风柜斗草提取物通过调控脂质代谢改善小鼠肝脂质沉积症

doi:10.11843/j.issn.0366-6964.2022.03.025

摘要/Abstract

摘要： 旨在探究风柜斗草提取物(Sarcopyramis napalensis Wall extract,SNE)对蛋氨酸胆碱缺乏(MCD)饮食诱导的肝脏脂质沉积的作用与机制。本研究将60只雄性C57BL/6小鼠随机分为6组,每组10只,设置MCS组(MCD饲料的对照饲料组)、MCS+风柜斗草提取物组(400 mg·(kg·bw)^-1)、MCD组、MCD+风柜斗草提取物低、中、高剂量组(100、200、400 mg·(kg·bw)^-1),造模及药物干预6周。进行肝组织切片HE染色、天狼猩红染色,检测血清碱性磷酸酶(AKP)、谷草转氨酶(AST)、谷丙转氨酶(ALT),肝组织谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活力水平及丙二醛(MDA)含量以评估肝脂质沉积、肝纤维化以及肝抗氧化情况。使用Western blot技术检测腺苷酸激活蛋白激酶(AMPK)蛋白的激活情况,使用qRT-PCR技术检测固醇调节元件结合蛋白-1C (SREBP-1c)、肝型脂肪酸结合蛋白(L-FABP)、脂肪酸转运蛋白-3(FATP-3)、脂肪酸转运蛋白-4(FATP-4)、细胞间黏附分子-l (ICAM-1)、血管细胞黏附分子-l (VCAM-1)、基质金属蛋白酶-2(MMP-2)、组织金属蛋白酶抑制剂-2(TIMP-2) mRNA表达水平,同时采用LC-MS/MS技术进行肝组织非靶向脂质组学分析。结果表明,给予200、400 mg·(kg·bw)^-1剂量的风柜斗草提取物时均可显著改善MCD饲料饲喂的小鼠肝脂肪变性,减少炎症簇以及肝纤维化程度。给予风柜斗草提取物100、200、400 mg·(kg·bw)^-1时均可显著降低MCD饲料饲喂的小鼠血清中ALT和AKP的活力以及肝组织中MDA的含量,显著提高小鼠肝中GSH-Px的活力。Western blot及qRT-PCR检测结果显示,风柜斗草提取物显著激活MCD饲料饲喂的小鼠肝组织AMPK;显著抑制MCD饲料饲喂的小鼠肝组织SREBP-1c、L-FABP、FATP-3、FATP-4、ICAM-1、VCAM-1、MMP-2、TIMP-2的mRNA表达水平。肝脂质组学检测结果显示,风柜斗草提取物显著降低MCD饲料饲喂的小鼠肝中甘油三酯(TG)、磷脂酰甘油(PG)以及神经酰胺(Cer)的相对含量;显著提高鞘磷脂(SM)以及辅酶Q9相对含量。结果提示,风柜斗草提取物可通过AMPK/SREBP-1c途径抑制TG合成,以及通过抑制FATP与L-FABP表达来减少肝对脂肪酸的摄取,从而改善MCD饲料饲喂的小鼠肝脂肪变性;通过提高MCD饲料饲喂的小鼠肝组织中SM和辅酶Q9相对含量以及降低Cer相对含量,改善肝细胞氧化应激损伤情况,进而改善MCD饮食诱导的小鼠肝炎症和纤维化,最终达到改善肝脂质沉积症的作用。

