基于RNA-Seq技术研究枸杞多糖对环磷酰胺致雏鸡免疫抑制的拮抗机制

doi:10.11843/j.issn.0366-6964.2023.08.036

摘要/Abstract

摘要： 本试验旨在基于RNA-Seq技术研究枸杞多糖对环磷酰胺致雏鸡免疫抑制的拮抗机制，分析脾中与免疫相关差异表达基因的变化，为枸杞多糖拮抗雏鸡免疫抑制机制提供参考依据。将120只7日龄海兰褐蛋鸡随机分成3个组，分别为空白组（NC）、环磷酰胺组（CY）及枸杞多糖组（CYLbGp），CY和CYLbGp两组连续3 d每日胸肌注射80 mg·kg^-1环磷酰胺建立免疫抑制雏鸡模型，NC组肌注等体积生理盐水，CYLbGp组在模型建立后通过饮水的方式每天给予5 mg·kg^-1枸杞多糖，连续30 d。给药结束后，每组随机选取6只雏鸡采集脾，采用RNA-Seq技术检测3组在给予枸杞多糖后的差异表达基因。通过KEGG通路富集分析和蛋白质互作（PPI）网络分析，筛选关键差异基因和通路。结果表明，枸杞多糖干预后共有178个差异表达基因显著回调，显著富集在线粒体自噬-动物、趋化因子信号通路、C型凝集素受体信号通路、B细胞受体信号通路、血小板活化、自然杀伤细胞介导的细胞毒性和Th1和Th2细胞分化等免疫相关通路上。PPI分析结果表明，磷酸肌醇3激酶调节亚基（PIK3CD）、Janus激酶3（JAK3）、GATA结合蛋白3（GATA3）、低氧诱导因子（HIF1A）、NK2同源异型框5（NKX2-5）和肌醇多磷酸磷酸酶样1（INPPL1）为其中的关键基因。RT-qPCR结果与转录组学数据变化趋势基本一致，进一步表明转录组数据有较高的可靠性。综上，枸杞多糖通过作用于PIK3CD、JAK3、GATA3、HIF1A、和INPPL1等关键靶点，参与线粒体自噬、趋化因子信号通路、C型凝集素受体信号通路、B细胞受体信号通路、血小板活化、自然杀伤细胞介导的细胞毒性、Th1和Th2细胞分化等信号通路的调控，进而起到缓解雏鸡免疫抑制的作用。

关键词: RNA-Seq技术, 免疫抑制, 枸杞多糖, 环磷酰胺, 脾

Abstract: The aim of this research was to study the antagonistic mechanism of Lycium barbarum polysaccharide (LBP) on cyclophosphamide induced immunosuppression in chicks based on RNA-Seq technology, and to analyze the changes of immune-related differentially expressed genes in spleen, so as to provide reference for the antagonistic mechanism of LBP on immunosuppression of chicks. One hundred and twenty 7 days old Hy-line brown laying hens were randomly divided into three groups:blank group (NC), cyclophosphamide group (CY) and Lycium barbarum polysaccharide group (CYLbGp). The immunosuppressive chick model was established by injecting 80 mg·kg^-1 cyclophosphamide everyday into the pectoralis muscle for three consecutive days, and the same volume of normal saline was injected to the hens in NC group. After the establishment of the model, the chicks in the CYLbGp group were supplemented with Lycium barbarum polysaccharide (LBP) 5 mg·kg^-1 by drinking water everyday for 30 consecutive days. At the end of administration, spleens were collected from 6 chicks in each group, and the differentially expressed genes (DEGs) in the three groups were detected by RNA-Seq. KEGG pathway enrichment analysis and protein-protein interaction (PPI) network analysis were used to screen key differentially expressed genes and pathways. The results showed that after LBP intervention a total of 178 DEGs significantly reduced, and were significantly enriched in mitochondrial autophagy-animals, chemokine signaling pathways, C-type lectin receptors signaling pathways, B cell receptor signaling pathways, platelet activation, natural killer cell mediated cytotoxicity and Th1 and Th2 cells differentiation and other related immune pathways. PPI analysis showed that PIK3CD, JAK3, GATA3, HIF1A, NKX2-5 and INPPL1 were the key genes. The results of RT-qPCR were consistent with the trend of transcriptomic data, which further indicated the high reliability of transcriptomic data. It can be concluded that Lycium barbarum polysaccharide can act on key targets such as PIK3CD, JAK3, GATA3, HIF1A and INPPL1. Lycium barbarum polysaccharide is involved in the regulation of mitochondrial autophagy, chemokine signaling pathway, C-type lectin receptor signaling pathway, B-cell receptor signaling pathway, platelet activation, natural killer cell-mediated cytotoxicity, Th1 and Th2 cell differentiation and other signaling pathways, and thus it can relieve the immune suppression of chicks.

