姜黄素对非酒精性脂肪肝小鼠代谢组学的调控研究
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摘要
目的探讨姜黄素(CUR)对非酒精性脂肪肝病(NAFLD)的代谢调节作用。方法小鼠适应性饲养3 d后,随机分为3组:对照(C)组、高脂饮食模型(HFD)组、CUR(50 mg/kg)组。C组饲喂普通饲料,HFD和CUR组饲喂高脂饲料,CUR同时ig给药10周,C组和HFD组ig给予0.5%CMC-Na溶液,给药体积均为10 mL/kg。给药结束后,取血和肝脏,肝组织切片后进行HE染色和油红O染色,光学显微镜观察病理变化;采用基于气相色谱-质谱联用(GC/MS)的代谢组学方法检测血清和肝脏组织样品,数据通过Simca-P13.0及Metaboanalysis网络工具进行分析,找出代谢组变化趋势和相关差异化合物。此外,制备小鼠肝原代细胞,棕榈酸(PA)100 mmol/L诱导细胞NAFLD体外模型,观察CUR(10μmol/L)对代谢组变化的影响。结果油红O染色结果显示,HFD小鼠肝脏组织存在大量红染油滴,CUR组小鼠肝脏组织中的脂质油滴明显减少。HE染色分析显示,与C组比较,HFD组小鼠肝脏组织有空泡状,CUR组小鼠肝脏组织与C组比较没有明显区别。对血清样和肝组织样品的GC-MS数据进行PLS-DA分析发现C组与HFD组偏离较远,而CUR组与HFD组相比出现向C组偏移的趋势,进一步S-plot分析发现影响较大的代谢为胆固醇的合成、酮体的生成以及氨基酸代谢,细胞实验得到了类似的结果;经通路MetPA分析和代谢产物富集,血清和细胞中各组间差异化合物代谢主要集中在氨基酸代谢,肝脏中代谢差异主要集中在氨基酸代谢和部分脂质代谢通路;进一步进行差异代谢物的筛选,HFD组生糖氨基酸和生酮氨基酸较对照组均有不同程度的升高,CUR能够降低这些氨基酸的水平。结论高脂饮食会造成小鼠三大代谢的异常,姜黄素能够一定程度上改善小鼠代谢紊乱。
Objective To investigate the metabolic regulation of curcumin(CUR) on mice with nonalcoholic fatty liver disease(NAFLD). Methods After 3 days of adaptive feeding, mice were randomly divided into three groups: control group(C), high fat diet model(HFD) group and CUR(50 mg/kg) group. Group C was fed with common diet, group HFD and CRU were fed with high-fat diet, and Ig was given for 10 weeks. Group C and HFD were given 0.5% CMC-Na solution, the volume of administration was 10 mL/kg. After administration, blood and liver were taken, liver tissue sections were stained with HE and oil red O, and pathological changes were observed by optical microscopy. Serum and liver tissue samples were detected by metabolomics method based on gas chromatography-mass spectrometry(GC/MS). The data were analyzed by Simca-P13.0 and Metaboanalysis network tools to find the trend of metabolome changes and related differential compounds. In addition, primary hepatocytes of mice were prepared and NAFLD was induced by palmitic acid(PA) 100 mmol/L in vitro. The effects of CUR(10 micromol/L) on metabolites were observed. Results Oil red O staining results showed that there were a large number of red oil droplets in liver tissue of HFD mice,and lipid oil droplets in liver tissue of CUR mice were significantly reduced. HE staining analysis showed that there were vacuoles in the liver tissue of HFD mice compared with group C, and there was no significant difference between the liver tissue of CUR mice and that of group C. PLS-DA analysis of GC-MS data of serum and liver tissue samples showed that group C deviated from group HFD, while group CUR deviated from group C. Further S-plot analysis showed that cholesterol synthesis, ketone body formation and amino acid metabolism were the most influential metabolites. Similar results were obtained in cell experiments through pathway MetPA analysis and metabolite enrichment. The metabolic differences between groups in serum and cells were mainly concentrated in amino acid metabolism, while those in liver were mainly concentrated in amino acid metabolism and some lipid metabolism pathways. Further screening of different metabolites showed that the levels of sugar-producing amino acids and ketogenic amino acids in HFD group were higher than those in control group, and CUR could reduce the levels of these amino acids.Conclusion High fat diet can cause three major metabolic abnormalities in mice, curcumin can improve the metabolic disorders in mice to a certain extent.
