Table_9_Research on the Mechanism of HRP Relieving IPEC-J2 Cells Immunological Stress Based on Transcriptome Sequencing Analysis.XLSX

Early weaning increased the economic benefits of piglets. However, early weaning damages the intestinal barrier of piglets and causes immunological stress. The mechanism by which Hippophae rhamnoides polysaccharide (HRP) alleviates lipopolysaccharide (LPS)-induced intestinal porcine epithelial cells (IPEC-J2) inflammatory damage was investigated using proteomics in our previous studies. In this study we employed RNA-sequencing (RNA-seq) to determine the level and function of differentially expressed genes (DEGs) and further explore the mechanism of the HRP anti-inflammatory and immune process. The differential expression analysis indicated that 3622, 1216, and 2100 DEGs in the IPEC-J2 cells were identified in C vs. L, L vs. H6-L, and C vs. H6-L, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis foundsix identified pathways related to the immune system. Additionally, we used the Science, Technology, Engineering, and Math (STEM) program to categorize the 3,134 DEGs that were differentially expressed in H2-L, H4-L and H6-L into eight possible expression profiles, in which 612 were clustered into two profiles. The accuracy and consistency of RNA-seq data were validated by the results of qRT-PCR of the nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (NFKB2), MAP kinase interacting serine/threonine kinase 2 (MKNK2), mitogen-activated protein kinase kinase 1 (MAP2K1), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), Ras-related protein R-Ras (RRAS), TNF receptor-associated factor 1 (TRAF1), NF-kappa-B inhibitor alpha (NFKBIA), interleukin 8 (IL8), tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), and transforming growth factor beta-1 (TGFB1). Transcriptome sequencing also indicated that HRP reduced the expression levels of related DEGs and inhibited the activation of the mitogen-activated protein kinase (MAPK)/nuclear factor kappa-B (NF-κB) signaling pathway. Our findings indicate that the application of HRP in piglet diets during the early weaning period can improve intestinal epithelial function and integrity, and relieve intestinal damage, and improve piglet health.

早期断奶可提升仔猪的经济效益，但该手段同时会损伤仔猪肠道屏障并引发免疫应激。本团队前期已借助蛋白质组学方法，探究了沙棘多糖（Hippophae rhamnoides polysaccharide, HRP）缓解脂多糖（lipopolysaccharide, LPS）诱导的猪肠道上皮细胞（IPEC-J2）炎性损伤的作用机制。本研究采用RNA测序（RNA-sequencing, RNA-seq）技术，分析差异表达基因（differentially expressed genes, DEGs）的表达水平与功能，以进一步解析HRP发挥抗炎及免疫调节作用的分子机制。差异表达分析结果显示，在对照组（C）与脂多糖处理组（L）、脂多糖处理组（L）与高剂量HRP组（H6-L）、对照组（C）与高剂量HRP组（H6-L）的对比中，IPEC-J2细胞分别筛选出3622、1216和2100个DEGs。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析共筛选出6条与免疫系统相关的通路。此外，本研究借助科学、技术、工程与数学（Science, Technology, Engineering, and Math, STEM）程序，将H2-L、H4-L及H6-L组中筛选出的3134个DEGs划分为8种潜在表达模式，其中612个DEGs被聚类至2个表达谱中。本研究通过实时荧光定量PCR（qRT-PCR）对B细胞κ轻链基因增强子结合因子2（nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, NFKB2）、丝裂原活化蛋白激酶互作丝氨酸/苏氨酸激酶2（MAP kinase interacting serine/threonine kinase 2, MKNK2）、丝裂原活化蛋白激酶激酶1（mitogen-activated protein kinase kinase 1, MAP2K1）、丝裂原活化蛋白激酶激酶激酶8（mitogen-activated protein kinase kinase kinase 8, MAP3K8）、Ras相关蛋白R-Ras（Ras-related protein R-Ras, RRAS）、肿瘤坏死因子受体相关因子1（TNF receptor-associated factor 1, TRAF1）、NF-κB抑制蛋白α（NF-kappa-B inhibitor alpha, NFKBIA）、白细胞介素8（interleukin 8, IL8）、肿瘤坏死因子α诱导蛋白3（tumor necrosis factor alpha-induced protein 3, TNFAIP3）及转化生长因子β1（transforming growth factor beta-1, TGFB1）进行验证，结果证实了RNA-seq数据的准确性与一致性。转录组测序结果同时显示，HRP可下调相关DEGs的表达水平，并抑制丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）/核因子κB（nuclear factor kappa-B, NF-κB）信号通路的激活。本研究结果表明，在仔猪早期断奶阶段的日粮中添加HRP，可改善肠道上皮功能与屏障完整性，缓解肠道损伤，进而提升仔猪健康水平。