柴胡皂苷D（SaikosaponinD）靶向STAT3发挥抗类风湿性关节炎作用的研究

Imbalance between T helper 17 (Th17) and regulatory T cell (Treg) is the most important factor causing rheumatoid arthritis (RA). Activation of signal transducer and activator of transcription 3 (STAT3) facilitates abundance of Th17 polarization which resulting in promotion of a balance shift to Th17 cells and causing severe arthritis. Tofacitinib, a pan-Janus kinase (JAK) inhibitor, is the only one FDA-approved drug for RA treatment which down-regulates STAT3 signaling main through inhibiting JAKs activation. Interestingly, even if tofacitinib is the only one FDA-approved drug which indirectly related to STAT3 target, it is still more effective than most of first-line agents for RA patients, especially in patients with methotrexate-resistant active RA. Therefore, STAT3 has been regarded as a novel molecular target for drugs development in RA treatment. However, studies on specific inhibitors of STAT3 in drugs discovery and development for RA treament are really scarce despite its indispensible roles in RA pathogenesis. Hence, targeting STAT3 for anti-RA drugs discovery and accelerating drugs approval processes are urgently needed to be performed. In our previous study, surface plasmon resonance (SPR) was used to screen candidates from 1200 natural compounds which could combine with STAT3 protein. Based on the dissociation constant in the SPR screening as well as the availability, activity and novelty of these components, saikosaponin D (SSD) was identified as the optimal candidate for the present study. In addition, SSD selectively binded to STAT3 at SH2 domain, and inhibited activation of STAT3 signaling stimulated by IL-6 in spleen cells, Th17 polarization and IL-17A level in vitro. In the present study, we will subsequently evaluate its effects on Treg polarization and cytokines levels in vitro; gene overexpression and siRNA interference will be employed for two-way authentication to validate that STAT3 is indeed the molecular target of SSD for suppressing inflammation through recovering balance between Th17 and Treg；in vivo experiment, collagen-induced arthritis model is engaged to evaluate its effects on antagonizing arthritis in vivo. All of our in vitro and in vivo findings will illuminate the effects and molecular signal pathways as well as the target of SSD for antagonizing RA. We believe that the data of our project will provide a strong support for SSD and other drugs discovery in RA treatment.

Th17/Treg平衡向Th17偏移是造成类风湿性关节炎(RA)的重要原因，信号转导转录活化因子3(STAT3)激活会诱导Th17分化而加重RA。Tofacitinib是通过间接抑制STAT3治疗RA仅有的上市药物，疗效显著，提示若靶向STAT3治疗RA疗效可能更佳。因此，本项目以STAT3为靶点切入，运用表面等离子共振分析（SPR）技术从1200个化合物中筛选STAT3结合物。前期首次发现柴胡皂苷D(SSD)特异性结合在STAT3的SH2分子域；体外研究发现SSD抑制STAT3的活化和Th17分化及IL-17A表达。后续将探讨SSD对Treg分化的影响；体外采用基因过表达和干扰脾细胞，并体内采用慢病毒表达和干扰胶原诱导关节炎小鼠，验证其靶向STAT3发挥抗RA作用。综合动物、细胞、蛋白和基因水平结果明确SSD抗RA药效作用、信号通路和靶点蛋白，为SSD进一步开发为治疗RA药物提供依据。

Th17/Treg平衡向Th17偏移是造成类风湿性关节炎（RA）的重要原因，信号转导转录活化因子3（STAT3）激活会诱导Th17分化而加重RA。Tofacitinib是通过间接抑制STAT3治疗RA仅有的上市药物，疗效显著，提示若靶向STAT3治疗RA疗效可能更佳。因此，本项目以STAT3为靶点切入，运用表面等离子共振分析（SPR）技术从1200个化合物中筛选STAT3结合物。SPR研究发现柴胡皂苷D（Saikosaponin D, SSD）与STAT3有特异性结合，分子对接进一步表明其结合在STAT3的SH2分子域，该分子域为STAT3磷酸化形成二聚体并入核发挥其转录功能的重要分子域。体外研究发现SSD抑制STAT3的活化和Th17分化及IL-17A表达，且SSD对Treg分化的无影响，从而显著下调Th17/Treg比例，恢复其平衡；进一步机制研究结果表明SSD抑制STAT3磷酸化，入核及转录活性；同时，体外脾细胞过表达及敲减STAT3，发现SSD抑制Th17分化及IL-17A表达作用减弱或消失。体内用胶原（bovine type II collagen）和完全弗氏佐剂（complete freund’s adjuvant）诱导DBA雄性小鼠关节炎模型评估SSD抗RA作用，结果表明，SSD显著降低关节炎小鼠临床评分，改善骨参数，抑制关节滑膜增生及炎症细胞浸润，且降低血清及关节组织炎症因子；另外，尾静脉注射shSTAT3慢病毒感染胶原佐剂诱导关节炎小鼠，发现SSD抑制血清炎症因子、改善骨参数、抑制关节滑膜增生及炎症细胞浸润作用减弱。本研究综合动物、细胞、蛋白和基因水平结果明确SSD靶向STAT3抗RA，为SSD进一步开发为治疗RA药物提供依据。

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