| Objective: The purpose of this study was to find the anti-PRRSV target of matrine on Marc-145 cells.Methods: According to the chemical structure of matrine,the biotinylated probe,probe 1 and probe 2,were synthesized by hydrolytic ring-opening and non-hydrolytic ring-opening respectively.The safe concentration of the small molecule probe on Marc-145 cells as well as the inhibition rate on PRRSV-infected Marc-145 cells were detected by MTT assay.Real-time q PCR(q PCR)was used to detect the effect of small molecule probes on PRRSV N gene expression in Marc-145 cells treated with PRRSV for 12 h,24 h,36 h,48 h and 72 h.The Western blot was used to detect the effect of small molecule probes on PRRSV N protein expression in Marc-145 cells treated with PRRSV for 12 h,24 h and 36 h.The inflammatory model of LPS on Marc-145 cells was established,and the activity of small molecule probes was verified by detecting the expression of IL-6 gene and protein by q PCR and Western blot.Based on the high affinity of biotin and streptavidin,the target of matrine on Marc-145 cells was obtained using the "target fishing" strategy and identified by SDS-PAGE-silver nitrate staining and high-resolution mass spectrometry.The antiviral targets of matrine were predicted by network pharmacology,and potential anti-PRRSV targets were selected by comparison with "target fishing" proteins.The protein interaction network was constructed through the database and the potential targets were subjected to GO analysis and KEGG analysis.Results: 1.The cell viability of probe 1 and probe 2 was above 90% in the range of 0.125-1.5 mg/m L.2.The inhibition rate of small molecular probe on PRRSV-infected Marc-145 cells was detected by MTT assay,and the results showed that the highest inhibition rate of probe 1 on PRRSV-infected Marc-145 cells was 3.3 %,and the highest inhibition rate of probe 2 on PRRSV-infected Marc-145 cells was 35.7 %.The results of detection of PRRSV N gene by q PCR showed that PRRSV infected Marc-145 cells for 12 h,compared with PRRSV group,the expression of PRRSV N gene in probe 1 group was significantly decreased(p<0.05),and PRRSV infected Marc-145 cells for 24-72 h,compared with PRRSV group,the expression of PRRSV N gene in probe 1 group was increased,and PRRSV infected Marc-145 cells for12-72 h,compared with PRRSV group,the expression level of PRRSV N gene in probe 2 group was significantly decreased(p<0.05).The results of detection of PRRSV N protein by western blot showed that PRRSV infected Marc-145 cells for 12-24 h,compared with PRRSV group,the expression level of PRRSV N protein in probe 1 and probe 2 group was significantly decreased(p<0.05),and PRRSV infected Marc-145 cells for 36 h,compared with PRRSV group,the expression level of PRRSV N protein in probe1 and probe 2 group was increased.3.The anti-inflammatory activity of probe 1 and probe 2 was detected by q PCR and Western blot.The results showed that probe 1 and probe 2 could significantly reduce the expression of IL-6 mRNA and protein(p<0.05).4.The SDS-PAGE-silver nitrate staining and high resolution mass spectrometry were used to identify the “target fishing” protein,and the silver staining results showed that there was protein enrichment at 17-26 k Da and 26-34 k Da,and mass spectrometry detection showed that a total of 819 proteins were fished by the probe.By comparing with the anti-PRRSV targets screened by the database,eight matrine anti-PRRSV candidate target proteins were obtained,namely ALB,HSPA8,AKR1B1,GSTP1,PTPN1,HSP90AB1,GART and TGF-β2.Conclusion:1.Matrine biotinylated small molecule probes have anti-PRRSV and anti-inflammatory activities.2.We screened eight potential targets of matrine against PRRSV,namely ALB,HSPA8,AKR1B1,GSTP1,PTPN1,HSP90AB1,GART,and TGF-β2... |
