College of Pharmacy, Second Military Medical University, Shanghai 200433, China.
College of Pharmacy, Second Military Medical University, Shanghai 200433, China; Department of Pharmacy, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
Int Immunopharmacol. 2018 Jan;54:254-260. doi: 10.1016/j.intimp.2017.11.018. Epub 2017 Nov 21.
Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, is reported as a potent suppressor of pro-inflammatory functions of macrophages. However, little is known about the target proteins of EsA for its anti-inflammatory activity. In the present study, to identify the intracellular target for EsA, affinity resins bearing immobilized EsA were used to capture binding proteins of EsA from RAW264.7 cell lysates. Mass spectrography and Western blot analysis of captured proteins indicated that ribosomal protein S3a preferentially bound to EsA affinity resin. Competition experiment further demonstrated that free EsA can disturb the specific interaction between recombinant RPS3a and affinity resin. Surface Plasmon Resonance analysis confirmed that EsA directly bound to RPS3a. Lentivirus-mediated RNAi RPS3a resulted in suppression of TNF-α and IL-6 production and impediment of signal transduction in LPS-stimulated RAW264.7 cells, indicating that RPS3a is required for LPS-triggered signaling during induction of pro-inflammatory cytokines. In addition, EsA inhibited the expression of inflammatory factors more strongly in the case of RPS3a interference. These results suggest that EsA exerts its anti-inflammatory activity by targeting RPS3a and impairing its signaling function. These new findings not only extended our understanding on the intracellular mechanisms of EsA, but also indicated RPS3a as an essential component for LPS-mediated pro-inflammatory signaling, thus implying RPS3a as a novel therapeutic target for anti-inflammatory therapy.
Esculentoside A（EsA）是从商陆中分离得到的一种皂苷，据报道它是一种强大的巨噬细胞促炎功能抑制剂。然而，对于其抗炎活性的靶蛋白知之甚少。在本研究中，为了鉴定 EsA 的细胞内靶标，使用固定有 EsA 的亲和树脂从 RAW264.7 细胞裂解物中捕获 EsA 的结合蛋白。捕获蛋白的质谱和 Western blot 分析表明核糖体蛋白 S3a 优先与 EsA 亲和树脂结合。竞争实验进一步表明，游离的 EsA 可以干扰重组 RPS3a 与亲和树脂的特异性相互作用。表面等离子体共振分析证实 EsA 直接与 RPS3a 结合。慢病毒介导的 RPS3a RNAi 导致 TNF-α和 IL-6 产生的抑制以及 LPS 刺激的 RAW264.7 细胞中信号转导的受阻，表明 RPS3a 是 LPS 触发诱导促炎细胞因子信号所必需的。此外，在 RPS3a 干扰的情况下，EsA 对炎症因子的表达抑制作用更强。这些结果表明，EsA 通过靶向 RPS3a 并损害其信号转导功能发挥抗炎活性。这些新发现不仅扩展了我们对 EsA 细胞内机制的理解，还表明 RPS3a 是 LPS 介导的促炎信号的必需组成部分，因此暗示 RPS3a 是抗炎治疗的新的治疗靶点。