关键词: 风柜斗草, 肝脂质沉积症, 小鼠

Abstract: The study aimed to investigate the effect of Sarcopyramis napalensis Wall extract (SNE) on hepatic lipidosis induced by methionine-choline-deficient (MCD) diet and its underlying mechanisms. Sixty male mice were divided into 6 groups, namely MCS (control diet of MCD diet) group, MCS+400 mg·(kg·bw)^-1 SNE group, MCD group, MCD+100 mg·(kg·bw)^-1 SNE group, MCD + 200 mg·(kg·bw)^-1 SNE group, MCD + 400 mg·(kg·bw)^-1 SNE group. Mice were fed with MCD diet and treated with SNE simultaneously for 6 weeks. The levels of hepatic lipidosis, liver fibrosis and the abilities of anti-oxidation were determined by HE and sirius red staining of liver section, and by the analysis of serum alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA). The activation of adenine monophosphate activated protein kinase (AMPK) was determined by Western blot. The mRNA expression levels of sterol regulatory element binding protein-1C (SREBP-1c), liver-type fatty acid-binding protein (L-FABP), fatty acid transport protein-3 (FATP-3), fatty acid transport protein-4 (FATP-4), intercellular adhesion molecule-l (ICAM-1), vascular cell adhesion molecule-l (VCAM-1), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2) were determined by real-time fluorescence quantitative PCR. Hepatic lipid metabolomics were conducted by LC-MS/MS. The results showed that 200 and 400 mg·(kg·bw)^-1 of SNE could significantly improve hepatic steatosis, reduce liver inflammation and liver fibrosis in MCD diet-fed mice. 100, 200, 400 mg·(kg·bw)^-1 of SNE could significantly reduce the activities of serum ALT and AKP and the levels of hepatic MDA, and significantly increase the activities of hepatic GSH-Px in MCD diet-fed mice. The results of Western blot and real-time fluorescence quantitative PCR showed that SNE significantly activated AMPK in liver tissue of MCD diet-fed mice, and significantly inhibited the mRNA expression levels of SREBP-1c, L-FABP, FATP-3, FATP-4, ICAM-1, VCAM-1, MMP-2, and TIMP-2. The results of hepatic lipid metabolomics showed that SNE significantly decreased the contents of triglyceride (TG), phosphatidylglycerol (PG) and ceramide (Cer), and increased the contents of sphingomyelin (SM) and coenzyme Q9 in MCD diet-fed mice. These results indicate that SNE can reduce the synthesis of hepatic TG by modulating AMPK/SREBP-1c pathway, and can also reduce the intake of fatty acids in the liver by inhibiting the expression of FATP and L-FABP mRNA, thus improve hepatic steatosis of MCD-diet fed mice. Moreover, SNE can attenuate the oxidative stress through increasing the content of SM and coenzymes Q9 and decreasing the content of Cer in liver tissue of MCD-diet fed mice, thus improves liver inflammation and fibrosis. Taken together, SNE therefore exerts the protective effect on hepatic lipidosis.

Key words: Sarcopyramis napalensis Wall, hepatic lipidosis, mice

中图分类号:

S856.5

王玥文, 蔡凤迎, 薛凯航, 华宝玉, 郭昌, 李焰, 邱龙新. 风柜斗草提取物通过调控脂质代谢改善小鼠肝脂质沉积症[J]. 畜牧兽医学报, 2022, 53(3): 925-937.

WANG Yuewen, CAI Fengying, XUE Kaihang, HUA Baoyu, GUO Chang, LI Yan, QIU Longxin. Sarcopyramis napalensis Wall Extract Improves Hepatic Lipidosis in Mice through Regulating Lipid Metabolism[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 925-937.

