一种使用串联质量标签标记蛋白质组学分析的从天然化合物中获得抗 PRRSV 的最佳成分配方的新策略。

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses.

机构信息

Shanxi key lab. for modernization of TCVM, College of Veterinary Medicine, Shanxi Agricultural University, Shanxi, 030801, Taigu, China.

Laboratory Animal Center, Shanxi Agricultural University, Shanxi, 030801, Taigu, China.

出版信息

BMC Vet Res. 2022 May 14;18(1):179. doi: 10.1186/s12917-022-03184-w.

DOI:10.1186/s12917-022-03184-w

PMID:35568854

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9106989/

Abstract

BACKGROUND

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV.

METHODS

A total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated.

RESULTS

MT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-β mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 μg/mL TS) showed synergistic anti-PRRSV effect.

CONCLUSIONS

The component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 μg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-β may be one of the targets of the component formula possessed anti-PRRSV activity.

摘要

背景

猪繁殖与呼吸综合征（PRRS）是一种最重要的猪病毒性疾病之一，一直威胁着中国的养猪业。目前，由于 PRRSV 株的高度遗传多样性，大多数商业疫苗未能提供完全保护。本研究旨在优化一种具有明确作用机制的中药（TCM）化合物配方，以对抗 PRRSV。

方法

使用猪肺泡巨噬细胞（PAMs）筛选了 13 种天然化合物的抗 PRRSV 活性。选择三种具有强抗 PRRSV 活性的化合物通过蛋白质组学分析鉴定其潜在的蛋白靶标。基于蛋白质组学结果，采用正交设计确定最佳的化合物配方。MTT 测定法用于确定每种化合物对 PAMs 的最大非细胞毒性浓度（MNTC）。QPCR 和 Western blot 分别用于检测 PRRSV N 基因和蛋白的表达。使用串联质量标签（TMT）相对定量蛋白质组学技术分别检测用 PRRSV、苦参碱（MT）、甘草酸（GA）和茶皂素（TS）处理的 PAMs 的差异蛋白表达。使用具有抗 PRRSV 活性的三种浓度的化合物进行正交设计。MTT 测定法筛选出四个高安全性的配方，并评价其抗 PRRSV 作用。

结果

MT、GA 和 TS 呈剂量依赖性抑制 PRRSV 复制。CCL8、IFIT3、IFIH1 和 ISG15 是 MT、GA 或 TS 处理细胞中表达水平变化最大的前四种蛋白。IFIT3、IFIH1、ISG15 和 IFN-β mRNAs 的相对表达与蛋白质组学结果一致。该配方（0.4 mg/mL MT+0.25 mg/mL GA+1.95 μg/mL TS）显示出协同抗 PRRSV 作用。