芍药苷-6-氧-苯磺酸酯(CP-25)是有抗炎免疫调节潜能，可靶向GRK2的新活性单体。自免肝(AIH)中CP-25调控树突细胞(DC)内GRK2对信号分子的作用及机制有待揭示。预实验表明：CP-25抑制GRK2、PI3K、AKT磷酸化及DC糖脂代谢。因此提出假设：CP-25抑制GRK2转膜，促进DC胞内其与PI3K、AKT结合及作用，负调控代谢相关PI3K-AKT-mTOR通路，抑制DC活性，缓解AIH。本实验拟用S-100诱导AIH模型，观察CP-25作用、检测DC表型及功能。seahorse分析DC线粒体功能，确定代谢关键节点。另构建DC负载肝癌细胞Hepa1-6新颖AIH模型，明确DC对CD8+T细胞的介导。采用GRK2 siRNA/分子对接/酶活性/钓靶/CO-IP实验，探究DC中CP-25靶向GRK2调控PI3K-AKT的作用。为CP-25治疗AIH提供实验依据、阐明分子机制。

Paeoniflorin-6′-O-benzene sulfonate (code: CP-25), a potential anti-inflammatory and immune regulation drug, is a novel ester derivative of paeoniflorin (Pae). But in autoimmune hepatitis (AIH), the mechanism of CP-25 regulating the effect of G protein-coupled receptor kinases 2 (GRK2) to signal molecules in dendritic cells (DCs) is not clear. Previous results showed that the activation of DCs is associated with a dramatic increase of metabolism in DCs for the experimental AIH. CP-25 inhibited the phosphorylation of GRK2, PI3K and AKT, which interfered the glucose uptake and non-esterified fatty acid (NEFA) secretion of DCs. Based on these observations, we hypothesize that CP-25 inhibits the phosphorylation and translocation of GRK2 to the plasma membrane and increases the expression of GRK2 in the cytoplasm, which up-regulates the effect of GRK2 to PI3K and/or AKT in DCs. Then CP-25 down-regulates the metabolism-related PI3K-AKT-mTOR pathway to suppress DCs metabolism and alleviate AIH. To test this hypothesis, we are going to use S-100 to induce AIH mouse model for observing the effect of CP-25 on AIH and DCs. And we will investigate the mitochondrial function of DCs by seahorse. Additionally, we are going to generate a novel mouse model of AIH by immunization of C57BL/6 mice with DCs loaded with well-differentiated murine hepatocellular carcinoma cells (Hepa1-6) to demonstrate that DCs is critical for CD8+T cells differentiation in AIH after CP-25 treatment. Finally, The small interfering RNA (siRNA) targeting GRK2 will be used to silence the mRNA expression of GRK2 in DCs. Then 3D molecular docking, ADP-Glo kinase assay, target fishing and co-immunoprecipitation (CO-IP) will be used to explore the targeted inhibition of CP-25 on GRK2 regulating PI3K-AKT. The central purpose of this study is to investigate the effects and molecular mechanism of CP-25 in experimental AIH.