芍药苷-6-氧-苯磺酸酯调控GRK2对PI3K-AKT的作用在DC参与自身免疫性肝炎中的机制

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.

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。

项目背景.自身免疫性肝炎(AIH)是自身免疫反应介导的慢性进行性肝脏炎症性疾病。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。树突细胞(DC)作为重要抗原提呈细胞，细胞内代谢改变会影响其功能。AIH中CP-25靶向DC内GRK2，介导代谢相关PI3K-AKT信号，可为AIH治疗提供新策略，为阐明CP-25药理作用机制及临床转化提供实验依据。.研究内容.1.CP-25对AIH的药效及对免疫细胞表型、功能、代谢的调控.2.CP-25靶向GRK2介导代谢相关PI3K-AKT通路调控免疫细胞机制.3.GRK2作为抗AIH药靶潜能及介导免疫调控机制.4.DC代谢介导肝脏炎症的免疫学机制.5.CP-25对代谢及肠道菌群的影响.6.CP-25对肾脏炎症的药理作用.重要结果及关键数据.1.CP-25对AIH有良好药理作用，抑制DC成熟，CD8+T细胞分化，促进Treg转化.2.CP-25靶向GRK2介导代谢相关PI3K-AKT信号，促进下游FoxO1核转位及与PD-L1结合互作，诱发DC代谢抑制，下调免疫功能.3.自免性疾病患者GRK2及PI3K-AKT-mTOR信号高表达，靶向GRK2可下调DC或T细胞活化及功能，发挥免疫抑制作用.4.AIH中GRK2膜转位介导免疫细胞PI3K-AKT-mTOR信号活化，调节糖脂质代谢；免疫细胞分化及功能；JAK2-STAT3-SOCS3、AMPK信号的交叉对话.5.PI3K-AKT-FoxO1轴介导DC糖酵解和IL-12分泌，通过调控DC代谢及功能，在小鼠纤维炎症中发挥抗炎免疫抑制；过继转移未成熟、代谢活力低的DC缓解疾病进展.6.CP-25抑制肠道菌群丰度和多样性，对变形菌、乳酸杆菌作用最显著，有通过肝肠循环调控免疫的潜能.7.CP-25通过炎症免疫调控，对肾脏炎症有一定缓解作用.科学意义.1.CP-25对AIH的药效显著，机制与调节DC、T细胞代谢、免疫应答密切相关.2.DC的抗原提呈及中介作用，在肝脏炎症及纤维形成中发挥重要功能，靶向DC治疗，有抗AIH潜能.3.GRK2在自免性疾病患者体内高表达，其作为CP-25的靶点，是AIH治疗新靶标.4.CP-25免疫抑制及代谢调节作用在肾脏炎症、类风湿性关节炎等其他免疫炎症性疾病中也有一定药效