Gene expression profile analysis of ileum transcriptomes in pigs fed Gelsemium elegans plants

Gelsemium elegans is a flowering plant in the Loganiaceae. Because it can promote the growth of pigs and sheep, it is widely used, including in veterinary clinics, but little information is available about its biological effects. Here, we used high-throughput sequencing to characterize the differentially expressed genes (DEGs) in the ileums of pigs between a control group and a group fed Gelsemium elegans for 45 days. We found that Gelsemium elegans affected many inflammatory and immune pathways, including biological processes such as defense responses, inflammation and immune responses. Moreover, this study identified several important genes related to the anti-inflammatory activity of Gelsemium elegans (e.g., CXCL-8, IL1A, and CSF2), which will be beneficial for further study of the pharmacological mechanisms and clinical applications of Gelsemium elegans.


Results
Transcriptome sequencing data quality testing. Transcriptome sequencing was performed on a total of 6 samples from the experimental group and the control group (Table 1), and an average of 52.168 M reads were obtained. After removing the linker fragments and the low-quality fragments, a total of 51.171 M high-quality clean reads were obtained; the Q20% of the clean reads in each sample was above 96.80%, and the Q30% was above 91.60%, indicating that the sequencing quality was good and provided good original data for subsequent data assembly. Upon comparing the clean reads to the reference genome, an average of 49.135 M could be mapped, which accounted for 96% of the total number of sequences. The comparison showed that the alignment efficiency was good.
Gene expression statistics. HTSeq 11 was used to quantify the expression level of each sample gene to obtain the raw counts, which were the input data for the next differential expression analysis. Then, the raw counts for each sample were standardized to obtain the FPKM value of each sample gene, and the expression levels of six sample genes were determined ( Table 2). The number of highly expressed genes (FPKM > 50) in each ileum sample was maintained at approximately 800, while the number of genes with low expression or no expression (FPKM < 1) was above 7000 in all samples.
Screening of differentially expressed genes. Taking a |log2 (fold change)| ≥ 1, a P-value ≤ 0.05 and an FDR (adjusted for P-value) ≤ 0.05 as the screening conditions, the experimental group and the control group were compared and analyzed. A total of 446 differentially expressed genes (DEGs) were screened in this experiment, of which 237 genes were upregulated and 209 genes were downregulated. GO classification of differentially expressed genes. GO annotation was conducted for the DEGs, and a total of 30 significant GO terms were enriched. As shown in Fig. 1, the DEGs were mainly enriched in biological processes such as defense responses, humoral immune responses, inflammatory responses, and immune system processes, which indicates that intake of Gelsemium elegans leads to changes in immune and inflammatory response processes in animal bodies. In the cellular component category, the cell periphery, plasma membrane, extracellular region and plasma membrane part terms were most prominent; in the molecular function category, receptor activity/receptor binding was the main term.
Enrichment of differentially expressed gene pathways. As shown in Fig. 2, 446 DEGs between the experimental group and the control group were enriched in signaling pathways related to inflammation and the immune response, including the complement and coagulation cascade pathway, the IL-17 signaling pathway, the rheumatoid arthritis pathway and the hematopoietic cell lineage pathway, with relatively high enrichment rates.  www.nature.com/scientificreports www.nature.com/scientificreports/ The genes enriched in the pathways are shown in Table 3. Statistical analyses showed that CXCL-8, IL1A, C3, C5, CSF2, C2, CXCL-2, FOS, AMCF-II, and C8G were involved in at least three of the above pathways, suggesting that these genes may be important hubs for communication between different signaling pathways. Further statistical analyses showed that CXCL-8, CSF2 and AMCF-II are all involved in the cytokine-cytokine receptor interaction pathway and the rheumatoid arthritis pathway, which are related to inflammation, suggesting that these two pathways may be key pathways in inflammation-related reaction processes.
