Kaempferol inhibits Pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways

doi:10.1016/S2095-3119(20)63477-3

摘要/Abstract

摘要：

目的：伪狂犬病是危害养猪业最为严重的病毒性疾病之一。疫苗是目前预防伪狂犬病毒感染的主要方式。2011年以来中国许多疫苗免疫过的猪场再次爆发了伪狂犬病，表明现有的疫苗不能提供充足的保护。因此，本研究旨在探究山奈酚在体外对伪狂犬病毒的抑制作用及其机制，为狂犬病的防控提供新的思路。

方法：以阿昔洛韦为阳性对照，用CCK-8法测定不同浓度山奈酚对PK-15细胞的毒性，计算半数毒性浓度（CC₅₀）以及山奈酚对病毒所致细胞病变的抑制率，计算半数抑制浓度（IC₅₀）。通过荧光定量PCR法测定山奈酚在病毒感染不同阶段（对细胞的保护作用和抗病毒吸附、穿入和复制）以及在感染后不同时间点（2、4、8和16 h）分别加入药物后对病毒复制的抑制活性。采用Western blotting 和荧光定量PCR法检测分别检测山奈酚对NF-κB和MAPKs信号通路的影响及其下游靶基因的转录水平的变化。

结果：山奈酚在PK-15细胞上的CC₅₀和IC₅₀分别是0.213和 0.026 mol L^-1，其选择指数（SI）为8.33，抗病毒活性优于阿昔洛韦（IC₅₀为0.055 mol L^-1；SI为6.34）。作用方式研究表明山奈酚显著抑制病毒的穿入和复制阶段，与病毒组相比，病毒基因拷贝数分别减少了约4倍和30倍。山奈酚在病毒感染后16 h内加入均能显著抑制病毒的增殖，与病毒组相比，在病毒感染后2h加入，病毒基因拷贝数减少约15倍。山奈酚显著调节病毒感染导致NF-κB和MAPKs信号通路的激活及其下游靶基因MAPKs（ATF-2和c-Jun）和NF-κB （IL-1α，IL-1β和IL-2）的转录水平。

结论：山奈酚能显著抑制病毒感染，作用方式为抑制病毒的穿入和复制阶段，作用机制可能为调节NF-κB和MAPKs信号通路及其下游靶基因。

创新性：初次评价山奈酚对伪狂犬病毒的抑制活性，结果显示其活性优于抗疱疹病毒常用药物阿昔洛韦，表明山奈酚在防控伪狂犬病毒以及其它疱疹病毒感染具有一定的应用前景。

Abstract:

Pseudorabies virus (PRV), in the family Herpesviridae, is a pathogen of Aujeszky’s disease, which causes great economic losses to the pig industry. Recent outbreaks of Pseudorabies imply that new control measures are urgently needed. The present study shows that kaempferol is a candidate drug for controlling PRV infection, as it possesses the ability to inhibit PRV replication in a dose-dependent manner in vitro. Kaempferol at a concentration of 52.40 μmol L^–1 could decrease PRV-induced cell death by 90%. With an 50% inhibitory concentration (IC₅₀) value of 25.57 μmol L^–1, kaempferol was more effective than acyclovir (positive control) which has an IC₅₀ value of 54.97 μmol L^–1. A mode of action study indicated that kaempferol inhibited viral penetration and replication stages, decreasing viral loads by 4- and 30-fold, respectively. Addition of kaempferol within 16 h post infection (hpi) could significantly inhibit virus replication, and viral genome copies were decreased by almost 15-fold when kaempferol was added at 2 hpi. Kaempferol regulated the NF-κB and MAPKs signaling pathways involved in PRV infection and changed the levels of the target genes of the MAPKs (ATF-2 and c-Jun) and NF-κB (IL-1α, IL-1β and IL-2) signaling pathways. The findings of the current study suggest that kaempferol could be an alternative measure to control PRV infection.

Key words: kaempferol , antiviral activity , Pseudorabies virus

. [J]. Journal of Integrative Agriculture, 2021, 20(8): 2227-2239.

CHEN Xu, CHEN Ya-qin, YIN Zhong-qiong, WANG Rui, HU Huai-yue, LIANG Xiao-xia, HE Chang-liang, YIN Li-zi, YE Gang, ZOU Yuan-feng, LI Li-xia, TANG Hua-qiao, JIA Ren-yong, SONG Xu. Kaempferol inhibits Pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways[J]. Journal of Integrative Agriculture, 2021, 20(8): 2227-2239.

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