Fuzhengjiedu San inhibits porcine reproductive and respiratory syndrome virus by activating the PI3K/AKT pathway

Abstract

Background: Traditional Chinese medicine (TCM) has been garnering ever-increasing worldwide attention as the herbal extracts and formulas prove to have potency against disease. Fuzhengjiedu San (FZJDS), has been extensively used to treat viral diseases in pigs, but its bioactive components and therapeutic mechanisms remain unclear.

Methods: In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the mechanisms underlying FZJDS's action in treating porcine reproductive and respiratory syndrome virus (PRRSV). We constructed PPI network and screened the core targets according to their degree of value. GO and KEGG enrichment analyses were also carried out to identify relevant pathways. Lastly, qRT-PCR, flow cytometry and western blotting were used to determine the effects of FZJDS on core gene expression in PRRSV-infected monkey kidney (MARC-145) cells to further expand the results of network pharmacological analysis.

Results: Network pharmacology data revealed that quercetin, kaempferol, and luteolin were the main active compounds of FZJDS. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway was deemed the cellular target as it has been shown to participate most in PRRSV replication and other PRRSV-related functions. Analysis by qRT-PCR and western blotting demonstrated that FZJDS significantly reduced the expression of P65, JNK, TLR4, N protein, Bax and IĸBa in MARC-145 cells, and increased the expression of Bcl-2, consistent with network pharmacology results. This study provides that FZJDS has significant antiviral activity through its effects on the PI3K/AKT signaling pathway.

Conclusion: We conclude that FZJDS is a promising candidate herbal formulation for treating PRRSV and deserves further investigation.

Fig 1. Venn diagram showing intersection of common targets of FuZhengJieDu (drug) and PRRSV (disease).

Fig 2. Protein-protein interaction (PPI) analysis of FZJDS against PRRSV.

(A) PPI networks of 30 intersecting shared targets between FZJDS and PRRSV analyzed by STRING. (B) Protein interaction network of identified core targets related to the action of FZJDS against PRRSV by topology selection. Light blue nodes were regular targets while yellow nodes were core targets.

Fig 3. Construction of network of 30 core targets of FZJDS (components) and viral disease (PRRSV).

The nodes denoting the compounds are shown as hexagons representing the intersecting ingredients of the three herbs. The names of the herbs are indicated by ellipses. Different colors represent different compounds; targets are indicated by purple diamonds, and red V’s represent disease. Node size is shown in ascending order according to degree.

Fig 4. Functional characterization of FZJDS against PRRSV intersecting genes.

(a) Gene ontology analysis of intersecting genes of FZJDS and PRRSV. (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of intersecting genes between FZJDS and PRRSV.

Fig 5. Network illustrating the interaction among herbs, disease, core targets and corresponding pathways.

The blue diamonds indicate FZJDS, while the green V’s represent PRRSV. The purple ellipses indicate the pathway-related targets, while, the orange triangles show the corresponding pathway. Node size is in ascending order according to degree.

Fig 6. Cytotoxicity of FZJDS on MARC-145 cells.

(a) Cell viability of FZJDS. (b) CC50 curves of FZJDS. (c) IC50 curves of FZJDS.

Fig 7. Effect of FZJDS on the growth of porcine reproductive and respiratory syndrome virus (PRRSV) in MARC-145 cells.

(a) Effect of FZJDS dose on mRNA expression of the PRRSV N gene as measured by qRT-PCR. (b) Effect of time of FZJDS exposure on mRNA expression of PRRSV N gene as measured by qRT-PCR. (c) Effect of FZJDS dose on PRRSV N protein expression as determined by western blotting. (d) Results of greyscale analysis of Fig 7C. (e) Effect of time of FZJDS exposure on PRRSV N protein expression as measured by western blotting. (f) Results of greyscale analysis of Fig 7E. Data shown in a-f are means ± SEM from four individual wells per group in one experiment. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. control group.

Fig 8. Effect of FZJDS on PI3K/Akt pathway in MARC-145 cells.

(a) Western blotting analysis shows the level of TLR-4 in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (b) Quantification of protein levels from the western blotting in panel a. (c) Western blotting analysis shows the level of P-p65 and P65 in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (d, e) Quantification of protein levels from the western blotting in panel c. (f) Western blotting analysis shows the level of P-JNK and JNK in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (g, h) Quantification of protein levels from the western blotting in panel c. (f) Western blotting analysis shows the level of P-JNK and JNK in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (g,h) Quantification of protein levels from the western blotting in panel f. (i) Western blotting analysis shows the level of P-IkBa and IkBa in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (j,k) Quantification of protein levels from the western blotting in panel i. (l) Western blotting analysis shows the level of Bcl-2 in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (n) Quantification of protein levels from the western blotting in panel l. (m) Western blotting analysis shows the levels of Bax in MARC-145 cells infected with PRRSV (100 TCID50) after treatment with FZJDS. (o) Quantification of protein levels from the western blotting in panel m. For b, d, e, g, h, j, k, n and o, results are means ± SEM from three independent experiments, each of which was performed in triplicate. *P < 0.05; **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig 9. Effect of FZJDS on apoptosis rate in PRRSV-infected MARC-145 cells.

The apoptosis rate in PRRSV-infected MARC-145 cells treated with different FZJDS concentrations was determined by flow cytometry with annexin V/propidium iodide (PI) dual staining (a,b,c,d). The percentage of apoptotic cells is shown in (e). *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 10. FZJDS inhibits activation of JNK in MARC-145 cells.

The effect of P-JNK inhibition on apoptosis in MARC-145 cells induced by PRRSV was determined by flow cytometry (a,b,c,d). The percentage of apoptotic cells was also determined (e). (f) Western blotting analysis shows the levels of P-JNK after inhibitor treatment in MARC-145 cells. (g) Quantification of P-JNK protein levels from the western blotting data in panel f. For e and g, results are shown as the mean ± SEM from three independent experiments, each of which was performed in triplicate. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.