[1]苏静,胡以平.聚集素对肝脂肪变性细胞的作用及机制[J].新乡医学院学报,2023,40(1):001-10.[doi:10.7683/xxyxyxb.2023.01.001]
　SU Jing,HU Yiping.Effect and mechanism of clusterin on hepatic steatosis cells[J].Journal of Xinxiang Medical University,2023,40(1):001-10.[doi:10.7683/xxyxyxb.2023.01.001]
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聚集素对肝脂肪变性细胞的作用及机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年1期
页码:
001-10
栏目:
基础研究
出版日期:
2023-01-05

文章信息/Info

Title:
Effect and mechanism of clusterin on hepatic steatosis cells
作者:
苏静; 胡以平
（海军军医大学细胞生物学教研室，上海　200082）
Author(s):
SU Jing; HU Yiping
（Department of Cell Biology,Naval Medical University,Shanghai 200082，China）
关键词:
脂肪变性; 聚集素; 游离脂肪酸
Keywords:
steatosis; clusterin; free fatty acid
分类号:
R575.5
DOI:
10.7683/xxyxyxb.2023.01.001
文献标志码:
A
摘要:
目的　探讨聚集素（CLU）对脂肪变性Hep1-6细胞中脂沉积、脂代谢、糖代谢及氧化应激的影响及作用机制。方法　将Hep1-6细胞随机分为对照组、CLU组、短发夹(Sh)组和绿色荧光蛋白(GFP)组，对照组细胞不给予任何处理，CLU组细胞加入过表达CLU慢病毒上清，Sh组细胞加入敲减CLU慢病毒上清，GFP组细胞加入对照病毒上清。分别采用蛋白免疫印迹法和实时荧光定量聚合酶链式反应法检测细胞中CLU蛋白和mRNA表达来验证稳定过表达和敲减CLU细胞株的成功建立。待对照组、CLU组、Sh组和GFP组细胞培养至密度约80%时，加入游离脂肪酸工作液1 mmol诱导24 h构建脂肪变性细胞模型。细胞模型建立成功后，Sh组细胞随机分为Sh-CLU低剂量组和Sh-CLU高剂量组，Sh-CLU低剂量组每毫升培养基中加入400 ng CLU，Sh-CLU高剂量组每毫升培养基中加入800 ng CLU，继续培养24 h。另取Hep1-6细胞以每孔4×105个接种于12孔培养板，细胞贴壁后，加入含有体积分数1%牛血清白蛋白(BSA)的达尔伯克改良伊格尔无血清培养基培养24 h（BSA组）。油红染色观察对照组、BSA组、CLU组、Sh组和GFP组细胞中脂滴沉积情况，并采用酶标仪检测细胞中脂滴沉积量。采用实时荧光定量聚合酶链式反应法检测对照组、CLU组、Sh组、Sh-CLU低剂量组和Sh-CLU高剂量组细胞中脂肪酸合成、脂肪酸氧化、脂滴形成、脂转运、脂解、氧化应激和糖代谢相关基因mRNA表达水平。结果　与对照组相比，CLU组细胞中CLU蛋白和mRNA表达水平均显著升高，Sh组细胞中CLU蛋白和mRNA表达水平均明显降低。与对照组相比，CLU组细胞中脂滴沉积显著减少，Sh组细胞中呈现更多的红色脂滴沉积。与对照组相比，CLU组、Sh-CLU低剂量组和Sh-CLU高剂量组细胞中脂肪酸合成相关基因、脂肪酸氧化相关基因、脂解相关基因、氧化应激相关基因、脂转运和糖代谢相关基因mRNA表达水平总体显著升高，脂滴形成相关基因mRNA表达水平总体显著降低。与CLU组、Sh-CLU低剂量组和Sh-CLU高剂量组相比，Sh组细胞中脂肪酸氧化、脂转运、脂解和氧化应激相关基因mRNA表达水平总体显著降低，脂滴形成相关基因mRNA表达水平总体显著升高。结论　CLU在脂肪变性细胞中的主要作用可能是通过增强脂肪酸氧化和脂解调控脂质平衡，提高细胞抗氧化损伤能力。
Abstract:
Objective　To explore the effect and mechanism of clusterin (CLU) on lipid accumulation,lipid metabolism,glucose metabolism and oxidative stress in steatosis cells of Hep1-6.Methods　Hep1-6 cells were randomly divided into control group,CLU group,short hairpin (Sh) group and green fluorescent protein (GFP) group.The cells in control group were not treated with any treatment;the cells in CLU group were added with CLU overexpression lentivirus supernatant;the cells in Sh group were added with CLU knockdown lentivirus supernatant;the cells in GFP group were added with control virus supernatant.The expressions of CLU protein and mRNA were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction method respectively to verify the successful establishment of stable overexpression and knockdown CLU cell lines.When the cell density in the control group,CLU group,Sh group and GFP group was