Transcriptome analysis on the inflammatory cell infiltration of nonalcoholic steatohepatitis in bama minipigs induced by a long-term high-fat, high-sucrose diet

Abstract

Long-term adherence to a high-fat, high-calorie diet influences human health and causes obesity, metabolic syndrome and nonalcoholic steatohepatitis (NASH). Inflammation plays a key role in the development of NASH; however, the mechanism of inflammation induced by over-nutrition remains largely unknown. In this study, we fed Bama minipigs a high-fat, high-sucrose diet (HFHSD) for 23 months. The pigs exhibited characteristics of metabolic syndrome and developed steatohepatitis with greatly increased numbers of inflammatory cells, such as lymphocytes (2.27-fold, P<0.05), Kupffer cells (2.59-fold, P<0.05), eosinophils (1.42-fold, P<0.05) and neutrophils (2.77-fold, P<0.05). High-throughput RNA sequencing (RNA-seq) was performed to explore the systemic transcriptome of the pig liver during inflammation. Approximately 18.2 gigabases of raw sequence data were generated, and over 303 million high-quality reads were assembled into 21,126 unigenes. RNA-seq data analysis showed that 822 genes were differentially expressed in liver (P<0.05) between the HFHSD and control groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the process of inflammation involved the inflammatory signal transduction-related toll-like receptor, MAPK, and PPAR signaling pathways; the cytokine-related chemokine signaling, cytokine-cytokine receptor interaction, and IL2, IL4, IL6, and IL12 signaling pathways; the leukocyte receptor signaling-related T cell, B cell, and natural killer cell signaling pathways; inflammatory cell migration and invasion- related pathways; and other pathways. Moreover, we identified several differentially expressed inflammation-related genes between the two groups, including FOS, JUN, TLR7, MYC, PIK3CD, VAV3, IL2RB and IL4R, that could be potential targets for further investigation. Our study suggested that long-term HFHSD induced obesity and liver inflammation, providing basic insight into the molecular mechanism of this condition and laying the groundwork for further studies on obesity and steatohepatitis.

Figure 1. Extensive inflammatory cell infiltration within the dilated hepatic sinusoids in the HFHSD group (H&E stain).

A: HFHSD group portal area; B: Control group portal area; C: HFHSD group hepatic lobule; D: HFHSD group hepatic lobule. A, B, C, D, bar  = 100 µm. Note: a, Ito cell; b, lymphocyte; c, Kupffer cell; d, eosinophil; e, neutrophil.

Figure 2. The ultrastructure of inflammatory cells in the hepatic sinusoids of the HFHSD group.

There were a large number of phagocytic granules in the Kupffer cells, neutrophils and eosinophils. A: Kupffer cell; B: lymphocyte; C: neutrophil; D: eosinophil; bar  = 2 µm.

Figure 3. Validation of the RNA-Seq data by qRT-PCR.

The 16 genes in minipig livers associated with lipid metabolism, chemokines and the immune response were selected for validation. The data are presented as the fold differences between the HFHSD group and the control group, and each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.

Figure 4. Distribution of the genes according to their expression profiles by RPKM.

Figure 5. Gene ontology (GO) analysis of the Bama minipig transcriptome data (GO level 2).

GO analysis of 822 genes differentially expressed between the HFHSD group and the control group, which was used to predict the genes' involvement in biological processes (A), molecular functions (B) and cellular components (C). The GO Id and name are shown with different colors.

Figure 6. List of genes involved in the chemokine signaling pathway.

The genes in the red box represent differentially expressed genes between the HFHSD group and the control group in RNA-seq analysis. All other genes had no obvious differences in this experiment.

Figure 7. List of genes involved in the natural killer cell-mediated cytotoxicity pathway.

The genes in the red box illustrate differentially expressed genes between the HFHSD group and the control group in the RNA-seq analysis. All other genes had no obvious differences in this experiment.

Figure 8. List of genes involved in the T cell receptor signaling pathway.

The genes in the red box represent differentially expressed genes between the HFHSD group and the control group in the RNA-seq analysis. All other genes had no obvious differences in this experiment.

Figure 9. List of genes involved in the leukocyte transendothelial migration pathway.

The genes in the red box illustrate differentially expressed genes between the HFHSD group and the control group in the RNA-seq analysis. All other genes had no obvious differences in this experiment.