染料木素对油酸诱导HepG2细胞脂质蓄积的影响
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摘要
目的:探讨染料木素改善油酸诱导的HepG2细胞脂质蓄积的作用及机制。方法:采用不同浓度的油酸处理HepG2细胞24 h诱导建立脂质蓄积模型,12. 5,25,50μmol·L~(-1)的染料木素和油酸共同作用于细胞24 h,采用噻唑蓝(MTT)比色法测定细胞存活率,尼罗红和DAPI双染观察细胞内脂滴蓄积情况,并采用试剂盒测定细胞内甘油三酯(TG)含量,蛋白免疫印迹法(Western blot)测定染料木素对脂肪甘油三酯脂肪酶(ATGL),激素敏感性脂肪酶(HSL),磷酸化HSL(p-HSL),沉默信息调节因子1(SIRT1),过氧化物酶体增殖物激活受体α(PPARα),肉毒碱棕榈酰转移酶-1(CPT-1)蛋白表达的影响。结果:0. 5 mmol·L~(-1)油酸及12. 5,25,50μmol·L~(-1)染料木素作用细胞24 h对细胞存活率无明显影响;与正常组比较,模型组细胞中TG含量显著升高(P <0. 01),细胞内脂滴显著增多(P <0. 01),细胞中HSL的磷酸化水平明显下调(P <0. 05);与模型组比较,染料木素可明显降低细胞中TG含量(P <0. 05,P <0. 01),明显减少细胞内脂滴(P <0. 05),上调细胞中ATGL,p-HSL/HSL,SIRT1,PPARα,CPT-1蛋白表达水平(P <0. 05,P <0. 01)。结论:染料木素对HepG2细胞脂质蓄积模型有明显的改善作用,其机制可能通过上调ATGL,p-HSL/HSL,SIRT1,PPARα,CPT-1蛋白表达水平,进而促进脂质的水解和氧化代谢。
Objective: To explore the effect and mechanism of genistein on oleic acid-induced lipid accumulation in HepG2 cells. Method: Lipid accumulation model in HepG2 cells was induced by different concentrations of oleic acid for 24 h,and 12. 5,25,50 μmol·L~(-1) genistein and oleic acid acted on cells for 24 h.Cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide( MTT) assay. Double staining with Nile red and DAPI was used to observe the intracellular lipid droplets. Intracellular triglyceride( TG) content was determined by kit. The protein expression levels of triglyceride lipase( ATGL), hormone-sensitive fatty acid( HSL),phosphorylation HSL( p-HSL),silent information regulator 1( STRT1),peroxisome proliferatoractivated receptor α( PPARα),carnitine palmityl transferase 1( CPT-1) in oleic acid-induced HepG2 cells were detected by Western blot. Result: 0. 5 mmol·L~(-1) oleic acid and 12. 5,25,50 μmol·L~(-1) genistein had no significant effect on cell viability after treated cells for 24 h. Compared with normal group,the TG content and lipid droplets in oleic acid-induced HepG2 cells was significantly increased( P < 0. 01),and HSL phosphorylation in oleic acid-induced HepG2 cells was significantly down-regulated( P < 0. 05); compared with model group,the TG content and intracellular lipid droplets in HepG2 cells was significantly decreased after administration of genistein( P < 0. 05,P < 0. 01). Genistein can significantly up-regulate the protein expression levels of ATGL,p-HSL/HSL,SIRT1,PPARα,and CPT-1 compared with model group( P < 0. 05,P < 0. 01). Conclusion: Genistein can significantly improve the lipid accumulation in oleic acid-induced HepG2 cells,and its mechanism may be related to up-regulating the protein expression levels of ATGL,p-HSL/HSL,SIRT1,PPARα,CPT-1,and thus promoting lipid hydrolysis and oxidative metabolism.
Objective: To explore the effect and mechanism of genistein on oleic acid-induced lipid accumulation in HepG2 cells. Method: Lipid accumulation model in HepG2 cells was induced by different concentrations of oleic acid for 24 h,and 12. 5,25,50 μmol·L~(-1) genistein and oleic acid acted on cells for 24 h.Cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide( MTT) assay. Double staining with Nile red and DAPI was used to observe the intracellular lipid droplets. Intracellular triglyceride( TG) content was determined by kit. The protein expression levels of triglyceride lipase( ATGL), hormone-sensitive fatty acid( HSL),phosphorylation HSL( p-HSL),silent information regulator 1( STRT1),peroxisome proliferatoractivated receptor α( PPARα),carnitine palmityl transferase 1( CPT-1) in oleic acid-induced HepG2 cells were detected by Western blot. Result: 0. 5 mmol·L~(-1) oleic acid and 12. 5,25,50 μmol·L~(-1) genistein had no significant effect on cell viability after treated cells for 24 h. Compared with normal group,the TG content and lipid droplets in oleic acid-induced HepG2 cells was significantly increased( P < 0. 01),and HSL phosphorylation in oleic acid-induced HepG2 cells was significantly down-regulated( P < 0. 05); compared with model group,the TG content and intracellular lipid droplets in HepG2 cells was significantly decreased after administration of genistein( P < 0. 05,P < 0. 01). Genistein can significantly up-regulate the protein expression levels of ATGL,p-HSL/HSL,SIRT1,PPARα,and CPT-1 compared with model group( P < 0. 05,P < 0. 01). Conclusion: Genistein can significantly improve the lipid accumulation in oleic acid-induced HepG2 cells,and its mechanism may be related to up-regulating the protein expression levels of ATGL,p-HSL/HSL,SIRT1,PPARα,CPT-1,and thus promoting lipid hydrolysis and oxidative metabolism.