QRT-PCR verification results. The correlation analysis results of the qRT-PCR and RNA-seq data were similar (Fig. 3), which suggests that the transcriptome sequencing data are highly reproducible and accurate.  www.nature.com/scientificreports www.nature.com/scientificreports/ Discussion RNA-seq technology is an ideal high-throughput sequencing method that greatly improves the speed and efficiency of identifying new genes or biomarkers 12,13 . Therefore, in this study, the RNA-seq technique was used to determine the transcriptomic effects of Gelsemium elegans on the pig ileum, and 446 DEGs were obtained. A total of 237 genes were upregulated, and 209 genes were downregulated in the ileum tissue of the experimental group compared with that of the control group. At the same time, we found 14 important pathways. These results provide the basis for a better understanding of the molecular mechanisms of Gelsemium elegans in pigs.
The results of expression analysis of inflammatory response-related genes showed that the inflammatory genes in the experimental group were generally downregulated. First, genes associated with inflammatory-related  Table 3. Pathway enrichment gene expression. www.nature.com/scientificreports www.nature.com/scientificreports/ receptors and signaling pathways, such as the cytokine-cytokine receptor interaction pathway and the IL-17 signaling pathway, suggesting that cytokine-cytokine receptor action is inhibited and that intercellular signal transmission is weakened under Gelsemium treatment 14,15 . Most of the genes enriched in inflammatory response-related pathways, such as the rheumatoid arthritis pathway, and related metabolic processes in inflammation, such as the complement and coagulation cascade pathways, were downregulated, indicating that the intestinal inflammatory response of the experimental group was inhibited.
The downregulated genes associated with the complement and coagulation cascades pathway in the experimental group compared with the control group were C4BPB, C4BPA, F11, C2, C3 and C5, among which C3 is a local inflammatory reaction process mediator that can induce smooth muscle contraction, increase vascular permeability and promote mast cells and leukocytes to release histamine. C3 also plays the role of an inflammatory cell chemical inducer in chronic inflammation. In addition, C5 is an effective chemokine that stimulates leukocyte movement and induces leukocytes to migrate to inflammatory sites. The complement and coagulation cascades were all downregulated, which may have been related to the inhibition of inflammatory reactions. This finding shows that Gelsemium elegans might reduce inflammatory cell infiltration by reducing the levels of cytokines related to the complement and coagulation cascades.
The IL-17 signaling pathway is involved in the pathogenesis of many inflammatory and autoimmune diseases, including rheumatoid arthritis 16 and psoriasis. S100A8 is a calcium-zinc binding protein (with significantly downregulated expression) belonging to the low-molecular weight S100 protein family that participates in the migration process of neutrophils to inflammatory sites 1 , can bind with TLR4 and AGER receptors, and activates the MAP kinase and NF-K-B signaling processes, thus leading to expansion of the proinflammatory cascade and playing an important role in inflammatory process and immune response regulation.
In the rheumatoid arthritis pathway, a total of 5 DEGs were enriched, among which IL-8/CXCL-8 is a key mediator related to inflammation belonging to the CXC family of chemokines that is usually released in response to inflammatory stimulation. IL-8/CXCL-8 plays a key role in neutrophil recruitment, and degranulation 17 is considered to be an inflammatory mediator of gingivitis 18 and psoriasis. In a study of Gelsemium elegans, some researchers developed an injectable solution and oral liquid from a water extract of Gelsemium elegans and applied it to the treatment of psoriasis and neurodermatitis 19 . The downregulation of the CXCL-8 gene is consistent with the anti-inflammatory activities of Gelsemium elegans and emphasizes the role of CXCL-8 in the pharmacological activities of Gelsemium elegans. CSF2, also known as granulocyte-macrophage colony-stimulating factor (GM-CSF), is a monomeric glycoprotein. Clinical tests have revealed its proinflammatory effect. Studies have shown that CSF2 is highly expressed in joints with rheumatoid arthritis, and inflammation and injury can be reduced by blocking CSF2. IL-1α/IL1A, which are members of the IL-1 cytokine family together with IL-1β, can induce the release of IL-2 and mainly play a role in regulating and initiating inflammatory reactions. Studies have found that blocking the activity of IL-1α is likely to be useful for the treatment of skin diseases such as acne 20 . IL-1β is an important mediator of inflammatory reactions that can induce many kinds of self-inflammatory diseases. Experiments have found that intestinal ecological imbalance can induce osteomyelitis in an IL-1β-dependent manner 21 .