参考文献 40

[1]	WEBB C B.Hepatic lipidosis:clinical review drawn from collective effort[J].J Feline Med Surg,2018,20(3):217-227.
[2]	CENTER S A.Feline hepatic lipidosis[J].Vet Clin North Am Small Anim Pract,2005,35(1):225-269.
[3]	VALTOLINA C,FAVIER R P.Feline hepatic lipidosis[J].Vet Clin North Am Small Anim Pract,2017,47(3):683-702.
[4]	林晓兰,郑涛,何雪娇,等.习用风柜斗草研究进展[J].福建热作科技,2020,45(4):63-66.LIN X L,ZHENG T,HE X J,et al.Study progress of sarcopyramis nepalensis wall,snepalensis wall[J].Fujian Science&Technology of Tropical Crops,2020,45(4):63-66.(in Chinese)
[5]	GUO J R,ZHANG J W,YAO G M,et al.Hepatoprotective activity of the ethanol extract of Sarcopyramis nepalensis[J].J Huazhong Univ Sci Technol Med Sci,2012,32(6):844-848.
[6]	郑晓艳,陈育青.风柜斗草黄酮治疗大鼠非酒精性脂肪肝的研究[J].江西中医药大学学报,2016,28(2):84-87.ZHENG X Y,CHEN Y Q.Study of flavonoids from sarcopyramis nepalensis wall for treating non-alcoholic fatty liver disease[J].Journal of Jiangxi University of Traditional Chinese Medicine,2016,28(2):84-87.(in Chinese)
[7]	朱文俊,陈兴勇,刘乐,等.番鸭产蛋各期肝脂肪酸组成及AMPK信号通路基因表达研究[J].畜牧兽医学报,2021, 52(9):2429-2438.ZHU W J,CHEN X Y,LIU L,et al.Fatty acid composition and gene expression of AMPK signaling pathway in liver of muscovy duck at different egg-laying stages[J].Acta Veterinaria et Zootechnica Sinica,2021,52(9):2429-2438.(in Chinese)
[8]	BOUDABA N,MARION A,HUET C,et al.AMPK re-activation suppresses hepatic steatosis but its downregulation does not promote fatty liver development[J].EBioMedicine,2018,28:194-209.
[9]	HUANG H,LEE S H,SOUSA-LIMA I,et al.Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition[J].J Clin Invest,2018,128(12):5335-5350.
[10]	GARCIA D,HELLBERG K,CHAIX A,et al.Genetic liver-specific AMPK activation protects against diet-induced obesity and NAFLD[J].Cell Rep,2019,26(1):192-208.E6.
[11]	LI C Z,DONG X,DU W J,et al.LKB1-AMPK axis negatively regulates ferroptosis by inhibiting fatty acid synthesis[J].Signal Transduct Target Ther,2020,5(1):187.
[12]	KOHJIMA M,HIGUCHI N,KATO M,et al.SREBP-1c,regulated by the insulin and AMPK signaling pathways,plays a role in nonalcoholic fatty liver disease[J].Int J Mol Med,2008,21(4):507-511.
[13]	段素素,雷娜,吴海妹,等.脂质代谢调控机制及相应药物研究进展[J].山东医药,2021,61(20):93-96.DUAN S S,LEI N,WU H M,et al.Research progress on regulation mechanism of lipid metabolism and related drugs[J].Shandong Medical Journal,2021,61(20):93-96.(in Chinese)
[14]	FANG D L,WAN Y,SHEN W,et al.Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells[J].Mol Cell Biochem,2013,381(1-2):127-137.
[15]	MOSLEHI A,HAMIDI-ZAD Z.Role of SREBPs in liver diseases:a mini-review[J].J Clin Transl Hepatol,2018,6(3):332-338.
[16]	CHEN Q,WANG T T,LI J,et al.Effects of natural products on fructose-induced nonalcoholic fatty liver disease (NAFLD)[J]. Nutrients,2017,9(2):96.
[17]	IPSEN D H,LYKKESFELDT J,TVEDEN-NYBORG P.Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease[J].Cell Mol Life Sci,2018,75(18):3313-3327.
[18]	魏苏宁,苏雪莹,徐国恒.肝细胞甘油三酯代谢途径异常与脂肪肝[J].中国生物化学与分子生物学报,2016,32(2):123-132.WEI S N,SU X Y,XU G H.Anomaly of triglyceride metabolism in liver lead to NAFLD[J].Chinese Journal of Biochemistry and Molecular Biology,2016,32(2):123-132.(in Chinese)
[19]	RUI L Y.Energy metabolism in the liver[J].Compr Physiol,2014,4:177-197.
[20]	张震,富文俊,邢宇锋,等.L-FABP/PPARα信号通路与非酒精性脂肪性肝炎关系的研究进展[J].山东医药,2016,56(8):98-100.ZHANG Z,FU W J,XING Y F,et al.Research progress in the relationship between L-FABP/PPARα signaling pathway and nonalcoholic steatohepatitis[J].Shandong Medical Journal,2016,56(8):98-100.(in Chinese)
[21]	NEWBERRY E P,XIE Y,KENNEDY S,et al.Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene[J].J Biol Chem,2003,278(51):51664-51672.
[22]	NEWBERRY E P,XIE Y,KENNEDY S M,et al.Protection against Western diet-induced obesity and hepatic steatosis in liver fatty acid-binding protein knockout mice[J].Hepatology,2006,44(5):1191-1205.
