丹苘软胶囊干预大鼠非酒精性脂肪肝细胞模型基因芯片的组学分析
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摘要
目的:通过筛选mRNA(Clariom~(TM) S Assay)芯片及miRNA芯片的显著差异性基因,预测microRNA相关靶基因,探究丹苘软胶囊改善肝细胞脂肪代谢的调控机制。方法:提取丹苘软胶囊灌胃SD大鼠的含药血清,并对BRL大鼠肝细胞取对数生长期细胞进行实验,以阴性对照血清做对照,提取细胞总RNAs,采用Clariom S表达谱芯片及microRNA芯片检测细胞mRNA及miRNA的表达水平。运用生物信息学方法预测miRNA的靶基因,对脂肪代谢相关基因进行富集分析和通路分析。结果:经筛选和预测得到5629个预测靶基因参与20种生物学过程,7种细胞成分,有20种分子作用,神经营养素信号通路、丙型肝炎、PI3K-Akt信号通路被富集;rno-miR-26b-5p、rno-miR-129b-5p、rno-miR-21b-5p为调控网络中心;IPA数据库共检出31个与实验有关的脂肪代谢基因。结论:丹苘软胶囊在治疗非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)的机制可能与rno-miR-26b-5p、rno-miR-129b-5p、rno-miR-21b-5p,神经营养素信号通路、丙型肝炎、PI3K-Akt信号通路相关性较高。
Objective: To predict and investigate the regulatory mechanism of Danqing Soft Capsule improving fat metabolism of hepatocyte based on screening the difference between mRNA(Clariom~(TM) S Assay)and miRNA expression. Methods:We extracted blood-serum of the SD rats which received intragastric administration of Danqing Soft Capsule. BRL rat hepatocytes extracted logarithmic growth phase cells to perform experiments. Then Danqing Soft Capsule serum was used as experimental group and negative control serum was used as control group, respectively.Total RNAs were extracted and differently expressed miRNAs were detected by Clariom S expression profile chip for expressions level of mRNA and miRNA.Bioinformatics method was used to predict target genes of differential miRNAs regulation. Enrichment of functions and signaling pathways of the target genes was conducted by fat metabolism related genes. Result: In screening and predicting,5629 target genes were involved in 19 kinds of biological processes,11 kinds of cellular components,and 10 kinds of molecular functions.Neurotrophin signaling pathway, Hepatitis C and PI3 K-Akt signaling pathway were enriched.The rno-miR-26 b-5 p, rno-miR-129 b-5 p, rno-miR-21 b-5 p were the center of the molecular network. According to IPA database, 31 genes with fat metabolism effective interaction were screened out. Conclusion: The results shows rno-miR-26 b-5 p, rno-miR-129 b-5 p, rno-miR-21 b-5 p and neurotrophin signaling pathway. Hepatitis C and PI3 K-Akt signaling pathway may play a role in the differentiation process of non-alcoholic fatty liver induced by Danqing Soft Capsule.
Objective: To predict and investigate the regulatory mechanism of Danqing Soft Capsule improving fat metabolism of hepatocyte based on screening the difference between mRNA(Clariom~(TM) S Assay)and miRNA expression. Methods:We extracted blood-serum of the SD rats which received intragastric administration of Danqing Soft Capsule. BRL rat hepatocytes extracted logarithmic growth phase cells to perform experiments. Then Danqing Soft Capsule serum was used as experimental group and negative control serum was used as control group, respectively.Total RNAs were extracted and differently expressed miRNAs were detected by Clariom S expression profile chip for expressions level of mRNA and miRNA.Bioinformatics method was used to predict target genes of differential miRNAs regulation. Enrichment of functions and signaling pathways of the target genes was conducted by fat metabolism related genes. Result: In screening and predicting,5629 target genes were involved in 19 kinds of biological processes,11 kinds of cellular components,and 10 kinds of molecular functions.Neurotrophin signaling pathway, Hepatitis C and PI3 K-Akt signaling pathway were enriched.The rno-miR-26 b-5 p, rno-miR-129 b-5 p, rno-miR-21 b-5 p were the center of the molecular network. According to IPA database, 31 genes with fat metabolism effective interaction were screened out. Conclusion: The results shows rno-miR-26 b-5 p, rno-miR-129 b-5 p, rno-miR-21 b-5 p and neurotrophin signaling pathway. Hepatitis C and PI3 K-Akt signaling pathway may play a role in the differentiation process of non-alcoholic fatty liver induced by Danqing Soft Capsule.
引文
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[13] 蒲鹏.野黄芩苷对代谢综合征的防治及作用机制[D].武汉:武汉大学,2013.
[14] Parrales A,Iwakuma T.p53 as a Regulator of Lipid Metabolism in Cancer[J].International Journal of Molecular Sciences,2016,17(12).
[15] Han JW,Zhan XR,Li XY,et al.Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats[J].World Journal of Gastroenterology,2010,16(48):6111.
[16] Prokesch A,Graef FA,Madl T,et al.Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis[J].Faseb Journal,2017,31(2):732-742.
[17] Han C,Wei S,He F,et al.The regulation of lipid deposition by insulin in goose liver cells is mediated by the PI3K-AKT-mTOR signaling pathway[J].PloS one,2015,10(5):e0098759.
[18] PLOS Genetics Staff.Correction:Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism[J].PLoS genetics,2015,11(3):e1005055.
[19] Roe B,Kensicki E,Mohney R,et al.Metabolomic profile of hepatitis C virus-infected hepatocytes[J].Plos One,2011,6(8):e23641.