Objective To investigate the metabolic regulation of curcumin(CUR) on mice with nonalcoholic fatty liver disease(NAFLD). Methods After 3 days of adaptive feeding, mice were randomly divided into three groups: control group(C), high fat diet model(HFD) group and CUR(50 mg/kg) group. Group C was fed with common diet, group HFD and CRU were fed with high-fat diet, and Ig was given for 10 weeks. Group C and HFD were given 0.5% CMC-Na solution, the volume of administration was 10 mL/kg. After administration, blood and liver were taken, liver tissue sections were stained with HE and oil red O, and pathological changes were observed by optical microscopy. Serum and liver tissue samples were detected by metabolomics method based on gas chromatography-mass spectrometry(GC/MS). The data were analyzed by Simca-P13.0 and Metaboanalysis network tools to find the trend of metabolome changes and related differential compounds. In addition, primary hepatocytes of mice were prepared and NAFLD was induced by palmitic acid(PA) 100 mmol/L in vitro. The effects of CUR(10 micromol/L) on metabolites were observed. Results Oil red O staining results showed that there were a large number of red oil droplets in liver tissue of HFD mice,and lipid oil droplets in liver tissue of CUR mice were significantly reduced. HE staining analysis showed that there were vacuoles in the liver tissue of HFD mice compared with group C, and there was no significant difference between the liver tissue of CUR mice and that of group C. PLS-DA analysis of GC-MS data of serum and liver tissue samples showed that group C deviated from group HFD, while group CUR deviated from group C. Further S-plot analysis showed that cholesterol synthesis, ketone body formation and amino acid metabolism were the most influential metabolites. Similar results were obtained in cell experiments through pathway MetPA analysis and metabolite enrichment. The metabolic differences between groups in serum and cells were mainly concentrated in amino acid metabolism, while those in liver were mainly concentrated in amino acid metabolism and some lipid metabolism pathways. Further screening of different metabolites showed that the levels of sugar-producing amino acids and ketogenic amino acids in HFD group were higher than those in control group, and CUR could reduce the levels of these amino acids.Conclusion High fat diet can cause three major metabolic abnormalities in mice, curcumin can improve the metabolic disorders in mice to a certain extent.
引文
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[14]Fiehn O.Metabolomics-the link between geno-types and phenoty pes[J].Plant Mol Biol,2002,48(1/2):155-171.
[15]阿基业.代谢组学数据处理方法:主成分分析[J].中国临床药理学与治疗学,2010,15(5):481-489.
[16]Wang Z X,Xu D,She L L,et al.Curcumin restrains hepatic glucose production by blocking cAMP/PKAsignaling and reducing acetyl CoA accumulation in highfat diet(HFD)-fed mice[J].Mol Cell Endocrinol,2018,474:127-136.
[17]He J,Gao H X,Yang N,et al.The aldose reductase inhibitor epalrestat exerts nephritic protection on diabetic nephropathy in db/db mice through metabolic modulation[J].Acta Pharmacol Sin,2019,40:86-97.
[18]Bugianesi E,McCullough A J,Marchesini G.Insulin resistance:a metabolic pathway to chronic liver disease[J].Hepatology,2005,42(5):987-1000.
[19]查锡良.生物化学第7版[M].北京:人民卫生出版社,2008.
[20]Adeva M M,Calvi?o J,Souto G,et al.Insulin resistance and the metabolism of branched-chain amino acids in humans[J].Amino Acids,2012,43(1):171-181.
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[22]Xiao F,Huang Z Y,Li H K,et al.Leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways[J].Diabetes,2011,60(3):746-756.
[23]宋文琪,李时莲,张美和,等.单纯性肥胖儿童血清氨基酸水平分析[J].中国儿童保健杂志,2011,19(4):324-327.
[24]Felig P.Amino acid metabolism in man[J].Annu Rev Biochem,1975,144:905-952.
[25]Sookoian S,Pirola C J.Alanine and aspartate aminotransferase and glutamine-cycling pathway:their roles in pathogenesis of metabolic syndrome[J].World JGastroenterol,2012,18(29):3775-3781.
[26]叶春,房宇.非酒精性脂肪肝大鼠血清氨基酸谱的改变及分析[J].南京师大学报:自然科学版,2016,39(1):90-94.
[27]江向洋.肠道菌群与NAFLD发生发展的关联代谢机制研究[D].杭州:浙江大学,2017.
[28]Sahebkar A.Curcuminoids for the management of hypertriglyceridaemia[J].Nat Rev Cardiol,2014,11(2):123.
[29]Sahebkar A.A systematic review and meta-analysis of randomized controlled trials investigating the effects of curcumin on blood lipid levels[J].Clin Nutr,2014,33(3):406-414.
[30]Amin F,Islam N,Anila N,et al.Clinical efficacy of the co-administration of Turmeric and Black seeds(Kalongi)in metabolic syndrome-a double blind randomized controlled trial-TAK-MetS trial[J].Complement Ther Med,2015,23(2):165-174.
[2]Neuschwander-Tetri B A,Caldwell S H.Nonalcoholic steatohepatitis:summary of an AASLD single topic conference[J].Hepatology,2003,37(5):1202-1219.
[3]Willebrords J,Pereira I V,Maes M,et al.Strategies,models and biomarkers in experimental non-alcoholic fatty liver disease research[J].Prog Lipid Res,2015,59:106-125.
