高投饲率对圆口铜鱼幼鱼肝脏抗氧化、免疫功能和脂质代谢的影响

Effects of High Feeding Rate on Hepatic Antioxidant,Immune Function and Lipid Metabolism in Juvenile Largemouth Bronze Gudgeon(Coreius guichenoti)

本试验通过研究高投饲率对圆口铜鱼(Coreius guichenoti)幼鱼肝脏抗氧化、免疫功能和脂质代谢的影响,来探究高投饲率导致圆口铜鱼脂肪肝形成的原因。设置2%(对照组)和5%(高投饲率组)2个投饲率,每组设3个重复,每个重复投放30尾圆口铜鱼,养殖期为8周。结果显示:与对照组相比,高投饲率显著提高圆口铜鱼幼鱼肝脏甘油三酯(TG)、总胆固醇(TC)、游离脂肪酸(NEFA)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(AKP)和总胆汁酸(TBA)含量/活性(P<0.05);高投饲率显著提高圆口铜鱼幼鱼肝脏活性氧(ROS)、还原性谷胱甘肽(GSH)和丙二醛(MDA)含量以及总抗氧化能力(T-AOC)(P<0.05),显著降低过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性(P<0.05),且显著升高免疫球蛋白G(IgG)、免疫球蛋白M(IgM)含量与溶菌酶(LYS)活性(P<0.05),显著上调促炎因子基因肿瘤坏死因子α(TNFα)和白细胞介素1β(IL1β)的表达(P<0.05)。肝脏转录组测序结果显示,脂质代谢相关的过氧化物酶体增殖物激活受体(PPAR)信号通路、甘油酯代谢、脂肪酸生物合成和类固醇生物合成通路与脂肪肝的形成密切相关。对与脂质代谢相关的差异表达基因(DEGs)进行分析发现,脂肪肝中脂合成相关基因甘油-3-磷酸酰基转移酶3(GPAT3)、脂素1(LPIN1)、磷脂酸磷酸酶1(PLPP1)、脂肪酸合成酶(FASN)、乙酰辅酶A羧化酶1(ACC1)和线粒体酰基载体蛋白(ACPM)显著上调,脂解相关基因含Patatin样磷脂酶域包含蛋白2(PNPLA2)、单甘油酯脂肪酶(MGLL)、脂蛋白脂肪酶(LPL)、二酰基甘油激酶β(DGKB)和长链脂肪酸乙酰辅酶A连接酶6(ACSL6)显著下调;此外,脂质代谢相关的差异表达转录因子芳香烃受体1(AHR1)和核受体亚家族B2(NR0B2)与脂合成相关基因呈正相关,与脂解相关基因呈负相关,而核受体亚家族D1(NR1D1)、Krueppel样因子9(KLF9)和Krueppel样因子11(KLF11)与脂合成相关基因呈负相关,与脂解相关基因呈正相关。以上结果表明,高投饲率引起圆口铜鱼幼鱼肝脏脂肪合成的增加、脂肪分解的降低,从而导致肝脏脂肪蓄积,最终诱导脂肪肝的形成,并引发氧化应激和炎症反应,加剧肝脏的损伤。

In order to investigate the causes of fatty liver formation in juvenile largemouth bronze gudgeon(Coreius guichenoti),this experiment was conducted to investigate the effects of high feeding rate on hepatic antioxidant,immune function and lipid metabolism of largemouth bronze gudgeon.Two feeding rates of 2%(control group)and 5%(high feeding rate group)were set up,with 3 replicates in each group and 30 fish in each replicate.The culture period was 8 weeks.The results showed that,compared with the control group,the high feeding rate significantly increased the hepatic triglyceride(TG),total cholesterol(TC),non-esterified fatty acid(NEFA),alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phospha-tase(AKP)and total bile acid(TBA)contents/activities(P<0.05);the high feeding rate also significantly increased the hepatic reactive oxygen species(ROS),reducing glutathione(GSH),malondialdehyde(MDA)contents and total antioxidant capacity(T-AOC)(P<0.05),significantly decreased catalase(CAT)superoxide dismutase(SOD)activities(P<0.05),significantly increased immunoglobulin G(IgG),immu-noglobulin M(IgM)contents and lysozyme(LYS)activity(P<0.05),and significantly up-regulated the ex-pression of pro-inflammatory factor genes tumor necrosis factorα(TNFα)and interleukin 1β(IL1β)(P<0.05).The results of liver RNA-seq revealed that fatty liver formation was closely associated with lipid metab-olism,in particular the peroxisome proliferator-activated receptor(PPAR)signaling pathway,glycerolipid me-tabolism,fatty acid biosynthesis,and steroid biosynthesis pathway.Moreover,the analysis of differentially ex-pressed genes(DEGs)related to lipid metabolism showed that the lipid synthesis-related genes glycerol-3-phosphate acyltransferase 3(GPAT3),lipin 1(LPIN1),phospholipid phosphatase 1(PLPP1),fatty acid synthase(FASN),acetyl-CoA carboxylase 1(ACC1)and mitochondrial acyl carrier protein(ACPM)were significantly up-regulated(P<0.05),while the lipolysis-related genes Patatin-like phospholipase domain-con-taining protein 2(PNPLA2),monotriglyceride lipase(MGLL),lipoprotein lipase(LPL),diacylglycerol ki-naseβ(DGKB)and long-chain fatty acid acetyl-CoA ligase 6(ACSL6)were significantly down-regulated in fatty liver(P<0.05).In addition,differentially expressed transcription factors related to lipid metabolism,aryl hydrocarbon receptor 1(AHR1)and nuclear receptor subfamily 0 group b member 2(NR0B2)were positively correlated with lipogenesis related genes and negatively correlated with lipolysis related genes,while nuclear re-ceptor subfamily 1 group D member 1(NR1D1),Krueppel transcription factor 9(KLF9)and Krueppel tran-scription factor 11(KLF11)were negatively correlated with lipogenesis related genes and positively correlated with lipolysis related genes.The above results indicate that high feeding rate can accelerate liver fat accumula-tion by increasing lipid synthesis and decreasing lipolysis,and finally induce the formation of fatty liver in ju-venile largemouth bronze gudgeon,which may induce oxidative stress and inflammatory responses to aggravate liver damage.