[20] McRae S,Iqbal J,Sarkar-Dutta M,et al.The hepatitis C virus-induced NLRP3 inflammasome activates the sterol regulatory element-binding protein (SREBP) and regulates lipid metabolism[J].Journal of Biological Chemistry,2016,291(7):3254-3267.
[21] Gurcel L,Abrami L,Girardin S,et al.Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival[J].Cell,2006,126(6):1135-1145.
[22] Pham DD,Do HT,Bruelle C,et al.p75 neurotrophin receptor signaling activates sterol regulatory element-binding protein-2 in hepatocyte cells via p38 mitogen-activated protein kinase and caspase-3[J].Journal of Biological Chemistry,2016,291(20):jbc.M116.722272.
[23] Baezaraja B,Sachs BD,Li P,et al.p75 Neurotrophin Receptor Regulates Energy Balance in Obesity[J].Cell Reports,2016,14(2):255-268.
[24] Genzer Y,Chapnik N,Froy O.Effect of brain-derived neurotrophic factor (BDNF) on hepatocyte metabolism[J].International Journal of Biochemistry & Cell Biology,2017,88(5):69-74.
[2] 刘锐,李劲平,伍娟娟,等.丹苘软胶囊对非酒精性脂肪肝模型大鼠肝脏SOCS-3 mRNA表达的影响[J].中华中医药学刊,2012,30(1):191-193.
[3] 刘锐,李劲平,伍娟娟,等.丹苘软胶囊对非酒精性脂肪肝模型大鼠胰岛素抵抗及瘦素抵抗的影响[J].中国实验方剂学杂志,2012,18(1):188-191.
[4] 谭朝晖,邹立华,刘荣火,等.丹参对非酒精性脂肪性肝炎大鼠脂质代谢及脂肪细胞因子影响[J].辽宁中医药大学学报,2016(9):40-42.
[5] 张乃键.miR-26b影响脂肪细胞胰岛素敏感性的机制分析[D].蚌埠:蚌埠医学院,2015.
[6] 杨蕾,徐广峰,季晨博,等.hsa-miR-26b表达沉默对脂肪细胞蛋白分泌的影响[J].江苏医药,2014,40(13):1489-1491.
[7] Zanchi C,Macconi D,Trionfini P,et al.MicroRNA-184 is a downstream effector of albuminuria driving renal fibrosis in rats with diabetic nephropathy[J].Diabetologia,2017,60(6):1114-1125.
[8] 凌宏艳.3T3-L1脂肪细胞胰岛素抵抗相关miRNAs的筛选及功能研究[D].长沙:中南大学,2009.
[9] Song C,Yao J,Cao C,et al.PPARγ is regulated by miR-27b-3p negatively and plays an important role in porcine oocyte maturation[J].Biochem Biophys Res Commun,2016,479(2):224-230.
[10] Kim KH,Song MJ,Yoo EJ,et al.Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c[J].Journal of Biological Chemistry,2004,279(50):51999-52006.
[11] 刘锐,李劲平,伍娟娟,等.丹苘软胶囊对NAFLD模型大鼠肝脏ABCA1蛋白表达的影响[J].中医学报,2011,26(9):1062-1064.
[12] Martel C,Allouche M,Esposti DD,et al.Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation[J].Hepatology,2013,57(1):93-102.
[13] 蒲鹏.野黄芩苷对代谢综合征的防治及作用机制[D].武汉:武汉大学,2013.
[14] Parrales A,Iwakuma T.p53 as a Regulator of Lipid Metabolism in Cancer[J].International Journal of Molecular Sciences,2016,17(12).
[15] Han JW,Zhan XR,Li XY,et al.Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats[J].World Journal of Gastroenterology,2010,16(48):6111.
[16] Prokesch A,Graef FA,Madl T,et al.Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis[J].Faseb Journal,2017,31(2):732-742.
[17] Han C,Wei S,He F,et al.The regulation of lipid deposition by insulin in goose liver cells is mediated by the PI3K-AKT-mTOR signaling pathway[J].PloS one,2015,10(5):e0098759.
[18] PLOS Genetics Staff.Correction:Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism[J].PLoS genetics,2015,11(3):e1005055.
[19] Roe B,Kensicki E,Mohney R,et al.Metabolomic profile of hepatitis C virus-infected hepatocytes[J].Plos One,2011,6(8):e23641.
[20] McRae S,Iqbal J,Sarkar-Dutta M,et al.The hepatitis C virus-induced NLRP3 inflammasome activates the sterol regulatory element-binding protein (SREBP) and regulates lipid metabolism[J].Journal of Biological Chemistry,2016,291(7):3254-3267.
[21] Gurcel L,Abrami L,Girardin S,et al.Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival[J].Cell,2006,126(6):1135-1145.
[22] Pham DD,Do HT,Bruelle C,et al.p75 neurotrophin receptor signaling activates sterol regulatory element-binding protein-2 in hepatocyte cells via p38 mitogen-activated protein kinase and caspase-3[J].Journal of Biological Chemistry,2016,291(20):jbc.M116.722272.
[23] Baezaraja B,Sachs BD,Li P,et al.p75 Neurotrophin Receptor Regulates Energy Balance in Obesity[J].Cell Reports,2016,14(2):255-268.
[24] Genzer Y,Chapnik N,Froy O.Effect of brain-derived neurotrophic factor (BDNF) on hepatocyte metabolism[J].International Journal of Biochemistry & Cell Biology,2017,88(5):69-74.