Transcriptome analysis reveals the alleviating effect of Polysaccharide of Atractylodes macrocephala Koidz on thymic involution in Magang geese

Abstract

Thymic involution is one of the important causes of decreased immunity in the body. Noncoding RNAs (miRNAs and lncRNAs) play crucial roles in regulating organ growth and development. Polysaccharide of Atractylodes macrocephala Koidz (PAMK) is widely acknowledged for its anti-oxidant, anti-aging, and immune-enhancing effects. However, its potential application in preventing the age-related thymic involution of Magang geese has not been previously reported. In this study, 54 4-month-old Magang geese were randomly divided into 3 groups, the thymus and serum of 18 geese were collected aseptically after 3 days of prefeeding period, and the remaining geese were randomly divided into control and PAMK groups (3 replicates per group and 6 Magang geese per replicate). Geese in the control group were fed a basal diet, and geese in the PAMK group were fed a basal diet supplemented with 400 mg/kg PAMK. The thymus and serum were collected 1 month later. The results of thymus index measurement showed that PAMK could alleviate thymus index. Furthermore, compared with the M5-Control group, HE staining showed that PAMK made the proportion of thymus medulla increased, and the boundary between cortex and medulla was clearer. Antioxidant function and cytokine content detection showed that, compared with the M5-Control group, PAMK increased T-AOC and GSH-Px levels in thymus, increased T-AOC level and SOD activity in serum, decreased MDA content in thymus and serum, and decreased IL-1β, IL-6 and TNF-α levels. To further explore the mechanism, 3 samples from the control and PAMK groups were selected for RNA-Seq. Through transcriptome analysis and prediction, a triple regulatory ceRNA network of 9 mRNAs, 11 miRNAs and 32 lncRNAs associated with alleviating thymic involution was constructed. Moreover, these genes were respectively enriched in the PPAR, Cytokine-cytokine receptor interaction, WNT, Apelin and MAPK signaling pathways. In summary, PAMK may alleviate age-related thymic involution in Magang geese by alleviating the thymus index, increasing the antioxidant level and regulating the cytokine content, potentially via the PPAR, Cytokine-cytokine receptor interaction, WNT, Apelin, and MAPK signaling pathways.

Keywords: Goose; Polysaccharides of Atractylodes macrocephala Koidz; Thymic involution; Transcriptome analysis.

Fig. 1

Alleviation of the decrease in the thymic index and improvement in the histological structure of Magang geese by PAMK. (a) Morphology observation of thymus. (b) Weight of the thymus. (c) Weights of Magang Geese. (d) Thymic index. (e) Histological observation of the thymus (50 ×; 100 ×; 200 ×). TM stands for thymic medulla, and TC stands for thymic cortex. Data are presented as the means ± standard errors of the mean (SEMs), and data columns labeled with different lowercase letters indicate significant differences (P < 0.05), the same letter indicates that the differences are not statistically significant (P > 0.05). The same applies to the figures below.

Fig. 2

Effects of PAMK on the thymus and serum antioxidant capacity. (a) Thymus T-AOC level. (b) Thymus MDA content. (c) Thymus GSH-Px content. (d) Serum T-AOC level. (e) Serum MDA content. (f) Serum SOD activity.

Fig. 3

Effects of PAMK on thymus and serum cytokines. (a) IL-1β level in the thymus. (b) IL-6 levels in the thymus. (c) TNF-α levels in the thymus. (d) Serum IL-1β levels. (e) Serum IL-6 levels. (f) Serum TNF-α levels.

Fig. 4

Identification of DEGs in the thymus of Magang geese from the M5-Control and M5-PAMK group. (a) M5-PAMK vs M5-Control DEG volcano map. (b) M5-PAMK vs M5-Control DEG heatmap. (c) GO histogram of M5-PAMK vs M5-Control DEGs showing 50 significantly enriched GO terms. Horizontal coordinates indicate enriched GO terms and vertical coordinates indicate -log₁₀ (P value). (d) KEGG scatter plot of PAMK vs Control DEGs showing 10 significantly enriched pathways. The size of the points represents the gene number, and the color represents the P value.

Fig. 5

Identification of DEMs in the thymus of Magang geese from the M5-Control and M5-PAMK groups and DEM-DEG analysis. (a) M5-PAMK vs M5-Control DEM volcano map. (b) M5-PAMK vs M5-Control DEM heatmap. (c) miRNA‒mRNA regulatory network. The ellipsoid indicates mRNAs and the rectangle indicates miRNAs. Green represents downregulation, and red represents upregulation.

Fig. 6

Identification of DELs in the thymus of Magang geese from the M5-Control and M5-PAMK group and DEL-DEM analysis. (a) M5-PAMK vs M5-Control DEL volcano map. (b) lncRNA‒miRNA regulatory network. The green diamonds represent miRNAs, and the pink triangles represent lncRNAs.

Fig. 7

The ceRNA regulatory network associated with thymic involution in Magang geese. (a) The lncRNA-miRNA-mRNA network structure. The ellipsoids represent mRNAs, the diamonds represent miRNAs, and the triangles represent lncRNAs. Green represents down-regulation and red represents up-regulation. (b) The distribution of ceRNA network node degrees. (c) Difference in betweenness centrality among mRNAs, miRNAs and lncRNAs. (d) Clossness centrality differences among mRNAs, miRNAs and lncRNAs. (e) The degree centrality differences among mRNAs, miRNAs and lncRNAs.

Fig. 8

The qRT‒PCR method was used to verify the differentially expressed RNAs in the process of alleviating age-related thymus involution in Magang geese by PAMK. (a) DEGs. (b) DEMs. (c) DELs. (d) Correlation between RNA-Seq and qRT-PCR results. Log₂ fold change is represented by the mean ± SD, with n = 3, and the differences between each gene are all statistically significant with P < 0.05.

Fig. 9

Key genes and pathways in PAMK for alleviating thymus involution. (a) Venn diagram of genes in DEGs associated with thymus involution and DEGs from ceRNA network. (b) Sankey bubble map between genes and pathways. (c) CeRNA regulatory network of 9 key genes associated with mitigation of thymus involution.