引文
[1] CAO P,HUANG G,YANG Q,et al. The effect of chitooligosaccharides on oleic acid-induced lipid accumulation in Hep G2 cells[J]. Saudi Pharm J,2016,24(3):292-298.
[2] Kwon E,KANG M,Kim S,et al. Zanthoxylum ailanthoides suppresses oleic acid-induced lipid accumulation through an activation of LKB1/AMPK pathway in Hep G2 cells[J]. Evid-Based Compl Alt,2018,doi:10. 1155/2018/3140267.
[3] ZENG L,TANG W,YIN J,et al. Alisol a 24-acetate prevents hepatic steatosis and metabolic disorders in Hep G2 cells[J]. Cell Physiol Biochem,2016,40(3/4):453-464.
[4] HUANG W,CHEN Y,LIU H,et al. Ginkgolide C reduced oleic acid-induced lipid accumulation in Hep G2cells[J]. Saudi Pharm J,2018,26(8):1178-1184.
[5]徐立,符晶,方芳,等.加味茵陈五苓散治疗湿热蕴结型非酒精性脂肪性肝病的疗效及对肠道菌群的影响[J].中国实验方剂学杂志,2019,25(12):127-132.
[6] SHEN H,HUANG S,Kung C,et al. Genistein ameliorated obesity accompanied with adipose tissue browning and attenuation of hepatic lipogenesis in ovariectomized rats with high-fat diet[J]. J Nutr Biochem,2019,67:111-122.
[7] Behloul N,WU G. Genistein:a promising therapeutic agent for obesity and diabetes treatment[J]. Eur J Pharmacol,2013,698(1/3):31-38.
[8] GAN M,ZHENG T,SHEN L,et al. Genistein reverses isoproterenol-induced cardiac hypertrophy by regulating miR-451/TIMP2[J]. Biomed Pharmacother,2019,112:108618.
[9] ZHONG H,LIU H,JIANG Z. Genistein ameliorates fat accumulation through AMPK activation in fatty acidinduced BRL cells[J]. J Food Sci,2017,82(11):2719-2725.
[10] Mohamed S S,Nallasamy P,Muniyandi P,et al.Genistein improves liver function and attenuates nonalcoholic fatty liver disease in a rat model of insulin resistance[J]. J Diabetes,2009,1(4):278-287.
[11] WANG W,CHEN J,MAO J,et al. Genistein ameliorates non-alcoholic fatty liver disease by targeting the thromboxane A2pathway[J]. J Agric Food Chem,2018,66(23):5853-5859.
[12]吴丹,邓颖勋,王宇琦,等.染料木黄酮对脂肪变Hep G2细胞甘油三酯水平和Lipin1表达的影响[J].营养学报,2013,35(4):353-356.
[13] Rafiei H,Omidian K,Bandy B. Dietary polyphenols protect against oleic acid-induced steatosis in an in vitro model of NAFLD by modulating lipid metabolism and improving mitochondrial function[J]. Nutrients,2019,11(3):541.
[14] Day C P,James O F. Steatohepatitis:a tale of two"hits"?[J]. Gastroenterology,1998,114(4):842-845.
[15] El-Kader S M A. Non-alcoholic fatty liver disease:the diagnosis and management[J]. World J Hepatol,2015,7(6):846.
[16]孙秀芹. 2型糖尿病合并非酒精性脂肪肝相关因素分析[J].中国预防医学杂志,2019,20(1):78-80.
[17] Bechmann L P,Hannivoort R A,Gerken G,et al. The interaction of hepatic lipid and glucose metabolism in liver diseases[J]. J Hepatol,2012,56(4):952-964.
[18] Schweiger M,Schreiber R,Haemmerle G,et al. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism[J]. J Biol Chem,2006,281(52):40236-40241.
[19] Ong K T,Mashek M T,BU S Y,et al. Adipose triglyceride lipase is a major hepatic lipase that regulates triacylglycerol turnover and fatty acid signaling and partitioning[J]. Hepatology,2011,53(1):116-126.
