石胆酸诱导的LO2细胞胰岛素抵抗模型及其应用
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摘要
目的利用石胆酸刺激人正常肝细胞(LO2)建立胰岛素抵抗模型,并用于筛选具有改善胰岛素抵抗降血糖活性的化合物。方法用不同浓度石胆酸(LCA)处理LO2细胞不同时间。通过MTT法检测细胞活性,并用不同浓度胰岛素刺激细胞,采用荧光标记2-脱氧葡萄糖(2-NBDG)作为光学探针检测细胞的葡萄糖摄取能力,明确建立稳定的LO2细胞胰岛素抵抗模型所需LCA的最适浓度及时间。Western blot检测LCA对胰岛素信号通路关键蛋白:磷酸化胰岛素受体底物1(p-IRS1)、胰岛素受体底物1(IRS1)、磷酸化蛋白激酶B(p-AKT)和蛋白激酶B(AKT)表达的影响。模型建立后,以二甲双胍为阳性药,对5种黄酮类化合物奥卡宁、异奥卡宁、马里苷、黄诺马苷、紫铆因及1种环烯醚萜类化合物梓醇,进行改善胰岛素抵抗降血糖活性筛选,以2-NBDG荧光标记葡萄糖法评估细胞糖摄取情况,以MTT法评估细胞存活率。结果 LCA作用于LO2细胞可显著降低细胞糖吸收,并产生明显的胰岛素耐受,而25μmol·L~(-1) LCA作用于LO2细胞24 h,模型稳定性最好。与对照组相比,12.5和25μmol·L~(-1) LCA组IRS1的蛋白磷酸化水平显著降低(P <0.05),且12.5和50μmol·L~(-1) LCA能明显减少AKT蛋白的磷酸化水平(P <0.01)。活性筛选发现,奥卡宁、异奥卡宁及紫铆因均能显著提高细胞糖摄取能力,并且其改善细胞胰岛素抵抗降血糖活性显著优于阳性药二甲双胍。结论 LCA可诱导LO2细胞建立稳定、可靠的胰岛素抵抗模型并用于药物筛选。
Objective To establish the insulin resistance model of human hepatocyte cells(LO2) by lithocholic acid(LCA), and screen the active compounds. Methods LO2 cells were treated with different concentrations of LCA for different time. Cell viability was measured by MTT assay. Cells were stimulated with different concentrations of insulin, and glucose uptake was measured by fluorescence-labeled glucose(2-NBDG) method. The effective concentration and time of LCA to the insulin resistance cell model were ascertained. Western blot was used to detect the effect of LCA on the expression of key proteins involving in insulin signaling pathway: phosphated insulin receptor substrate~(-1)(p-IRS1), insulin receptor substrate~(-1)(IRS1), phosphated protein kinase B(p-AKT)and protein kinase B(AKT). The insulin resistance model was used to screen the hypoglycemic activity of 6 compounds: okanin, isookanin, marein, flavanomarein, butein and catalpol. Results LCA significantly reduced the cellular glucose uptake and induced severe insulin tolerance in LO2 cells. While 25 μmol·L~(-1) LCA was applied to establish insulin resistance model in LO2 cells for 24 h. Compared with the control group, the phosphorylation level of IRS1 was significantly decreased in the 12.5 and 25 μmol·L~(-1) LCA groups(P < 0.05), and the phosphorylation level of AKT was significantly decreased in the 12.5 and 50 μmol·L~(-1) LCA groups(P < 0.01).Drug screening showed that okanin, isookanin and butein increased the glucose uptake. Their effects were better than metformin in LCA-induced insulin resistance cell model. Conclusion LCA can induce LO2 cells to establish a stable and reliable insulin resistance model, and can be used for drug screening.
Objective To establish the insulin resistance model of human hepatocyte cells(LO2) by lithocholic acid(LCA), and screen the active compounds. Methods LO2 cells were treated with different concentrations of LCA for different time. Cell viability was measured by MTT assay. Cells were stimulated with different concentrations of insulin, and glucose uptake was measured by fluorescence-labeled glucose(2-NBDG) method. The effective concentration and time of LCA to the insulin resistance cell model were ascertained. Western blot was used to detect the effect of LCA on the expression of key proteins involving in insulin signaling pathway: phosphated insulin receptor substrate~(-1)(p-IRS1), insulin receptor substrate~(-1)(IRS1), phosphated protein kinase B(p-AKT)and protein kinase B(AKT). The insulin resistance model was used to screen the hypoglycemic activity of 6 compounds: okanin, isookanin, marein, flavanomarein, butein and catalpol. Results LCA significantly reduced the cellular glucose uptake and induced severe insulin tolerance in LO2 cells. While 25 μmol·L~(-1) LCA was applied to establish insulin resistance model in LO2 cells for 24 h. Compared with the control group, the phosphorylation level of IRS1 was significantly decreased in the 12.5 and 25 μmol·L~(-1) LCA groups(P < 0.05), and the phosphorylation level of AKT was significantly decreased in the 12.5 and 50 μmol·L~(-1) LCA groups(P < 0.01).Drug screening showed that okanin, isookanin and butein increased the glucose uptake. Their effects were better than metformin in LCA-induced insulin resistance cell model. Conclusion LCA can induce LO2 cells to establish a stable and reliable insulin resistance model, and can be used for drug screening.
