Role of R-Spondin1 in the activation of hepatic stellate cells / 中华肝脏病杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of R-Spondinl in the activation of hepatic stellate cells (HSCs).</p><p><b>METHODS</b>Twenty-four healthy male Kunming mice were randomly divided into the following two groupsfibrosis model group (n=16) and control group (n=8). Hepatic fibrosis was induced by subcutaneous injections of CC14 (20% in olive oil) at a dose of 5 ml/kg twice per week. After 10 weeks, the animals were sacrificed by CO(2) over-exposure and liver tissues were harvested.The protein and mRNA levels of R-Spondin1, alphat-SMA,and collagen I were examined by Western blot assay and real-time PCR respectively. Additionally,HSCs were isolated from the mice liver tissues to examine the time-series expression changes of R-Spondinl, alpha-SMA, and nuclear beta-catenin.TCF activity was analyzed by luciferase reporter assay.Moreover,HSCs were cocultured with recombinant R-Spondin1 and DKK1 to evaluate dose-response.</p><p><b>RESULTS</b>R-Spondinl expression was significantly higher in the fibrosis model group than in the control group (protein level3.16 ± 0.18 vs. 0.99 ± 0.16, t =13.31, P < 0.01; mRNA level4.36 ± 0.26 vs. 0.98 ± 0.12, t =21.46, P < 0.01).The culture-activated mouse HSCs showed up-regulated TCF activity (5.33 ± 0.34 vs. non-activated 1.03 ± 0.09, t =20.93, P < 0.01), nuclear beta-catenin expression (4.47 ± 0.21 vs. 0.97 ± 0.14, t =25.25, P < 0.01), and R-Spondin1 expression (protein level 4.54 ± 0.18 vs. 1.04 ± 0.12, t =31.17, P < 0.01; mRNA level5.13 ± 0.15 vs. 1.01 ± 0.16, t=38.06, P < 0.01). Exogenous stimulation of freshly isolated mouse HSCs with recombinant R-Spondin1 induced a dose-dependent increase in both TCF activity and the expression of nuclear beta-catenin and alphat-SMA. DKK1 down-regulated activities of factors in the WNT signaling pathway and repressed activation of HSCs. Conclusion R-Spondin1 may promote HSC activation by enhancing the canonical WNT signaling pathway.</p>