Zhang Y, Han H, Sun L, Qiu H, Lin H, Yu L, Zhu W, Qi J, Yang R, Pang Y, Wang X, Lu G, Yang Y
State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of Plant Molecular Biology, Nanjing University, Nanjing, China.
Suzhou Industrial Park Center for Disease Control and Prevention, Suzhou, China.
Braz J Med Biol Res. 2017 Aug 17;50(10):e6586. doi: 10.1590/1414-431X20176586.
Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 μg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.
人肠道病毒71型（EV71）是手足口病（HFMD）的主要病原体，尤其是在4岁以下的婴幼儿中。紫草素是一种具有抗炎、抗病毒和抗菌活性的生物活性化合物，源自中药紫草的根部。本研究旨在检测紫草素酯衍生物PMM - 034对横纹肌肉瘤（RD）细胞中EV71的抗病毒活性。使用WST - 1法测定PMM - 034对RD细胞的细胞毒性。使用蚀斑减少试验测定PMM - 034对RD细胞中EV71复制的剂量和时间依赖性作用。通过实时RT - PCR测定EV71/VP1和促炎细胞因子（IL - 1β、IL - 6、IL - 8和TNF - α）的mRNA表达水平，通过蛋白质印迹分析测定EV71/VP1和磷酸化p65蛋白表达。PMM - 034对RD细胞仅表现出微弱的细胞毒性。然而，PMM - 034对RD细胞中的EV71表现出显著的抗病毒活性，50%抑制浓度为2.31μg/mL。用PMM - 034处理的细胞中VP1 mRNA和蛋白水平显著降低。此外，用PMM - 034处理的细胞中IL - 1β、IL - 6、IL - 8和TNF - α的相对mRNA表达水平显著降低，而处理细胞中磷酸化p65蛋白表达也显著降低。这些结果表明，PMM - 034抑制了RD细胞中促炎细胞因子的表达，表现出对EV71的抗病毒活性，PMM - 034处理细胞中VP1 mRNA和蛋白水平降低证明了这一点。因此，PMM - 034作为一种EV71抑制剂具有进一步开发的潜力。
