丹参多糖抑制氟苯尼考致肉鸡肝脏损伤的分子机制研究

Study on the Molecular Mechanism of Salvia miltiorrhiza Polysaccharide in Inhibiting Florfenicol-induced Liver Injury in Broilers

本试验旨在探究丹参多糖缓解氟苯尼考致肉鸡肝脏损伤的转录组学靶点与通路。将30只1日龄肉鸡随机平均分成3组:空白对照组(LA组)饲喂自来水、氟苯尼考造模组(LB组)给予含0.15 g/L氟苯尼考的自来水、丹参多糖治疗组(LC组)给予含0.15 g/L氟苯尼考和5 g/L丹参多糖的自来水。从1日龄开始,连续给药5 d。于第6天各组分别随机取3只鸡,处死,无菌摘取其肝脏组织。肝脏组织经过RNA抽提、纯化和建库后,采用IlluminaHiSeq测序平台,对这些文库进行双末端测序。然后对原始下机数据进行过滤,将过滤后得到的高质量序列比对到该物种的参考基因组上。根据比对结果,计算每个基因的表达量。分别构建3组肉鸡肝脏的数字化基因表达谱(DGE)文库,比较DGE文库的差异表达基因,寻找基因表达差异最显著的关键基因,并进行GO功能注释、KEGG通路分析。结果显示:LA vs LB、LA vs LC及LB vs LC比较组分别筛选出1989(上调基因495个,下调基因1494个)、1278(上调基因344个,下调基因934个)、380个(上调基因165个,下调基因215个)表达差异显著的基因。LA vs LB、LA vs LC及LB vs LC比较组差异表达基因的GO功能注释主要归类为生物过程和细胞组成两个分支。KEGG通路分析结果表明,LA vs LB的差异表达的基因主要富集到药物代谢-细胞色素P450,细胞外基质(ECM)受体相互作用、细胞因子与细胞因子受体的相互作用、细胞色素P450对异生物的代谢,谷胱甘肽代谢,过氧化物酶体增殖物激活受体(PPAR)信号通路,甘氨酸、丝氨酸和苏氨酸代谢,丙氨酸、天冬氨酸和谷氨酸代谢等通路。LB vs LC差异表达的基因主要富集到药物代谢-细胞色素P450和甘氨酸、丝氨酸和苏氨酸代谢及PPAR信号通路等。丹参多糖可通过调节与细胞色素P450代谢通路有关的细胞色素P4503A4酶(CYP3A4)、细胞色素P4502C23b酶(CYP2C23b)、谷胱甘肽S-转移酶(GSTA3)、类谷胱甘肽S-转移酶α2(GSTAL2)等mRNA的表达以及与PPAR信号通路有关的脂肪酸结合蛋白1(FABP1)和长链脂肪酸辅酶A连接酶(ACSBG2)等mRNA的表达,从而抑制氟苯尼考产生的有害代谢产物,促进肝脏脂质的正常代谢,进而缓解氟苯尼考引起的肝脏损伤。

This experiment was conducted to explore transcriptome targets and pathways of Salvia miltiorrhiza polysaccharides in alleviating liver injury induced by flufenicol in broilers.Thirty one-day-old broilers were randomly divided into three groups:blank control group(LA group)were fed tap water,florfenicol model group(LB group)was given tap water containing 0.15 g/L florfenicol,and Salvia miltiorrhiza polysaccharides treatment group(LC group)was given tap water containing 0.15 g/L florfenicol and 5 g/L Salvia miltiorrhiza polysaccharides tap water.The drug was administered continuously for 5 days from the age of 1 day.On day 6,three chickens were randomly selected from each group and killed,and their liver tissues were aseptically extracted.After RNA extraction,purification and library construction of liver tissues,the Illumina HiSeq libraries were sequenced using the Illumina HiSeq platform.Then the original offline data was filtered and the high-quality sequences obtained after filtering were aligned to the reference genome of the species.The expression level of each gene was calculated according to the comparison results.3 groups chicken liver digital gene expression patterns(DGE)library were built respectively,the DGE library of differentially expressed genes were compared,looking for the key genes with most significant difference in gene expression,and GO function annotation,KEGG pathway were analyzed.The results showed that 1989(495 up-regulated,1494 down-regulated),1278(344 up-regulated,934 down-regulated)and 380 genes(165 up-regulated,215 down-regulated)were screened out in the comparison groups of LA vs LB,LA vs LC and LB vs LC,respectively.The GO functional annotations of LA vs LB,LA vs LC and LB vs LC comparison groups were mainly classified into two branches:biological process and cell composition.The results of the KEGG pathway analysis showed that differentially expressed genes of LA vs LB were mainly enriched in drug metabolism-cytochrome P450,extracellular matrix receptor interaction,cytokine-cytokine receptor interaction,metabolism of xenobiotics by cytochrome P450,glutathione metabolism,peroxidase body growth activated receptor(PPAR)signaling pathway,glycine,serine and threonine metabolism,alanine,aspartate and glutamate metabolism pathways.The genes differentially expressed in LB vs LC were mainly concentrated in drug metabolism-cytochrome P450 and glycine,serine and threonine metabolism,PPAR signaling pathway.Salvia miltiorrhiza polysaccharides could be adjusted by the cytochrome P450 metabolic pathways related cytochrome P4503A4 enzyme(CYP3A4),cytochrome P4502C23b enzyme(CYP2C23b),glutathione S transferase(GSTA3),kind of glutathione S transferase alpha 2(GSTAL2)mRNA expression and fatty acid binding protein 1(FABP1)associated with PPAR signaling pathways and ACSBG2 coa ligase(long chain fatty acids)mRNA expression,thus inhibiting fluorobenzene nicol to produce harmful metabolites,promoting the normal metabolism of liver lipids and alleviating the liver injury caused by flufenicol.