A large number of studies have indicated that Gelsemium elegans has strong anti-inflammatory activity [22][23][24] . Researchers have used transcriptome analysis to study SH-SY5Y neurons in humans treated with low concentrations of Gelsemium alkaloids and found significant changes in related regulatory genes of the inflammatory response signaling pathway 23 . Some researchers have also found that medium and high doses of Gelsemium could significantly inhibit the mitosis process in vaginal epithelial cells and promote the formation of epidermal granules in mouse tail scales. At the same time, the level of IL-2 in the serum of experimental mice was found to be significantly reduced in three dose groups 24 . Koumine, which is one of the major Gelsemium alkaloids, has been found to reduce the levels of the proinflammatory factors TNF and IL-1, attenuate collagen-induced increases in fibrillary protein content, and inhibit astrocyte activation and proinflammatory factor-mediated pain 25,26 .
Using qPCR and Western blot analysis, Yuan et al. found that koumine can inhibit the release of inflammatory mediators and cytokines such as TNF-α, IL-6 and IL-1β 27 . Similarly, studies have shown that IL-1α 28 , CXCL8 29 , CSF2 30 , C3 31 , C5 32 and S100A8 33 were consistent at the mRNA and protein levels. However, Yang et al. found that koumine can selectively inhibit the proliferation of T cells, especially CD4+ T (Th) cells, reducing the levels of inflammatory cytokines (Th1 cytokines IFN-γ, IL-1β, 2, 6, 12, TNF-α and Th17-α cytokines IL-17A) 34 . Aggarwal A. et al. found that rheumatoid arthritis is closely related to polarity deviation in Th cells and systemic disorder of inflammatory and anti-inflammatory cell networks 35 . It is worth noting that macrophages can release IL-1α, which acts on Th cells to regulate their activity. These studies suggest that Gelsemium elegans may regulate the polarity deviation of Th cells by inhibiting the expression of IL-1α, thus reducing the production of inflammatory factors. In this study, the expression of many inflammatory cytokine genes was inhibited, and the levels of these cytokines entering the blood to reach corresponding lesions were decreased; these changes achieved an anti-inflammatory effect by restoring the balance of the network.

Conclusions
In this study, we reported the ileum transcriptome profiles of three-way hybrid weaned piglets fed two different diets (a complete diet without antibacterial drugs and a complete diet containing 2% Gelsemium elegans herb without antibacterial drugs) and carried out detection and DEG identification. The results showed that Gelsemium elegans affected many inflammatory and immune pathways, including biological processes such as defense responses, inflammation and immune responses. More importantly, this study identified several important genes (e.g., CXCL-8, IL1A, and CSF2). These data provide a clearer understanding for elucidating the molecular mechanisms of the pharmacological actions of Gelsemium, such as its anti-inflammatory effects. (2019) 9:15756 | https://doi.org/10.1038/s41598-019-52374-4 www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Animals and treatments. In this study, 6 healthy castrated male ternary hybrid piglets approximately 60 days old and weighing 20 ± 2 kg were obtained from Hunan Xinwufeng, Yong'an branch (Hunan, China), and randomly divided into control and Gelsemium elegans treatment groups. The control group was a blank control group fed a complete diet without antibacterial drugs, and the Gelsemium elegans group was an experimental group. The diet was prepared by adding 2% Gelsemium elegans powder to a complete diet without antibacterial drugs. The formula and nutritional level of the complete diet are shown in Table 4. After 45 days of continuous feeding, administration was stopped, and all piglets were fasted for 24 hours. The piglets were slaughtered within one day after stopping the drug, and a sterile blade was used to take ileum tissue from the control group and the experimental group. The tissue samples were washed in distilled water to remove blood and placed into centrifuge tubes. The tubes were sealed with sealing film and quickly placed in liquid nitrogen for storage. The samples were stored at −80 °C until RNA extraction. This experiment was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of China and approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (IACUC# 201302).
