State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, China.
Institute of Pharmaceutical Biotechnology, School of Biological and Food Engineering, Suzhou University, Suzhou, China.
Br J Pharmacol. 2017 Sep;174(17):2818-2831. doi: 10.1111/bph.13885. Epub 2017 Jul 18.
Non-muscular myosin heavy chain IIA (NMMHC IIA) plays a key role in tissue factor expression and venous thrombosis. Natural products might inhibit thrombosis through effects on NMMHC IIA. Here, we have shown that a natural saponin, D39, from Liriope muscari exerted anti-thrombotic activity in vivo, by targeting NMMHC IIA.
Expression and activity of tissue factor in endothelial cells were analysed in vitro by Western blot and simplified chromogenic assays. Interactions between D39 and NMMHC IIA were assessed by serial affinity chromatography and molecular docking analysis. D39-dependent interactions between NMMHC IIA and TNF receptor 2 (TNFR2) were measured by immunofluorescence, co-immunoprecipitation and proximity ligation assays. Anti-thrombotic activity of D39 in vivo was evaluated with a model of inferior vena cava ligation injury in mice.
D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior vena cava-ligated mice dose-dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3β-NF-κB signalling pathways.
D39 inhibited tissue factor expression and thrombus formation by modulating the Akt/GSK3β and NF-κB signalling pathways through NMMHC IIA. We identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.
非肌肉肌球蛋白重链 IIA（NMMHC IIA）在组织因子表达和静脉血栓形成中发挥关键作用。天然产物可能通过影响 NMMHC IIA 来抑制血栓形成。在这里，我们已经表明，来自石蒜的天然甾体皂苷 D39 通过靶向 NMMHC IIA，在体内发挥抗血栓活性。
通过 Western blot 和简化显色测定法在体外分析内皮细胞中组织因子的表达和活性。通过连续亲和层析和分子对接分析评估 D39 与 NMMHC IIA 之间的相互作用。通过免疫荧光、共免疫沉淀和接近连接测定法测量 D39 依赖性 NMMHC IIA 与 TNF 受体 2（TNFR2）之间的相互作用。通过小鼠下腔静脉结扎损伤模型评估 D39 在体内的抗血栓活性。
D39 剂量依赖性地抑制 HUVECs 中的组织因子表达和促凝活性，并降低下腔静脉结扎小鼠的血栓重量。连续亲和层析和分子对接分析表明，D39 与 NMMHC IIA 结合。在 HEK293T 细胞中，D39 抑制由 NMMHC IIA 过表达引起的组织因子表达。在 HUVECs 中，用 NMMHC IIA 敲低可阻断这种作用。D39 抑制 NMMHC IIA 与 TNFR2 的解离，从而调节 Akt/GSK3β-NF-κB 信号通路。
D39 通过调节 Akt/GSK3β 和 NF-κB 信号通路，通过 NMMHC IIA 抑制组织因子表达和血栓形成。我们鉴定了一种新的靶向 NMMHC IIA 的天然产物，作为治疗血栓形成障碍和其他血管病变的潜在药物。
