College of Animal Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, Yunnan, China.
College of Animal Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, Yunnan, China; Department of Oncology-Pathology, Karolinska Institute, 17176, Stockholm, Sweden.
Vet Microbiol. 2021 Apr;255:109016. doi: 10.1016/j.vetmic.2021.109016. Epub 2021 Feb 17.
Porcine Reproductive and Respiratory Syndrome (PRRS) is a devastating disease among the most notorious threats to the swine industry worldwide and is characterized by respiratory distress and reproductive failure. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with complicated genetic diversity make the current vaccination strategy far from cost-effective and thus urge identification of potent lead candidates to provide prevention and treatment approaches. From an in vitro small molecule screening with the TargetMol Natural Compound Library comprising 623 small molecules, cytopathic effect (CPE) observations and RT-qPCR analysis of viral ORF7 gene expression identified cepharanthine (CEP) to be one of the most protent inhibitors of PRRSV infection in Marc-145 cells. When compared with tilmicosin, which is one of the most commonly used antibiotics in swine industry to inhibit infections, CEP more prominently inhibited PRRSV infection represented by both RNA and protein levels, further reduced the TCID by 5.6 times, and thus more remarkably protected Marc-145 cells against PRRSV infection. Mechanistically, western blot analyses of the Marc-145 cells and the porcine alveolar macrophages (PAMs) with or without CEP treatment and PRRSV infection at various time points revealed that CEP can inhibit the expression of integrins β1 and β3, integrin-linked kinase (ILK), RACK1 and PKCα, leading to NF-κB suppression and consequent alleviation of PRRSV infection. Collectively, our small molecule screening identified cepharanthine as an inhibitor of PRRSV infection in vitro by suppressing Integrins/ILK/RACK1/PKCα/NF-κB signalling axis, which may enlighten the deeper understanding of the molecular pathogenesis of PRRSV infection and more importantly, suggested CEP as a potential promising drug for PRRS control in veterinary clinics.
猪繁殖与呼吸综合征（PRRS）是一种严重的疾病，是全球养猪业最著名的威胁之一，其特征是呼吸窘迫和繁殖失败。高度进化的猪繁殖与呼吸综合征病毒（PRRSV）株具有复杂的遗传多样性，使得当前的疫苗接种策略远非高性价比，因此迫切需要确定有效的候选药物，以提供预防和治疗方法。通过使用 TargetMol 天然化合物库（包含 623 种小分子）进行体外小分子筛选，对细胞病变效应（CPE）进行观察，并对病毒 ORF7 基因表达进行 RT-qPCR 分析，鉴定出石蒜碱（CEP）是抑制 Marc-145 细胞中 PRRSV 感染的最有效抑制剂之一。与替米考星（一种在养猪业中最常用的抗生素之一，用于抑制感染）相比，CEP 更显著地抑制了 PRRSV 感染，无论是在 RNA 还是蛋白水平上，进一步将 TCID 降低了 5.6 倍，因此更显著地保护 Marc-145 细胞免受 PRRSV 感染。从机制上讲，Western blot 分析表明，在 Marc-145 细胞和猪肺泡巨噬细胞（PAMs）中，无论是否用 CEP 处理和感染 PRRSV，CEP 均可抑制整合素β1 和β3、整合素连接激酶（ILK）、RACK1 和 PKCα 的表达，从而抑制 NF-κB 并减轻 PRRSV 感染。总之，我们的小分子筛选发现，石蒜碱通过抑制整合素/ILK/RACK1/PKCα/NF-κB 信号通路，抑制体外 PRRSV 感染，这可能会加深我们对 PRRSV 感染分子发病机制的理解，更重要的是，提示 CEP 可能成为兽医临床控制 PRRS 的一种有前途的药物。