Key words: RNA-Seq technology, immunosuppression, Lycium barbarum polysaccharide, cyclophosphamide, spleen

中图分类号:

S852.4

王建东, 唐玉林, 王敏, 张宝锁, 杨富强, 高海慧, 于洋, 郭延生. 基于RNA-Seq技术研究枸杞多糖对环磷酰胺致雏鸡免疫抑制的拮抗机制[J]. 畜牧兽医学报, 2023, 54(8): 3519-3532.

WANG Jiandong, TANG Yulin, WANG Min, ZHANG Baosuo, YANG Fuqiang, GAO Haihui, YU Yang, GUO Yansheng. The Mechanism of Lycium barbarum Polysaccharide against Immunosuppression Induced by Cyclophosphamide in Chicks Based on RNA-Seq Technique[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3519-3532.

参考文献 32

[1]	CHEN H L, LI D F, CHANG B Y, et al.Effects of Chinese herbal polysaccharides on the immunity and growth performance of young broilers[J].Poult Sci, 2003, 82(3):364-370.
[2]	BALAMURUGAN V, KATARIA J M.Economically important non-oncogenic immunosuppressive viral diseases of chicken-current status[J].Vet Res Commun, 2006, 30(5):541-566.
[3]	HOERR F J.Clinical aspects of immunosuppression in poultry[J].Avian Dis, 2010, 54(1):2-15.
[4]	FAN Y P, LU Y, WANG D Y, et al.Effect of epimedium polysaccharide-propolis flavone immunopotentiator on immunosuppression induced by cyclophosphamide in chickens[J].Cell Immunol, 2013, 281(1):37-43.
[5]	DOU W W, LI H M, CHENG Z Q, et al.Maternal antibody induced by recombinant gp85 protein vaccine adjuvanted with CpG-ODN protects against ALV-J early infection in chickens[J].Vaccine, 2013, 31(51):6144-6149.
[6]	TZIANABOS A O.Polysaccharide immunomodulators as therapeutic agents:structural aspects and biologic function[J].Clin Microbiol Rev, 2000, 13(4):523-533.
[7]	AYEKA P A, BIAN Y H, GITHAIGA P M, et al.The immunomodulatory activities of licorice polysaccharides (Glycyrrhiza uralensis Fisch.) in CT 26 tumor-bearing mice[J].BMC Complement Altern Med, 2017, 17(1):536.
[8]	牛忻, 闫娜, 李笃信, 等.枸杞多糖的分离鉴定与体外肠道代谢初步分析[J].天然产物研究与开发, 2019, 31(10):1682-1687, 1730.NIU X, YAN N, LI D X, et al.Separation and in vitro gut metabolism analysis of polysaccharides from the fruits of Lycium barbarum[J].Natural Product Research and Development, 2019, 31(10):1682-1687, 1730.(in Chinese)
[9]	PENG X M, TIAN G Y.Structural characterization of the glycan part of glycoconjugate LbGp2 from Lycium barbarum L.[J].Carbohydr Res, 2001, 331(1):95-99.
[10]	DING Y, YAN Y M, PENG Y J, et al.In vitro digestion under simulated saliva, gastric and small intestinal conditions and fermentation by human gut microbiota of polysaccharides from the fruits of Lycium barbarum[J].Int J Biol Macromol, 2019, 125:751-760.
[11]	KWOK S S, BU Y S, LO C Y, et al.A systematic review of potential therapeutic use of Lycium barbarum polysaccharides in disease[J].Biomed Res Int, 2019, 2019:4615745.
[12]	蔡松涛, 孙京涛, 魏瑄.枸杞多糖抑制核因子κB(NF-κB)通路降低骨关节炎软骨细胞炎性细胞因子水平[J].细胞与分子免疫学杂志, 2018, 34(11):989-993.CAI S T, SUN J T, WEI X.Lycium barbarum polysaccharide inhibits NF-κB pathway to reduce the level of inflammatory cytokines in osteoarthritis chondrocytes[J].Chinese Journal of Cellular and Molecular Immunology, 2018, 34(11):989-993.(in Chinese)
[13]	LIU B, WANG J L, WANG X M, et al.Reparative effects of Lycium barbarum polysaccharide on mouse ovarian injuries induced by repeated superovulation[J].Theriogenology, 2020, 145:115-125.
[14]	潘虹, 施真, 杨泰国, 等.枸杞多糖对糖尿病大鼠视网膜神经元的保护作用及其机制[J].中国应用生理学杂志, 2019, 35(1):55-59.PAN H, SHI Z, YANG T G, et al.The protective effects of Lycium barbarum polysaccharides on retinal neurons in diabetic rats and its mechanism[J].Chinese Journal of Applied Physiology, 2019, 35(1):55-59.(in Chinese)
