基于转录组测序分析枸杞多糖对免疫抑制雏鸡脾脏基因表达的影响

Effects of Lycium barbarum polysaccharide on genes expression in spleen of immunosuppressed chicks based on transcriptome sequencing

【目的】利用转录组测序技术分析枸杞多糖(LBP)对免疫抑制雏鸡脾脏基因表达的影响,挖掘LBP修复雏鸡免疫抑制的靶基因,为探究枸杞多糖对雏鸡免疫抑制的修复机制提供理论依据。【方法】将120羽7日龄海兰褐蛋鸡随机分成空白组(NC)、环磷酰胺(CTX)组(CY)和LBP组(CYLbGp),NC组肌注生理盐水,其余2组连续3 d肌注80 mg/(kg·d)CTX造模,造模后通过测定血清中IL-2、IL-6和INF-γ含量对免疫抑制雏鸡模型进行验证。造模成功后在CYLbGp组饮水中加入5 mg/(kg·d)LBP,给药结束后采集脾脏进行转录组测序,以P<0.05和|log2 Fold change|≥1为条件筛选出差异表达基因(DEGs),并进行GO功能注释分析和KEGG信号通路富集分析,以探究枸杞多糖对免疫抑制的修复作用。【结果】共获得1049301886个Raw reads,对其进行质控、过滤后,共获得1032097278个Clean reads。CY组和NC组之间共有701个DEGs,CYLbGp组和CY组之间共有748个DEGs。GO功能注释显示,LBP干预后显著回调的DEGs主要与结合、细胞过程、生物调节及细胞组分等功能相关。KEGG信号通路富集结果显示,LBP干预后显著回调的DEGs主要与神经活性配体-受体相互作用和视黄醇代谢通路相关,筛选出所富集的通路中关键的DEGs为人毒蕈碱型胆碱受体M5基因(CHRM5)、烟碱型乙酰胆碱受体β4基因(CHRNB4)、代谢型型谷氨酸受体1基因(GRM1)、胆囊收缩素基因(CCK)、神经肽Y1受体基因(NPY1R)、细胞色素P450家族成员2C18基因(CYP2C18)、血管紧张素Ⅱ受体1基因(AGTR1)、细胞色素P450家族成员3A5基因(CYP3A5)、丝氨酸蛋白酶2(PRSS2)和神经紧张素基因(NTS)。RT-qPCR结果与转录组学数据变化趋势基本一致,表明转录组数据有较高的可靠性。【结论】LBP修复雏鸡免疫抑制的作用机制可能与调节神经活性配体-受体相互作用和视黄醇代谢通路有关,其中CHRM5、CHRNB4、GRM1、CCK、NPY1R、CYP2C18、AGTR1、CYP3A5、PRSS2和NTS可作为LBP修复雏鸡免疫抑制的靶基因。

【Objective】This paper analyzed the effects of Lycium barbarum polysaccharide(LBP)on the gene expres-sion of spleen of immunosuppressed chicks via transcriptome sequencing,and explored the target genes of repair mecha-nism of LBP on immunosuppression of chicks.【Method】A total of 1207-day-old Hy-Line brown laying hens were ran-domly divided into negative control group(NC),cyclophosphamide(CTX)group(CY)and LBP group(CYLbGp).The NC group was intramuscularly injected with normal saline,while the other two groups were intramuscularly injected with 80 mg/(kg·d)CTX for 3 consecutive days for modeling.After modeling,the immunosuppressed chick model was veri-fied by measuring the levels of IL-2,IL-6 and INF-γin serum.After successful modeling,the CYLbGp group drinking water was added 5 mg/(kg·d)LBP,after the administration of CYLbGp group,spleen was collected to be used for transcriptome sequencing,diferentially expressed genes(DEGs)of P<0.05 and|log2 Fold Change|≥1 were screened,GO functional annotation analysis and KEGG pathway enrichment analysis were performed to explore the effects of LBP on immunosuppression repair.【Result】A total of 1049301886 Raw reads were obtained,and 1032097278 Clean reads were recieved after quality control and filtering.There were 701 and 748 DEGs between CY group and NC group and between CYLbGp and CY group respectively.GO functional annotation showed that the significantly returned DEGs after LBP intervention were mainly related to binding,cell process,biological regulation and cellular component functions.The KEGG pathway enrichment results showed that significantly returned DEGs after LBP intervention were mainly related to neuroactive ligand-receptor interaction and retinol metabolism pathway.The key DEGs in the enriched pathway were cholinergic receptor muscarinic 5(CHRM5),cholinergic receptor nicotinic beta 4 subunit(CHRNB4),glutamate metabotropic receptor 1(GRM1),cholecystokinin(CCK),neuropeptide Y receptor Y1(NPY1R),cytochrome P450 family 2 subfamily C member 18(CYP2C18),angiotensin II receptor type 1(AGTR1),cytochrome P450 family 3 subfamily A member 5(CYP3A5),serine protease 2(PRSS2)and neurotensin(NTS).The results of RT-qPCR were basically consistent with the trend of transcriptome data,which further indicated that transcriptome data had high reliability.【Conclusion】The repair mechanism of LBP on immunosuppression of chicks may be related to the regulation of neuroac-tive ligand-receptor interaction and retinol metabolism pathway.Among them,CHRM5,CHRNB4,GRM1,CCK,NPY1R,CYP2C18,AGTR1,CYP3A5,PRSS2and NTScan be used as target genes in the repair mechanism of LBP on immunosuppression of chicks.