Extraction of total RNA. Total RNA was isolated from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The integrity and purity of the RNA were detected by agarose gel electrophoresis (Tianneng, China) and with a NanoDrop (Thermo, USA), and the RNA concentration was accurately quantified with a Qubit instrument (Invitrogen, USA).
Construction of the sequencing library. The mRNA was enriched by magnetic beads with Oligo(dT), and then, the mRNA was randomly broken into short fragments of approximately 200 bp. Using mRNA as a template, first-strand cDNA was synthesized by reverse transcription using random primers. For synthesis of the second strand of cDNA, dTTP in the dNTP mixture was replaced by dUTP. The cDNA product was purified with AMPure XP beads (Beckman, USA); then, the double-stranded ends were blunted with End Repair Mix, and poly-A tails and sequencing linkers were added. The USER enzyme was used to digest the double-stranded cDNA to make a library containing only single-stranded cDNA. PCR (Life, USA) enrichment was performed, and the final cDNA library was obtained after 15 cycles of amplification.
Transcriptome sequencing. An Illumina HiSeq sequencing platform (Illumina, USA) was used, and the 2*150 sequencing mode was adopted to construct a qualified cDNA library for double-terminal (PE) sequencing by Shanghai Jingneng Biotechnology Limited (Shanghai, China).
Processing and analysis of sequencing results. According to the distribution feature that the low-mass fraction of Illumina sequencing data is usually concentrated at the end, the raw sequencing data were filtered to obtain clean reads. Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) software was used to remove the linker sequence fragments and low-mass fragments from the 3' ends of the raw data. Finally, high-quality clean reads were obtained, and the clean reads and pig reference genome sequences were  www.nature.com/scientificreports www.nature.com/scientificreports/ sequence-matched with STAR software 36 . The obtained alignment results were based on the positional information files of the known transcripts on the genome, and transcripts with expression values of 0 were removed with StringTie software 37 . Then, the transcripts for all samples assembled with StringTie were reassembled to obtain new transcripts. BLASTX software was used to carry out sequence alignment with the UniProt (http://www. uniprot.org/downloads), GO (http://geneontology.org/) and KEGG (http://www.genome.jp/) databases to obtain corresponding unigene functional annotation information.

Differential expression analysis.
The FPKM values were used to calculate the gene expression levels in the samples, and differential expression was screened between the experimental group samples and the control group samples using DESeq. 2 software. DEGs were identified by using a log2|fold change| ≥ 1, a P-value ≤ 0.05 and an FDR (adjusted for P-value) ≤ 0.05 as the screening conditions. GO functional enrichment analysis 38 and KEGG pathway enrichment analysis 39 for the DEGs were performed.
Validation of RNA-seq data by quantitative real-time PCR (qRT-PCR). Validation was performed by using the same RNA used for sequencing. qRT-PCR was performed on selected DEGs, including C3, CXCL-8, FOS, LTA, S100A8, IL1RL2, NOX1, KCNJ16, FMO1, MT2B and MT1A. Primer sequences were designed using the NCBI primer design tool and synthesized by Qingke Xinye Biotechnology Limited (Changsha, China). Fluorescence quantitative detection was performed using a TIANLONG TL988 fluorescence quantitative PCR instrument (Xi'an Tianlong Technology Limited). Each sample was analyzed in triplicate, and the expression of the target genes was standardized by GAPDH expression. The reaction protocol comprised one cycle of 95 °C for 1 min followed by forty cycles of 95 °C for 10 s, 60 °C for 5 s and 72 °C for 10 s. The gene expression was calculated by using the comparative Ct (2 −ΔΔCt ) method.

Data availability
The datasets analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) under BioProject ID: PRJNA 552943.