[4]Li Y R,Wang J,Tang Y H,et al.Bidirectional association between nonalcoholic fatty liver disease and type 2diabetes in Chinese population:Evidence from the Dongfeng-Tongji cohort study[J].PLoS One,2017,12(3):e0174291.
[5]Mikolasevic I,Milic S,Turk Wensveen T,et al.Nonalcoholic fatty liver disease-A multisystem disease?[J].World J Gastroenterol,2016,22(43):9488-9505.
[6]Kunnumakkara A B,Bordoloi D,Padmavathi G,et al.Curcumin,the golden nutraceutical:multitargeting for multiple chronic diseases[J].Br J Pharmacol,2017,174(11):1325-1348.
[7]常明向,吴梅梅,李瀚旻.姜黄素与甘草次酸联用对肝癌HepG-2细胞增殖的抑制作用[J].药物评价研究,2017,40(1):42-47
[8]李军,熊琨,龚元,等.基于信号转导通路的姜黄素抗氧化机制研究进展[J].中草药,2016,47(13):2373-2380.
[9]Sahebkar A.Why it is necessary to translate curcumin into clinical practice for the prevention and treatment of metabolic syndrome?[J].Biofactors,2013,39(2):197-208.
[10]Rahmani S,Asgary S,Askari G,et al.Treatment of nonalcoholic fatty liver disease with curcumin:a randomized placebo-controlled trial[J].Phytother Res,2016,30(9):1540-1548.
[11]Fujiwara H,Hosokawa M,Zhou X R,et al.Curcumin inhibits glucose production in isolated mice hepatocytes[J].Diabetes Res Clin Pract,2008,80(2):185-191.
[12]Nicholson J K,Lindon J C.Systems biology:metabonomics[J].Nature,2008,455(7216):1054-1056.
[13]Holmes E,Wilson I D,Nicholson J K.Metabolic phenotyping in health and disease[J].Cell,2008,134(5):714-717.
[14]Fiehn O.Metabolomics-the link between geno-types and phenoty pes[J].Plant Mol Biol,2002,48(1/2):155-171.
[15]阿基业.代谢组学数据处理方法:主成分分析[J].中国临床药理学与治疗学,2010,15(5):481-489.
[16]Wang Z X,Xu D,She L L,et al.Curcumin restrains hepatic glucose production by blocking cAMP/PKAsignaling and reducing acetyl CoA accumulation in highfat diet(HFD)-fed mice[J].Mol Cell Endocrinol,2018,474:127-136.
[17]He J,Gao H X,Yang N,et al.The aldose reductase inhibitor epalrestat exerts nephritic protection on diabetic nephropathy in db/db mice through metabolic modulation[J].Acta Pharmacol Sin,2019,40:86-97.
[18]Bugianesi E,McCullough A J,Marchesini G.Insulin resistance:a metabolic pathway to chronic liver disease[J].Hepatology,2005,42(5):987-1000.
[19]查锡良.生物化学第7版[M].北京:人民卫生出版社,2008.
[20]Adeva M M,Calvi?o J,Souto G,et al.Insulin resistance and the metabolism of branched-chain amino acids in humans[J].Amino Acids,2012,43(1):171-181.
[21]Krebs M,Krssak M,Bernroider E,et al.Mechanism of amino acid-induced skeletal muscle insulin resistance in humans[J].Diabetes,2002,51(3):599-605.
[22]Xiao F,Huang Z Y,Li H K,et al.Leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways[J].Diabetes,2011,60(3):746-756.
[23]宋文琪,李时莲,张美和,等.单纯性肥胖儿童血清氨基酸水平分析[J].中国儿童保健杂志,2011,19(4):324-327.
[24]Felig P.Amino acid metabolism in man[J].Annu Rev Biochem,1975,144:905-952.
[25]Sookoian S,Pirola C J.Alanine and aspartate aminotransferase and glutamine-cycling pathway:their roles in pathogenesis of metabolic syndrome[J].World JGastroenterol,2012,18(29):3775-3781.
[26]叶春,房宇.非酒精性脂肪肝大鼠血清氨基酸谱的改变及分析[J].南京师大学报:自然科学版,2016,39(1):90-94.
[27]江向洋.肠道菌群与NAFLD发生发展的关联代谢机制研究[D].杭州:浙江大学,2017.
[28]Sahebkar A.Curcuminoids for the management of hypertriglyceridaemia[J].Nat Rev Cardiol,2014,11(2):123.
[29]Sahebkar A.A systematic review and meta-analysis of randomized controlled trials investigating the effects of curcumin on blood lipid levels[J].Clin Nutr,2014,33(3):406-414.
[30]Amin F,Islam N,Anila N,et al.Clinical efficacy of the co-administration of Turmeric and Black seeds(Kalongi)in metabolic syndrome-a double blind randomized controlled trial-TAK-MetS trial[J].Complement Ther Med,2015,23(2):165-174.