Whole Transcriptome Analysis of Intervention Effect of Sophora subprostrate Polysaccharide on Inflammation in PCV2 Infected Murine Splenic Lymphocytes

Abstract

(1) Background: Sophora subprostrate, is the dried root and rhizome of Sophora tonkinensis Gagnep. Sophora subprostrate polysaccharide (SSP1) was extracted from Sophora subprostrate, which has shown good anti-inflammatory and antioxidant effects. Previous studies showed SSP1 could modulate inflammatory damage induced by porcine circovirus type 2 (PCV2) in murine splenic lymphocytes, but the specific regulatory mechanism is unclear. (2) Methods: Whole transcriptome analysis was used to characterize the differentially expressed mRNA, lncRNA, and miRNA in PCV2-infected cells and SSP1-treated infected cells. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and other analyses were used to screen for key inflammation-related differentially expressed genes. The sequencing results were verified by RT-qPCR, and western blot was used to verify the key protein in main enriched signal pathways. (3) Results: SSP1 can regulate inflammation-related gene changes induced by PCV2, and its interventional mechanism is mainly involved in the key differential miRNA including miR-7032-y, miR-328-y, and miR-484-z. These inflammation-related genes were mainly enriched in the TNF signal pathway and NF-κB signal pathway, and SSP1 could significantly inhibit the protein expression levels of p-IκB, p-p65, TNF-α, IRF1, GBP2 and p-SAMHD1 to alleviate inflammatory damage. (4) Conclusions: The mechanism of SSP1 regulating PCV2-induced murine splenic lymphocyte inflammation was explored from a whole transcriptome perspective, which provides a theoretical basis for the practical application of SSP1.

Keywords: Sophora subprostrate polysaccharide (SSP1); inflammation; murine splenic lymphocyte; porcine circovirus type 2 (PCV2); whole transcriptome analysis.

Figure 1

The structure of SSP1.

Figure 2

Histogram, cluster heat map and Venn chart of mRNA and LncRNA. 1607 differentially expressed mRNAs (A) and 549 lncRNAs (F) between C group and V group (FC ≥ 1.2, p < 0.05). 630 differentially expressed mRNAs (C) and 357 lncRNAs (H) between V group and SV group (FC ≥ 1.2, p < 0.05). Heat map generated by hierarchical clustering analysis of differentially expressed top 20 mRNAs (B,D) lncRNAs (G,I). 158 differentially co-expressed mRNAs (E) and 62 lncRNAs (J) were shown in Venn chart.

Figure 3

Histogram, cluster heat map and Venn chart of mRNA and LncRNA. 144 differentially expressed miRNAs (A) and 21 circRNAs (F) between C group and V group (FC ≥ 1.2, p < 0.05). 117 differentially expressed mRNAs (C) and 23 circRNAs (H) between V group and SV group (FC ≥ 1.2, p < 0.05). Heat map generated by hierarchical clustering analysis of differentially expressed top 20 miRNAs (B,D) circRNAs (G,I). 45 differentially co-expressed miRNAs (E) and 3 circRNAs (J) were shown in Venn chart.

Figure 4

Heat map of 19 differentially co-expression mRNAs related to inflammatory responses. Red represents higher expression and blue represents lower expression.

Figure 5

GO term analysis of DEGs. C group and V group: Gene Ontology annotation of mRNA in BP (A), MF (B), CC (C). V group and SV group: Top 20 enrichment genes in BP (D), MF (E), CC (F). GO, Gene Ontology; CC, cellular component; MF, molecular function; BP, biological process.

Figure 6

KEGG analysis of DE mRNAs. Enrichment and screening of KEGG pathway in C group and V group resulted in 325 signaling pathways, involving IL-17 signaling pathway, interaction of viral protein with cytokine and cytokine receptor, p53 signaling pathway, TNF signaling pathway, cytokine-cytokine receptor interaction, chemokine signaling pathway, C-type lectin receptor signaling pathway (A,B,E). 286 signaling pathways were obtained by enrichment and screening of the KEGG pathway in the V group and the SV group, which mainly involved primary immunodeficiency, natural killer cell-mediated cytotoxicity, NF-κB signaling pathway, B-cell receptor signaling pathway, FcγR-mediated phagocytosis, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway (C,D,F).

Figure 7

Trend cluster maps from DEGs by STEM analysis. (A) 100 DE mRNAs were down-regulated. (B) 397 DE mRNAs were down-regulated and then stabilized. (C) 257 DE mRNAs were down-regulated and then up-regulated. (D) 141 DE mRNAs were stabilized and then down-regulated. (E) 158 DE mRNAs were stabilized and then up-regulated. (F) 257 DE mRNAs were up-regulated and then down-regulated. (G) 686 DE mRNAs were up-regulated and then stabilized. (H) 129 DE mRNAs were up-regulated.

Figure 8

RT-qPCR validation of the expression of miRNA, lncRNA and mRNA (n = 3). Differentially expressed mRNA, LncRNA and miRNA were randomly chosen to validate the reliability of the RNA-seq analysis (n = 3). (A–R) The relative expression of partial mRNAs; (S–X) The relative expression of partial LncRNAs; (ZA–ZD) The relative expression of partial miRNAs. * represented significant or extremely significant difference compared with C group; # meant significant or extremely significant difference compared with V group.

Figure 9

Effect of SSP1 on NF-κB/TNF/ cytosolic DNA sensing pathway in PCV2-infected murine splenic lymphocytes. (A) The protein bands of p-p65, p65, p-IκB and IκB. (B) Analysis of protein gray value of p-p65 / p65 and p-IκB / IκB. (C) Analysis of protein gray value of GBP2. (D) The protein bands of p-SAMHD1, SAMHD1, TNF-α, IRF1, and GBP2. (E) Analysis of protein gray value of TNF-α and IRF1. (F) Analysis of protein gray value of p-SAMHD1/ SAMHD1. * or ** represented significant or extremely significant difference compared with C group; ## meant significant or extremely significant difference compared with V group.

Figure 10

Competing endogenous RNAs (ceRNAs) network construction. Differentially expressed mRNAs, miRNAs and lncRNAs, and in the C group and V group (A), and in the V group and SV group (B) were integrated to construct the ceRNA network. The up-regulated mRNA was represented as a red circle, while the down-regulated mRNA was represented as a blue circle. The up-regulated LncRNA was represented by a red diamond, while the down-regulated lncRNA was represented by a blue diamond. The up-regulated miRNA is represented by a red triangle, while the down-regulated miRNA is represented by a blue triangle.

Figure 11

LncRNA, mRNA and miRNA interaction network diagram in V group and SV group.