Open Access Anim Biosci Vol. 34, No. 1:143-153 January 2021 https://doi.org/10.5713/ajas.19.0874 pISSN 2765-0189 eISSN 2765-0235 Effect of JAK-STAT pathway in regulation of fatty liver hemorrhagic syndrome in chickens Yaling Zhu1,2,3,a, Huirong Mao1,a, Gang Peng1, Qingjie Zeng1, Qing Wei1, Jiming Ruan1,*, and Jianzhen Huang1,* * Corresponding Authors: Objective: To explore the molecular mechanisms of fatty liver hemorrhagic syndrome (FLHS) Jiming Ruan Tel: +86-791-83813080, Fax: +86-791-83900189, in laying hens, an experiment was conducted to reveal the differences in histopathological E-mail: [email protected] observation and gene expression between FLHS group and normal group. Jianzhen Huang Methods: We compared the histopathological difference using hematoxylin and eosin Tel: +86-791-83813080, Fax: +86-791-83900189, E-mail: [email protected] staining and proceeded with RNA sequencing of adipose tissue to search differentially expressed genes and enriched biological processes and pathways. Then we validated the 1 College of Animal Science and Technology, mRNA expression levels by real-time polymerase chain reaction and quantified protein Jiangxi Agricultural University, Nanchang, 330045, China levels in the circulation by enzyme-linked immunosorbent assay. 2 Department of Pathophysiology, Anhui Results: We identified 100 differentially expressed transcripts corresponding to 66 genes Medical University, Hefei 230000, China (DEGs) were identified between FLHS-affected group and normal group. Seven DEGs were 3 Laboratory Animal Research Center, College of Basic Medical Science, Anhui Medical significantly enriched in the immune response process and lipid metabolic process, including University, Hefei 230000, China phospholipase A2 group V, WAP kunitz and netrin domain containing 2, delta 4-desaturase sphingolipid 2, perilipin 3, interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF), and a These authors contributed equally to this suppressor of cytokine signaling 3 (SOCS3). And these genes could be the targets of immune work. response and be involved in metabolic homeostasis during the process of FLHS in laying ORCID hens. Based on functional categories of the DEGs, we further proposed a model to explain Yaling Zhu the etiology and pathogenesis of FLHS. IL-6 and SOCS3 mediate inflammatory responses https://orcid.org/0000-0001-8544-1507 Huirong Mao and the satiety hormone of leptin, induce dysfunction of Jak-STAT signaling pathway, leading https://orcid.org/0000-0003-2588-1521 to insulin resistance and lipid metabolic disorders. Conversely, CNTF may reduce tissue Gang Peng destruction during inflammatory attacks and confer protection from inflammation-induced https://orcid.org/0000-0002-5881-7297 Qingjie Zeng insulin resistance in FLHS chickens. https://orcid.org/0000-0001-7739-6793 Conclusion: These findings highlight the therapeutic implications of targeting the JAK- Qing Wei STAT pathway. Inhibition of IL6 and SOCS3 and facilitation of CNTF could serve as a https://orcid.org/0000-0002-9198-7378 Jiming Ruan favorable strategy to enhance insulin action and improve glucose homoeostasis, which are https://orcid.org/0000-0002-2387-1525 of importance for treating obesity-related disorders for chickens. Jianzhen Huang https://orcid.org/0000-0001-8086-1860 Keywords: Fatty Liver Hemorrhagic Syndrome; RNA-Seq; JAK-STAT Pathway; Differentially Submitted Nov 12, 2019; Revised Jan 13, 2020; Expressed Genes; Chicken Accepted Feb 10, 2020 INTRODUCTION Fatty liver hemorrhagic syndrome (FLHS) is a lipid metabolism disorder, which is mostly observed in commercial laying hens. FLHS causes a dramatic drop in egg production, even leads to an increase in mortality and results in considerable economic losses in the poultry industry [1]. For FLHS affected hens, the most obvious changes are enlarged and yellowish, putty-colored livers with various degrees of hemorrhages and an excessive amount of ab- dominal fat. Several factors have been implicated in the cause of FLHS, including nutrition, environment, heredity, toxic substances and hormones [2,3]. The major cause of FLHS is nutrition, as 97% of the affected birds were found to have large fat depots or were obese [4]. In modern poultry industry, an excessive intake of dietary energy is the major cause Copyright © 2021 by Animal Bioscience This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, www.animbiosci.org and reproduction in any medium, provided the original work is properly cited. 