about 80%,1 mmol of free fatty acid working solution was added for inducing 24 h to construct the steatosis cell model.After the cell model was established,the cells in the Sh group were randomly divided into Sh-CLU low-dose group and Sh-CLU high-dose group.The Sh-CLU low-dose group was added 400 ng CLU permilliliter of medium,the cells in the Sh-CLU high-dose group was added 800 ng CLU permilliliter of medium,and the cells in the above two groups were cultured for 24 h.Another Hep1-6 cells were taken and inoculated in 12-well culture plate with 4×105 cells per well.After the cells attached to the wall,Dulbecco′s modification of Eagle′s serum-free medium containing 1% bovine serum albumin(BSA) was added for culturing 24 h (BSA group).The lipid droplet deposition in cells in the control group,BSA group,CLU group,Sh group and GFP group was observed by oil red staining,and the amount of lipid droplet deposition in cells in the above five groups was detected by enzyme-labeled instrument.The expressions of fatty acid synthesis,fatty acid oxidation,lipid droplet formation,lipid transport,lipolysis,oxidative stress and glucose metabolism related gene mRNA in control group,CLU group,Sh group,Sh-CLU low-dose group and Sh-CLU high-dose group were detected by real-time fluorescence quantitative polymerase chain reaction method.Results　Compared with the control group,the expression levels of CLU protein and mRNA in the CLU group were significantly increased,while the expression levels of CLU protein and mRNA in the Sh group were significantly decreased.Compared with the control group,the lipid droplet deposition in cells in the CLU group was less,while there were more red lipid droplet deposition in the sh group.Compared with control group,the mRNA expression levels of fatty acid synthesis related genes,fatty acid oxidation related genes,lipolysis related genes,oxidative stress related genes,lipid transport and glucose metabolism related genes in CLU group,Sh-CLU low-dose group and Sh-CLU high-dose group were significantly increased overall,and the mRNA expression levels of lipid droplet formation related genes were significantly decreased overall.Compared with CLU group,Sh-CLU low-dose group and Sh-CLU high-dose group,the mRNA expression levels of fatty acid oxidation,lipid transport,lipolysis and oxidative stress related genes in Sh group were significantly decreased overall;while the mRNA expression levels of lipid droplet formation related genes were significantly increased overall.Conclusion　The main role of CLU in steatosis cells may be regulating lipid balance by enhancing fatty acid oxidation and lipolysis,and improve the anti oxidative damage ability of cells.

