Network pharmacology and experimental validation to reveal the target of matrine against PRRSV

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is an epidemic animal infectious disease worldwide. In our previous research it was suggested that matrine could inhibit PRRSV infection both in vitro and in vivo, but the antiviral mechanisms are still undecided. Network pharmacology can well solve the difficult problem of "multiple targets, multiple pathways" in the research of TCM action targets. The results of network pharmacology indicated that matrine exerts its anti-PRRSV effect by targeting HSPA8 and HSP90AB1. The results of real-time fluorescent quantitative PCR and western blot showed that infection with PRRSV induced a significant increase in the expression of HSPA8 and HSP90AB1 whereas matrine treatment could significantly reverse it, and the number of viruses of PRRSV also decreased. In this study, the method of network pharmacology was used to explore HSPA8 and HSP90AB1 which were the potential targets of matrine against PRRSV on Marc-145 cells.

Keywords: Biochemistry; Drugs; Virology.

Graphical abstract

Figure 1

Intersection of matrine targets and disease targets related to porcine reproductive and respiratory syndrome See also Tables S1 and S2.

Figure 2

PPI network (A) Protein interaction network diagram of 26 potential targets. (B) The 12 core target proteins that have been screened, highlighted in the red box. The core target screening criteria were set as Degree Un-Dir >11.04.

Figure 3

GO enrichment analysis and KEGG pathway analysis (A) GO enrichment analysis. (B) KEGG signal pathway analysis. The results of GO enrichment analysis and KEGG signal pathway analysis were arranged in ascending order of p value, and the top 10 objects were selected successively. All p-values were less than 0.01. See also Tables S3 and S4.

Figure 4

Network of “matrine-core target-pathway” The blue, yellow and green color represent matrine, the core targets and the signal pathways, respectively. The key targets screening criteria were set as Degree Un-Dir ＞ 5.619.

Figure 5

Schematic diagram of the interaction between matrine and target proteins (A) HSP90AB1, (B) GSK3B, (C) MAPK8, (D) MAPK14, (E) CASP3, (F) HSPA8. Molecular docking is done by AutodockTools.

Figure 6

Summary of docking free binding energy

Figure 7

The expression of PRRSV N, HSPA8 and HSP90AB1 mRNA in Marc-145 cells treated with different treatments Effects of high, middle and low doses of matrine on the expression of PRRSV N, HSPA8 and HSP90AB1 mRNA in Marc-145 infected with PRRSV for 24 h (A, B, C), 48 h (D, E, F) and 72 h (G, H, I). All data were expressed as mean ± standard errors of the mean (mean ± SEM), where n represented the number of groups in the experiment and n = 5. “a, b, c, d, e” represented a significant difference between different columns (p< 0.05).

Figure 8

Expression levels of PRRSV N, HSPA8 and HSP90AB1 after Marc-145 infection with PRRSV at different time points (A) 24 h, (B) 48 h and (C) 72 h. All data were expressed as mean ± standard errors of the mean (mean ± SEM), where n represented the number of groups in the experiment and n = 5. “a, b, c, d, e” represented a significant difference between different columns (p< 0.05).