[20]黄文钰,李向平.脂肪细胞三酰甘油分解代谢相关调节的研究进展[J].国际病理科学与临床杂志,2013,33(3):230-234.
[21] Sapiro J M,Mashek M T,Greenberg A S,et al. Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor alpha activity[J]. J Lipid Res,2009,50(8):1621-1629.
[22] Khan S A,Sathyanarayan A,Mashek M T,et al. ATGLcatalyzed lipolysis regulates SIRT1 to control PGC-1α/PPARαsignaling[J]. Diabetes,2015,64(2):418-426.
[2] Kwon E,KANG M,Kim S,et al. Zanthoxylum ailanthoides suppresses oleic acid-induced lipid accumulation through an activation of LKB1/AMPK pathway in Hep G2 cells[J]. Evid-Based Compl Alt,2018,doi:10. 1155/2018/3140267.
[3] ZENG L,TANG W,YIN J,et al. Alisol a 24-acetate prevents hepatic steatosis and metabolic disorders in Hep G2 cells[J]. Cell Physiol Biochem,2016,40(3/4):453-464.
[4] HUANG W,CHEN Y,LIU H,et al. Ginkgolide C reduced oleic acid-induced lipid accumulation in Hep G2cells[J]. Saudi Pharm J,2018,26(8):1178-1184.
[5]徐立,符晶,方芳,等.加味茵陈五苓散治疗湿热蕴结型非酒精性脂肪性肝病的疗效及对肠道菌群的影响[J].中国实验方剂学杂志,2019,25(12):127-132.
[6] SHEN H,HUANG S,Kung C,et al. Genistein ameliorated obesity accompanied with adipose tissue browning and attenuation of hepatic lipogenesis in ovariectomized rats with high-fat diet[J]. J Nutr Biochem,2019,67:111-122.
[7] Behloul N,WU G. Genistein:a promising therapeutic agent for obesity and diabetes treatment[J]. Eur J Pharmacol,2013,698(1/3):31-38.
[8] GAN M,ZHENG T,SHEN L,et al. Genistein reverses isoproterenol-induced cardiac hypertrophy by regulating miR-451/TIMP2[J]. Biomed Pharmacother,2019,112:108618.
[9] ZHONG H,LIU H,JIANG Z. Genistein ameliorates fat accumulation through AMPK activation in fatty acidinduced BRL cells[J]. J Food Sci,2017,82(11):2719-2725.
[10] Mohamed S S,Nallasamy P,Muniyandi P,et al.Genistein improves liver function and attenuates nonalcoholic fatty liver disease in a rat model of insulin resistance[J]. J Diabetes,2009,1(4):278-287.
[11] WANG W,CHEN J,MAO J,et al. Genistein ameliorates non-alcoholic fatty liver disease by targeting the thromboxane A2pathway[J]. J Agric Food Chem,2018,66(23):5853-5859.
[12]吴丹,邓颖勋,王宇琦,等.染料木黄酮对脂肪变Hep G2细胞甘油三酯水平和Lipin1表达的影响[J].营养学报,2013,35(4):353-356.
[13] Rafiei H,Omidian K,Bandy B. Dietary polyphenols protect against oleic acid-induced steatosis in an in vitro model of NAFLD by modulating lipid metabolism and improving mitochondrial function[J]. Nutrients,2019,11(3):541.
[14] Day C P,James O F. Steatohepatitis:a tale of two"hits"?[J]. Gastroenterology,1998,114(4):842-845.
[15] El-Kader S M A. Non-alcoholic fatty liver disease:the diagnosis and management[J]. World J Hepatol,2015,7(6):846.
[16]孙秀芹. 2型糖尿病合并非酒精性脂肪肝相关因素分析[J].中国预防医学杂志,2019,20(1):78-80.
[17] Bechmann L P,Hannivoort R A,Gerken G,et al. The interaction of hepatic lipid and glucose metabolism in liver diseases[J]. J Hepatol,2012,56(4):952-964.
[18] Schweiger M,Schreiber R,Haemmerle G,et al. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism[J]. J Biol Chem,2006,281(52):40236-40241.
[19] Ong K T,Mashek M T,BU S Y,et al. Adipose triglyceride lipase is a major hepatic lipase that regulates triacylglycerol turnover and fatty acid signaling and partitioning[J]. Hepatology,2011,53(1):116-126.
[20]黄文钰,李向平.脂肪细胞三酰甘油分解代谢相关调节的研究进展[J].国际病理科学与临床杂志,2013,33(3):230-234.
[21] Sapiro J M,Mashek M T,Greenberg A S,et al. Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor alpha activity[J]. J Lipid Res,2009,50(8):1621-1629.
[22] Khan S A,Sathyanarayan A,Mashek M T,et al. ATGLcatalyzed lipolysis regulates SIRT1 to control PGC-1α/PPARαsignaling[J]. Diabetes,2015,64(2):418-426.