参考文献/References:

［1］　GUSZYN＇SKA P,LEMANCEWICZ D,DZICIO J B,et al.Non-alcoholic fatty liver disease (NAFLD) and bariatric/metabolic surgery as its treatment option:a review［J］.J Clin Med,2021,10(24):5721.
［2］　KARLAS T,WIEGAND J,BERG,T.Gastrointestinal complications of obesity:non-alcoholic fatty liver disease (NAFLD) and its sequelae［J］.Best Pract Res Clin Endocrinol Metab,2013,27(2):195-208.
［3］　MICHELOTTI G A,MACHADO M V,DIEHL A M.NAFLD,NASH and liver cancer［J］.Nat Rev Gastroenterol Hepatol,2013,10(11):656-665.
［4］　DAY C P,JAMES O F.Steatohepatitis:a tale of two "hits"［J］.Gastroenterology,1998,114(4):842-845.
［5］　BROWNING J D,HORTON J D.Molecular mediators of hepatic steatosis and liver injury［J］.J Clin Invest,2004,114(2):147-152.
［6］　PARK S,MATHIS K W,LEE I K.The physiological roles of apolipoprotein J/clusterin in metabolic and cardiovascular diseases［J］.Rev Endocr Metab Disord,2014,15(1):45-53.
［7］　SATO N.The emerging role of the apolipoproteins APOE and APOJ in the interaction between diabetes and alzheimer disease［J］.J Clin Endocrinol Metab,2020,105(11):dgaa570.
［8］　ROSENBERG M E,SILKENSEN J.Clusterin:physiologic and pathophysiologic considerations［J］.Int J Biochem Cell Biol,1995,27(7):633-645.
［9］　STROCCHI P,SMITH M A,PERRY G,et al.Clusterin up-regulation following sub-lethal oxidative stress and lipid peroxidation in human neuroblastoma cells［J］.Neurobiol Aging,2006,27(11):1588-1594.
［10］　ZHOU W,GUAN Q,KWAN C C,et al.Loss of clusterin expression worsens renal ischemia-reperfusion injury［J］.Am J Physiol Renal Physiol,2010,298(3):F568-F578.
［11］　MCLAUGHLIN L,ZHU G,MISTRY M,et al.Apolipoprotein J/clusterin limits the severity of murine autoimmune myocarditis［J］.J Clin Invest,2000,106(9):1105-1113.
［12］　WITTWER J,BRADLEY D.Clusterin and its role in insulin resistance and the cardiometabolic syndrome［J］.Front Immunol,2021,12:612496.
［13］　HOOFNAGLE A N,WU M,GOSMANOVA A K,et al.Low clusterin levels in high-density lipoprotein associate with insulin resistance,obesity,and dyslipoproteinemia［J］.Arterioscler Thromb Vasc Biol,2010,30(12):2528-2534.
［14］　SEO J A,KANG M C,YANG W M.Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity［J］.Nat Commun,2020,11(1):2024.
［15］　KWON M J,JU T J,HEO J Y,et al.Deficiency of clusterin exacerbates high-fat diet-induced insulin resistance in male mice［J］.Endocrinology,2014,155(6):2089-2101.
［16］　LEE Y N,SHIM Y J,KANG B H,et al.Over-expression of human clusterin increases stress resistance and extends lifespan in drosophila melanogaster［J］.Biochem Biophys Res Commun,2012,420(4):851-856.
［17］　HAMADA N,MIYATA M,ETO H,et al.Loss of clusterin limits atherosclerosis in apolipoprotein E-deficient mice via reduced expression of Egr-1 and TNF-α［J］.J Atheroscler Thromb,2011,18(3):209-216.
［18］　MISHRA V K,PALGUNACHARI M N,HUDSON J S,et al.Structure and lipid interactions of an anti-inflammatory and anti-atherogenic 10-residue class G(\*) apolipoprotein J peptide using solution NMR［J］.Biochim Biophys Acta,2011,1808(1):498-507.
［19］　PARK J,LEE W,KIM H S,et al.Clusterin overexpression protects against western diet-induced obesity and NAFLD［J］.Sci Rep,2020,10(1):17484.
［20］　PARK J S,SHIM Y J,KANG B H,et al.Hepatocyte-specific clusterin overexpression attenuates diet-induced nonalcoholic steatohepatitis［J］.Biochem Biophys Res Commun,2018,495(2):1775-1781.
［21］　FABBRINI E,SULLIVAN S,KLEIN S.Obesity and nonalcoholic fatty liver disease:biochemical,metabolic,and clinical implications［J］.Hepatology,2010,51(2):679-689.
［22］　GREEN H,KEHINDE O.An established preadipose cell line and its differentiation in culture.II.Factors affecting the adipose conversion［J］.Cell,1975,5(1):19-27.
［23］　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.
［24］　BOZIC M,GUZMA·N C,BENET M,et al.Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis［J］.J Hepatol,2016,65(4):748-757.
［25］　WALTHER T C,CHUNG J,FARESE R J.Lipid droplet biogenesis［J］.Annu Rev Cell Dev Biol,2017,33:491-510.
［26］　MAKAROVA E,KAZANTSEVA A,DUBININA A,et al.Fibroblast growth factor 21 (FGF21) administration sex-specifically affects blood insulin levels and liver steatosis in obese A(y) mice［J］.Cells,2021,10(12):3440.
［27］　PILAR V M,PRIETO-HONTORIA P L,PARDO V,et al.Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes［J］.Free Radic Biol Med,2015,84:263-278.
［28］　TROUGAKOS I P,GONOS E S.Chapter 9:oxidative stress in malignant progression:the role of clusterin,a sensitive cellular biosensor of free radicals［J］.Adv Cancer Res,2009,104:171-210.
［29］　TROUGAKOS I P.The molecular chaperone apolipoprotein J/clusterin as a sensor of oxidative stress:implications in therapeutic approaches：a mini-review［J］.Gerontology,2013,59(6):514-523.
［30］　TAKASHIMA M,OGAWA W,HAYASHI K,et al.Role of KLF15 in regulation of hepatic gluconeogenesis and metformin action［J］.Diabetes,2010,59(7):1608-1615.
［31］　RODEN M,STINGTL H,CHANDRAMOULI V,et al.Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans［J］.Diabetes,2000,49(5):701-707.
［32］　PERRY R J,CAMPOREZ J G,KURSAWE R,et al.Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes［J］.Cell,2015,160(4):745-758.

更新日期/Last Update: 2023-01-05