143 Zhu et al (2021) Anim Biosci 34:143-153 of FLHS. Recently, a high-energy low-protein (HELP) diet (Approval number: JXAULL-20190019). induced FLHS model has been successfully established [5]. The metabolic disorders such as obesity and liver steatosis Experimental animals and tissue collection often result from the disruption of lipid homeostasis and over- All animal experiments were approved by the Jiangxi Agri- activation of immune pathways. And these damages are cultural University Animal Care and Use Committee. Ninety commonly localized around expanding volumes of adipose healthy 155-day-old Hyline Brown layers with an average tissue, and in particular visceral adipose tissue [6]. Adipose body weight of 1.5 kg were chosen in this study. All layers tissue is an important endocrine organ that controls energy were acclimatized for 7 days before the start of the experiment. hemostasis by storing excess energy in the form of triacylg- These layers were randomly divided into the experimental lycerol (TAG) and releasing energy in the form of free fatty and control groups, and each group contained 45 layers with acid. Excessive lipid storage in adipose tissue results in obe- 3 replicates. These chickens were fed the same basal diet that sity and ultimately its related complications including insulin was formulated to meet the nutritional requirements of layers resistance, non-alcoholic fatty liver disease and other lipid [11]. Layers in the control group were fed basal diet, and metabolic diseases [7]. Alternatively, adipose tissue is cur- layers in the experimental group were fed ‘HELP’ diet (Sup- rently considered as a hormonally active system in the control plementary Table S1). On experimental day 60, the livers of metabolism, not only as a store of excess energy. It has been were harvested from the tested chickens after they were sac- accepted there is a close link between chronic low-grade rificed humanely. Portions of the fresh liver samples were inflammation in adipose tissue and metabolic disorders [8]. stored at –80°C until further use. Accumulated evidence indicates that adipose tissue can release hormones and adipokines which affect glucose homeostasis, Histopathological examination lipid metabolism, and inflammation. For example, inter- Fresh liver samples were dissected, rinsed with saline and leukin-6 (IL-6) is a multifunctional cytokine that plays a fixed in 10% neutral buffered formalin. The subcutaneous major role in regulating immune responses, acute phase fat was immersion-fixed in 4% formaldehyde solution for reactions and hematopoiesis in chicken and human [9]. An 24 h, then dehydrated and embedded in paraffin using a adipocyte-derived hormone of leptin can activate the JAK- standard procedure [12]. The formalin-fixed samples were STAT signaling pathway through the chicken leptin receptor routinely processed, embedded in paraffin, stained with (chLEPR) to regulate energy homeostasis [10], and suppress- hematoxylin and eosin (H&E). The stained sections were ing cytokine signaling 3 (SOCS3) plays an essential role in observed using an optical microscope, and photographs affecting systemic inflammation, insulin resistance, and were taken. Diameters of adipocyte staining were determined leptin resistance [6]. Thereby, a better understanding of the using Image-Pro Plus 6.0 software. endocrine function of adipose tissue may shed insights into the lipid metabolic disorders and the importance of inflam- Statistical analyses mation as a pathogenic pathway in the development of many Phenotypic values were presented as mean±standard devia- hallmarks of the metabolic syndrome including insulin resis- tion (mean±SD). Statistical comparisons of phenotypic values tance, leading to more rational therapy for these increasingly between the experimental and control groups were conducted prevalent disorders. by Student t-test. The statistical difference was considered as To date, as far as we know, systematic studies to detect the significant at p≤0.05 and highly significant at p≤0.01. transcriptomic changes in abdominal adipose tissue from FLHS affected (experimental) hens in comparison with healthy RNA extraction and sequencing (control) individuals are rare. To this end, we explored the Total RNA was extracted from 100 mg of adipose tissue from RNA sequencing technology to compare gene expression three FLSH experimental individuals and three control indi- profiling in the abdominal fat tissue from FLHS experimen- viduals using the RiboPure kit (Ambion, Austin, TX, USA) tal and control chickens, and to identify candidate genes according to the manufacturer's protocol. RNA integrity was and pathways functionally related to FLHS in chickens. assessed by an Agilent Bioanalyser 2100 and RNA Nano 6000 Lab chip kit (Agilent Technologies, Santa Clara, CA, MATERIALS AND METHODS USA). Sequencing libraries were generated using the NEB- Next Ultra Directional RNA Library Prep