Wang Yuan, Yuan Ming-Ming, Zhou Jing, Zheng Xiao-Han, Yuan Chong-Jun, Chen Shuai, Luo Sen, Zhang Lei
Chengdu University of Traditional Chinese Medicine Chengdu 611137, China.
Sichuan Academy of Chinese Medicine Sciences Chengdu 610041, China.
Zhongguo Zhong Yao Za Zhi. 2022 Aug;47(16):4428-4435. doi: 10.19540/j.cnki.cjcmm.20220216.401.
The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 μg·mL(-1) and 251.78 μg·mL(-1), respectively, and TC_(50) was 1 749.98 μg·mL(-1) and 2 977.50 μg·mL(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 μg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.
本研究通过体外实验探讨了鸢尾黄素衍生物（SGY）对Ⅰ型单纯疱疹病毒（HSV-1）的抑制作用及机制。采用细胞计数试剂盒-8（CCK-8）法检测SGY及阳性药物阿昔洛韦（ACV）对非洲绿猴肾（Vero）细胞和小鼠小胶质细胞（BV-2）的细胞毒性，计算药物的最大无毒浓度和半数中毒浓度（TC50）。Vero细胞感染HSV-1后，通过细胞病变效应（CPE）测定病毒毒力以计算病毒滴度。检测受试药物对HSV-1感染Vero细胞所致细胞病变的抑制作用，并通过病毒吸附、复制及灭活初步探讨其作用方式。采用实时荧光定量PCR（RT-qPCR）检测药物对HSV-1感染后24 h BV-2细胞病毒载量及Toll样受体（TLR）mRNA表达的影响。SGY对Vero细胞和BV-2细胞的最大无毒浓度分别为382.804 μg·mL-1和251.78 μg·mL-1，TC50分别为1 749.98 μg·mL-1和2 977.50 μg·mL-1。在Vero细胞模型中，SGY对HSV-1的半数最大抑制浓度（IC50）为54.49 μg·mL-1，选择指数（SI）为32.12，其作用方式为显著抑制复制并直接灭活HSV-1。RT-qPCR结果显示，SGY显著降低细胞内病毒载量。病毒模型组与正常组相比，TLR2、TLR3及肿瘤坏死因子受体相关因子3（TRAF3）相对表达显著升高，TLR9相对表达降低，SGY干预后，TLR2、TLR3及TRAF3表达不同程度降低，TLR9表达增强。与正常组相比，病毒模型组诱导型一氧化氮合酶（iNOS）、肿瘤坏死因子-α（TNF-α）及白细胞介素-1β（IL-1β）等炎症因子表达显著升高，SGY干预后这些炎症因子水平下降。综上所述，SGY在体外对HSV-1具有显著抑制及直接灭活作用。此外，它还调节TLR2、TLR3及TLR9相关通路的表达，并抑制炎症因子水平升高。
