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

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

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

Hua Zhang et al. BMC Vet Res. .

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.

Keywords: Glycyrrhizic acid; IFN-β; Matrine; PRRSV; TMT; Tea saponin.

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Conflict of interest statement

The authors have no conflict of interest in the research.

Figures

Fig. 1
Fig. 1
Cytotoxicity (A) and anti-PRRSV activity (B and C) of the screened compounds on PAMs. A: The maximum non-cytotoxic concentrations of the screened compounds was determined by MTT assay for the cell viability. B and C: The detection of PRRSV N gene (B) and protein (C) expression by qPCR and western blot. Cropped bolts are displayed. Compared with PRRSV group, the differences in gene and protein were significant, * means p < 0.05, ** means p < 0.01, *** means p < 0.001
Fig. 2
Fig. 2
Venn diagram showing the overlap of commonly expressed proteins between three compound-treated groups and with control cell A and with virus alone group B
Fig. 3
Fig. 3
Jvenn diagrams showing the overlap of differentially expressed proteins among of up-regulated A or down-regulated B differential proteins in PRRSV group compared with cell group and the down-regulated (A) or up-regulated (B) differential proteins respectively in GA, MT and TS group compared with PRRSV group. The mRNA of top three of 28 overlapping proteins (IFIH1, ISG15 and IFIT3) and IFN-β were detected by qPCR C. * indicated that compared with cell group, the difference was significant, *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 4
Fig. 4
GO annotation and enrichment analysis of differentially expressed proteins of matrine, glycyrrhizic acid and tea saponin groups A. The enriched KEGG pathways of differentially quantified proteins of matrine, glycyrrhizic acid and tea saponin groups B
Fig. 5
Fig. 5
Cytotoxicity (A) and anti-PRRSV activity (B, C and D) of the component formula of Glycyrrhizic acid (GA), tea saponin (TS) and matrine (MT) on PAMs. Orthogonal L9 (34) experiment was conducted to generate the component formulas with different concentrations of MT, GA and TS. The cytotoxicity of these nine formulas was determined by MTT method A. The expression of PRRSV N gene and protein were determined by qPCR B and western blot C and D. Cropped bolts are displayed. Different letters (a-i) showed significant difference among groups, p < 0.05
Fig. 6
Fig. 6
The compound concentration in the formula were further optimized and the synergistic antiviral effect of GA, TS and MT were verified by qPCR (A) and western blot (B and C). Cropped bolts are displayed. Letters (a-i) showed significant difference among groups, p < 0.05
Fig. 7
Fig. 7
The component formula 6 inhibited the mRNA expression of IFN-β, IFIH1, ISG15 and IFIT3. Different letters (a-c) showed significant difference among groups, p < 0.05

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