[15]	LONG L N, KANG B J, JIANG Q, et al.Effects of dietary Lycium barbarum polysaccharides on growth performance, digestive enzyme activities, antioxidant status, and immunity of broiler chickens[J].Poult Sci, 2020, 99(2):744-751.
[16]	王思月.枸杞多糖对肉鸡肠道微生物区系、免疫功能及相关基因表达的影响[D].长春:吉林农业大学, 2019.WANG S Y.Effects of Lycium barbarum polysaccharides on intestinalmicroflora, immune function and expression of related genes in Broilers[D].Changchun:Jilin Agricultural University, 2019.(in Chinese)
[17]	王建东, 唐玉林, 王敏, 等.基于代谢组学技术研究枸杞多糖对雏鸡免疫抑制的拮抗机制[J].动物营养学报, 2022, 34(8):5354-5363.WANG J D, TANG Y L, WANG M, et al.Antagonistic mechanism of Lycium barbarum polysaccharides on immunosuppression of chicks based on metabonomics technology[J].Chinese Journal of Animal Nutrition, 2022, 34(8):5354-5363.(in Chinese)
[18]	王建东, 唐玉林, 王敏, 等.枸杞糖肽拮抗环磷酰胺诱导雏鸡免疫抑制的作用[J].西南农业学报, 2022, 35(5):1222-1227.WANG J D, TANG Y L, WANG M, et al.Effect of Lycium barbarum glycopeptide against the immunosuppression induced by cyclophosphamide in chicks[J].Southwest China Journal of Agricultural Sciences, 2022, 35(5):1222-1227.(in Chinese)
[19]	AHLMANN M, HEMPEL G.The effect of cyclophosphamide on the immune system:implications for clinical cancer therapy[J].Cancer Chemother Pharmacol, 2016, 78(4):661-671.
[20]	FENG L, XIAO X, LIU J, et al.Immunomodulatory effects of Lycium barbarum polysaccharide extract and its uptake behaviors at the cellular level[J].Molecules, 2020, 25(6):1351.
[21]	PALAZON A, GOLDRATH A W, NIZET V, et al.HIF transcription factors, inflammation, and immunity[J].Immunity, 2014, 41(4):518-528.
[22]	ENGELMAN J A, LUO J, CANTLEY L C.The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[J].Nat Rev Genet, 2006, 7(8):606-619.
[23]	TANGYE S G, BIER J, LAU A, et al.Immune dysregulation and disease pathogenesis due to activating mutations in PIK3CD-the goldilocks' effect[J].J Clin Immunol, 2019, 39(2):148-158.
[24]	何丽梅, 孙立哲, 李媛媛, 等.非重型再生障碍性贫血大鼠模型的构建方法[J].甘肃科学学报, 2022, 34(1):48-53.HE L M, SUN L Z, LI Y Y, et al.Establishment method of SD rat model with non-severe aplastic anemia[J].Journal of Gansu Sciences, 2022, 34(1):48-53.(in Chinese)
[25]	马慧娅.戊二醛聚合猪血红蛋白对化疗贫血小鼠治疗作用的初步研究[D].西安:西北大学, 2019.MA H Y.Studies on the therapeutic effects of pPolyHb onchemotherapy-induced anemia mice[D].Xi'an:Northwest University, 2019.(in Chinese)
[26]	LIM H D, LANE J R, CANALS M, et al.Systematic assessment of chemokine signaling at chemokine receptors CCR4, CCR7 and CCR10[J].Int J Mol Sci, 2021, 22(8):4232.
[27]	KHOURY O, ATALA A, MURPHY S V.Stromal cells from perinatal and adult sources modulate the inflammatory immune response in vitro by decreasing Th1 cell proliferation and cytokine secretion[J].Stem Cell Transl Med, 2020, 9(1):61-73.
[28]	DONG T Y, LIU Z L, ZHAO S, et al.The expression of CD9 and PIK3CD is associated with prognosis of follicular lymphoma[J].J Cancer, 2015, 6(12):1222-1229.
[29]	TRIPATHI D N, JENA G B.Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage:a study in mice[J].Chem Biol Interact, 2009, 180(3):398-406.
[30]	ZHOU Y L, CHEN X Y, YI R K, et al.Immunomodulatory effect of tremella polysaccharides against cyclophosphamide-induced immunosuppression in mice[J].Molecules, 2018, 23(2):239.
[31]	DEMPKE W C M, UCIECHOWSKI P, FENCHEL K, et al.Targeting SHP-1, 2 and SHIP pathways:a novel strategy for cancer treatment?[J].Oncology, 2018, 95(5):257-269.
[32]	ELICH M, SAUER K.Regulation of hematopoietic cell development and function through phosphoinositides[J].Front Immunol, 2018, 9:931.