Spinosin inhibits activated hepatic stellate cell to attenuate liver fibrosis by targeting Nur77/ASK1/p38 MAPK signaling pathway

Liver fibrosis remains a great challenge in the world. Spinosin (SPI), a natural flavonoid-C-glycoside, possesses various pharmacological activities including anti-inflammatory and anti-myocardial fibrosis effects. In this study, we investigate whether SPI can be a potential lead for the treatment of liver fibrosis and explore whether the orphan nuclear receptor Nur77, a negative regulator of liver fibrosis development, plays a critical role in SPI's action. A dual luciferase reporter system of α-SMA was established to evaluate the effect of SPI on hepatic stellate cell (HSC) activation in LX2 and HSC-T6 cells. A mouse model of CCl4-induced liver fibrosis was used to test the efficacy of SPI against liver fibrosis. The expression levels of Nur77, inflammatory cytokines and collagen were determined by Western blotting and qPCR. Potential kinase pathways involved were also analyzed. The affinity of Nur77 with SPI was documented by fluorescence titration. SPI can strongly suppress TGF-β1-mediated activation of both LX2 and HSC-T6 cells in a dose-dependent manner. SPI increases the expression of Nur77 and reduces TGF-β1-mediated phosphorylation levels of ASK1 and p38 MAPK, which can be reversed by knocking out of Nur77. SPI strongly inhibits collagen deposition (COLA1) and reduces inflammatory cytokines (IL-6 and IL-1β), which is followed by improved liver function in the CCl4-induced mouse model. SPI can directly bind to R515 and R563 in the Nur77-LBD pocket with a Kd of 2.14 μM. Spinosin is the major pharmacological active component of Ziziphus jujuba Mill. var. spinosa which has been frequently prescribed in traditional Chinese medicine. We demonstrate here for the first time that spinosin is a new therapeutic lead for treatment of liver fibrosis by targeting Nur77 and blocking the ASK1/p38 MAPK signaling pathway.