[23]	PEREZ V M,GABELL J,BEHRENS M,et al.Deletion of fatty acid transport protein 2(FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARα-regulated genes[J].J Biol Chem,2020,295(17):5737-5750.
[24]	KAZANTZIS M,STAHL A.Fatty acid transport proteins,implications in physiology and disease[J].Biochim Biophys Acta,2012, 1821(5):852-857.
[25]	冯爱娟,陈东风,樊丽琳,等.FATP4在大鼠非酒精性脂肪肝中的表达及相关性[J].重庆医学,2007,36(8):707-708,711.FENG A J,CHEN D F,FAN L L,et al.Effect of fatty acid transport protein 4 on rat non-alcoholic fatty liver[J].Chongqing Medicine,2007,36(8):707-708,711.(in Chinese)
[26]	MASUZAKI R,KANDA T,SASAKI R,et al.Noninvasive assessment of liver fibrosis:current and future clinical and molecular perspectives[J].Int J Mol Sci,2020,21(14):4906.
[27]	REN J J,HUANG T J,ZHANG Q Q,et al.Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic fibrosis via the regulation of MMPs/TIMPs in mice[J].Hepatobiliary Pancreat Dis Int,2019,18(1):38-47.
[28]	ASOKAN S M,HUNG T H,LI Z Y,et al.Protein hydrolysate from potato confers hepatic-protection in hamsters against high fat diet induced apoptosis and fibrosis by suppressing Caspase-3 and MMP2/9 and by enhancing Akt-survival pathway[J].BMC Complement Altern Med,2019,19(1):283.
[29]	HEMMANN S,GRAF J,RODERFELD M,et al.Expression of MMPs and TIMPs in liver fibrosis-a systematic review with special emphasis on anti-fibrotic strategies[J].J Hepatol,2007,46(5):955-975.
[30]	WANG X J,LI L,WANG H W,et al.Epoxyeicosatrienoic acids alleviate methionine-choline-deficient diet-induced non-alcoholic steatohepatitis in mice[J].Scand J Immunol,2019,90(3):e12791.
[31]	张敏,沈彤.神经酰胺与肥胖相关疾病防治的研究进展[J].安徽医科大学学报,2020,55(1):146-149.ZHANG M,SHEN T.Research progress of ceramides in the prevention and treatment of obesity-related diseases[J].Acta Universitatis Medicinalis Anhui,2020,55(1):146-149.(in Chinese)
[32]	韩海静,齐雪,牛春燕.神经酰胺在非酒精性脂肪性肝病发生发展中的作用[J].临床肝胆病杂志,2017,33(8):1584-1588.HAN H J,QI X,NIU C Y.Role of ceramide in development and progression of nonalcoholic fatty liver disease[J].Journal of Clinical Hepatology,2017,33(8):1584-1588.(in Chinese)
[33]	孙明谦,苗兰,张颖,等.基于液质联用技术对早期高脂血症金黄地鼠肝脏鞘脂的靶向代谢组学研究[J].实验动物科学,2021,38(2):30-34,40.SUN M Q,MIAO L,ZHANG Y,et al.Targeted metabonomics of sphingolipids in liver tissue from early stage hyperlipidemia hamsters using liquid chromatography-tandem mass spectrometry[J].Laboratory Animal Science,2021,38(2):30-34,40.(in Chinese)
[34]	LIN M L,LIAO W J,DONG M J,et al.Exosomal neutral sphingomyelinase 1 suppresses hepatocellular carcinoma via decreasing the ratio of sphingomyelin/ceramide[J].FEBS J,2018,285(20):3835-3848.
[35]	孟星星.酸性鞘磷脂酶/神经酰胺通路介导噪声刺激所致小鼠肝细胞损伤[D].西安:第四军医大学,2017.MENG X X.Acid sphingomyelinase/ceramide mediates hepatocytic injury of mice induced by noise exposure[D].Xi'an:Air Force Medical University,2017.(in Chinese)
[36]	RÉGNIER M,POLIZZI A,GUILLOU H,et al.Sphingolipid metabolism in non-alcoholic fatty liver diseases[J].Biochimie,2019, 159:9-22.
[37]	PREUSS C,JELENIK T,BODIS K,et al.A new targeted lipidomics approach reveals lipid droplets in liver,muscle and heart as a repository for diacylglycerol and ceramide species in non-alcoholic fatty liver[J].Cells,2019,8(3):277.
[38]	CHEN K,CHEN X,XUE H L,et al.Coenzyme Q10 attenuates high-fat diet-induced non-alcoholic fatty liver disease through activation of the AMPK pathway[J].Food Funct,2019,10(2):814-823.
[39]	DURAND M,COUÉ M,CROYAL M,et al.Changes in key mitochondrial lipids accompany mitochondrial dysfunction and oxidative stress in NAFLD[J].Oxid Med Cell Longev,2021,2021:9986299.
[40]	KOHLI R,KIRBY M,XANTHAKOS S A,et al.High-fructose,medium chain trans fat diet induces liver fibrosis and elevates plasma coenzyme Q9 in a novel murine model of obesity and nonalcoholic steatohepatitis[J].Hepatology,2010,52(3):934-944.