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Transcriptome Analysis on the Inflammatory Cell Infiltration of Nonalcoholic Steatohepatitis in Bama Minipigs Induced by a Long-Term High-Fat, High-Sucrose Diet

Significance Statement

 

Obesity, metabolic syndrome and the associated chronic inflammation are among the most prevalent diseases that impose enormous health and economic burdens on governments around the world and on the global economy. Inflammation is believed to be the driving force behind the development of NASH and also acts as a critical predictor of the histological progression to fibrosis and cirrhosis; thus, inflammation represents a potentially major therapeutic target in NASH. Several species have been used as models for obesity and NASH, and rodents have been established as the primary model for these diseases. However, there are metabolic and physiological differences between humans and rodents that have undoubtedly slowed progress and complicated this research. Pigs are rapidly emerging as a biomedical model for obesity and energy metabolism in humans because of their similar metabolic features, cardiovascular systems, and proportional organ sizes. We established the metabolic syndrome with Bama minipigs after being fed a high-fat, high-sucrose diet (HFHSD) for 23 months; the early signs of NASH, steatosis, oxidation stress, iron overload, lipid peroxidation and cellular damage all can be observed in the liver. Compared with the control group, there was a significant increase in the number of inflammatory cells, including lymphocytes, eosinophils, neutrophils, and Kupffer cells, in hepatic lobules in the HFHSD pigs. Through the analysis of the RNA-Seq data pathways, 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. The choice of animal disease model plays an important role in the study of the hepatic inflammation molecular mechanisms of NASH. From this study, the minipigs exhibited obesity, hyperinsulinemia, dyslipidemia, and inflammation accompanied by significantly increased levels of inflammatory cells. Differences in genes and pathways clearly indicated progressive signal transduction, directed toward the invasion of inflammatory cells, and toward leukocyte transendothelial migration. The results of this study indicated that the minipgs should be of good models for the future study and the development of new drugs of NASH.

Figure Legend: Extensive inflammatory cell infiltration within the dilated hepatic sinusoids in the HFHSD group (H&E stain and ultrastructure). 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 um. Note: a, Ito cell; b, lymphocyte; c, Kupffer cell; d, eosinophil; e, neutrophil. E: Kupffer cell; F: lymphocyte; G: neutrophil; H: eosinophil; bar=2um.

Transcriptome Analysis on the Inflammatory Cell Infiltration of Nonalcoholic Steatohepatitis in Bama Minipigs Induced by a Long-Term High-Fat, High-Sucrose Diet

Journal Reference

Jihan Xia, Jing Yuan, Leilei Xin, Yuanyuan Zhang, Siyuan Kong, Yaoxing Chen, Shulin Yang, Kui Li. PLoS ONE. 2014, 9(11): e113724.

Show Affiliations

State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, P. R. China, and

College of Animal Science, Yangtz University, Jinzhou, Hubei, P. R. China, and

College of Veterinary Medicine, China Agricultural University, Beijing, P. R. China

Electronic address: [email protected], [email protected]

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.

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