Potential mechanisms and therapeutic effect of dietary resveratrol supplementation on the spleen organ of chicken in chronic unpredictable mild stress transcriptomic analysis

Abstract

Chronic unpredictable mild stress (CUMS) affects chicken immune system and welfare, causing huge losses of growth performance and welfare. Resveratrol (RSV), an antioxidant and anti-inflammatory natural plant polyphenol, is widely used for the prevention of stress related disease. The aim of this study is to explore the therapeutic effect of RSV on spleen damage in CUMS. We successfully constructed a CUMS model. A total of 288 healthy one-day-old chicks were used in this study and were divided in 3 groups, control, CUMS and CUMS+RSV group. During 42 days of age, spleen tissue samples were collected and analyzed. Transmission electron microscope (TEM), Hematoxylin and eosin (H&E) staining, immunofluorescence, qRT- PCR, Western blots, immunohistochemical staining and RNA- sequencing (RNA-seq) technology was used to determine any changes and analyzed the mRNA and enrichment pathways. Histopathology and ultrastructure showed there was a severe damage of tissues. The results of RNA-seq showed that a total of 206, 267 and 211 DEGs were identified (log2 Fold Change| >1, P < 0.05) in control -vs- CUMS group, CUMS -vs- CUMS+RSV group and control -vs- CUMS+RSV group, respectively. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the SDEGs, two immune/stress- related pathways including PPAR signaling pathway and neuroactive ligand receptor interaction were selected. The genes related to PPAR signaling pathway identified were PLIN1, MMP1, ANGPTL4 and FABP4 and Neuroactive ligand-receptor interaction genes were GRPR, NTSR1, KNG1 and AGT. The PLIN1, MMP1, ANGPTL4, FABP4, GRPR, KNG1 and AGT were up regulated and NTSR1 was down regulated in CUMS group. When compared to CUMS -vs- CUMS+RSV group, PLIN1, FABP4, KNG1 and AGT were down regulated genes and NTSR1 was up regulated gene. Taken together, KEGG pathway analyses of DEGs, verified by qRT-PCR and Western blots, the current study suggested that these data reveal the promising role of RSV in the prevention and therapy of a wide variety of tissue damage and PPAR signaling pathway, neuroactive ligand-receptor interaction in chronic unpredictable mild stress.

Keywords: Chicken; Chronic unpredictable mild stress; Resveratrol; Spleen.

Fig. 1

Using a transmission electron microscope (magnification 25,000x), we observed ultra-structural changes in the spleen at 42 day of age. In the CUMS group the cell showed severe mitochondria damage, such as mitochondria swelled and, chromatic agglutination were observed (A). The results of H&E staining of the spleen at (50μm) (B), The number of splenic nodules was decreased and atrophic in CUMS group, and the germinal centre of splenic nodules was unclear, the boundary between the red and white pulp in the experimental group's spleens was unclear, additionally, the decreased splenic follicular, and blurred or ruptured splenic margins and irregular splenic trabecular.

Fig. 2

Differentially expressed genes (DEGs) between groups (A). Clustering map of differentially expressed genes (DEGs) between control_vs_ CUMS group (B), Clustering map of differentially expressed genes (DEGs) between CUMS_vs_ CUMS+RSV (C) and Clustering map of differentially expressed genes (DEGs) between control_vs_ CUMS+RSV (D) by RNA-seq analysis in spleen of chicken.

Fig. 3

Results of correlation analysis of spleen (A), Principal component analysis (PCA) of control, CUMS and CUMS+RSV by RNA-seq analysis of spleen (B). Results of volcano plot (C) and Venn diagram of common overlapping of different gene (D).

Fig. 4

Results of GO enrichment analysis of differentially expressed genes (DEGs) between control_vs_ CUMS group (A), GO enrichment analysis of different expressed genes (DEGs) between CUMS _vs_ CUMS+RSV group (B) by RNA-seq analysis in chicken spleen.

Fig. 5

Results of KEGG enrichment analysis of differentially expressed genes (DEGs) between control_vs_ CUMS group (A) and KEGG enrichment analysis of differentially expressed genes (DEGs) between CUMS_vs_CUMS+RSV group (B) by RNA-seq analysis in chicken.

Fig. 6

The representative images of NTSR1 in spleen detected with immunofluorescence confocal microscopy under 200 magnification.Scalebar:100 mm (A), the quantitate fluorescence intensity rate of NTSR1 (B). Result of immunohisochemical staining of spleen at (50 µm), expression of angiotensinogen (AGT) in the spleen, positive expression brown (arrow) of AGT occurred in the red pulp and white pulp spleen tissue in CUMS group (C).

Fig. 7

For validation of RNA seq analysis, qRT-PCR result of PPAR signaling pathway and Neuroactive ligand-receptor interaction related genes expression levels in spleen (A).The proteins expression of PPAR signalling pathway of different gene were analyzed by western blott (B), the quantitate rate of protein level analysed by western bolt (C).