The mRNA relative expression levels of α-SMA and FN in TGF-β group were higher than those in TGF-β+ CDKI-73 group, and the differences were statistically significant (P<0.001). However, the relative expression of TGF-β receptor Ⅱ protein in CDKI-73 group was higher than those in negative control group (4.68±0.63 vs. 1.00±0.22, P=0.004). The relative expression level of phosphorylated SMAD2 in TGF-β+ CDKI-73 group was lower than those in TGF-β group (1.67±0.24 vs. 3.99±0.44, P<0.001). Transwell assay showed that 0.5 μmol/L CDKI-73 could effectively inhibit the migration of LX2 cells, and the inhibitory ability became stronger with the increase of CDKI-73 concentration.

CDKI-73 can inhibit the activation of hepatic stellate cells and liver fibrosis by inhibiting Rab11-dependent TGF-β signaling pathway both in vivo and in vitro.

Hepatic fibrosis; Cyclin-dependent kinase inhibitor proteins; Hepatic stellate cells; TGF-β signaling pathway