引文
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[12]姜保平,乐亮,姚霞,等. HepG2细胞胰岛素抵抗模型的建立及在筛选两色金鸡菊活性化合物中的应用[J].中国现代中药,2017,19(2):165-173.
[13]Yang JS,Li WJ,Zhou GM,et al. Comparative study on radiosensitivity of various tumor cells and human normal liver cells[J]. World J Gastroenterol,2005,11(26):4098-4101.
[14]李倩,王继红,王欣,等.人肝细胞系L02胰岛素抵抗细胞模型的建立及鉴定[J].现代预防医学,2007,34(22):4233-4235.
[15]Yan F,Chen X,Zheng X. Protective effect of mulberry fruit anthocyanin on human hepatocyte cells(LO2)and Caenorhabditis elegans under hyperglycemic conditions[J].Food Res Int,2017,102:213-224.
[16]Geng S,Wang S,Zhu W,et al. Curcumin suppresses JNKpathwaytoattenuateBPA-inducedinsulinresistance in LO2 cells[J]. Biomed Pharmacother,2018,97:1538-1543.
[17]Nie X,Chen H,Zhang J,et al. Rutaecarpine ameliorates hyperlipidemia and hyperglycemia in fat-fed,streptozotocin-treated rats via regulating the IRS-1/PI3K/Akt and AMPK/ACC2 signaling pathways[J]. Acta Pharmacol Sin,2016,37(4):483-496.
[18]王槾,郭姣,胡旭光,等.复方贞术调脂方对HepG2细胞胰岛素抵抗的作用及机制研究[J].广东药学院学报,2014,30(2):202-206.
[19]李芬.小檗碱改善胰岛素抵抗作用分子机制的研究[D].武汉:华中科技大学,2016.
[2]El Kihel L,Clément M,Bazin MA,et al. New lithocholic and chenodeoxycholic piperazinylcarboxamides with antiproliferative and pro-apoptotic effects on human cancer cell lines[J]. Bioorg Med Chem,2008,16(18):8737-8744.
[3]doNascimentoPG,Lemos TL,AlmeidaMC,etal.Lithocholic acid and derivatives:Antibacterial activity[J].Steroids,2015,104:8-15.
[4]He XL,Xing Y,Gu XZ,et al. The synthesis and antitumor activity of lithocholic acid and its derivatives[J]. Steroids,2017,125:54-60.
[5]Qi Y,Jiang C,Cheng J,et al. Bile acid signaling in lipid metabolism:metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice[J]. Biochim Biophys Acta,2015,1851(1):19-29.
[6]姜保平.两色金鸡菊茶饮对2型糖尿病胰岛素抵抗的预防作用及机制研究[D].北京:北京协和医学院,2015.
[7]刘晓华,江湖,李海星,等.胰岛素诱导3T3-L1脂肪细胞胰岛素抵抗模型的建立[J].食品科学,2012,33(19):249-253.
[8]Bonora E,Capaldo B,Perin PC,et al. Hyperinsulinemia and insulin resistance are independently associated with plasma lipids,uric acid and blood pressure in non-diabetic subjects. The GISIR database[J]. Nutr Metab Cardiovasc Dis,2008,18(9):624-631.
[9]Reinke H,Asher G. Circadian clock control of liver metabolic functions[J]. Gastroenterology,2016,150(3):574-580.
[10]Lin H,Mao Q,Wang Y,et al. A study on the situations of human L02 hepatocytes in genetically tolerized immunocompetent rats[J]. Chin J Hepatol,2006,14(8):578-581.
[11]方红娟,冯琼,王强,等. S1P对人LO2肝细胞株胰岛素抵抗模型糖代谢的影响[J].基础医学与临床,2015,35(8):1025-1030.
[12]姜保平,乐亮,姚霞,等. HepG2细胞胰岛素抵抗模型的建立及在筛选两色金鸡菊活性化合物中的应用[J].中国现代中药,2017,19(2):165-173.
[13]Yang JS,Li WJ,Zhou GM,et al. Comparative study on radiosensitivity of various tumor cells and human normal liver cells[J]. World J Gastroenterol,2005,11(26):4098-4101.
[14]李倩,王继红,王欣,等.人肝细胞系L02胰岛素抵抗细胞模型的建立及鉴定[J].现代预防医学,2007,34(22):4233-4235.
[15]Yan F,Chen X,Zheng X. Protective effect of mulberry fruit anthocyanin on human hepatocyte cells(LO2)and Caenorhabditis elegans under hyperglycemic conditions[J].Food Res Int,2017,102:213-224.
[16]Geng S,Wang S,Zhu W,et al. Curcumin suppresses JNKpathwaytoattenuateBPA-inducedinsulinresistance in LO2 cells[J]. Biomed Pharmacother,2018,97:1538-1543.
[17]Nie X,Chen H,Zhang J,et al. Rutaecarpine ameliorates hyperlipidemia and hyperglycemia in fat-fed,streptozotocin-treated rats via regulating the IRS-1/PI3K/Akt and AMPK/ACC2 signaling pathways[J]. Acta Pharmacol Sin,2016,37(4):483-496.
[18]王槾,郭姣,胡旭光,等.复方贞术调脂方对HepG2细胞胰岛素抵抗的作用及机制研究[J].广东药学院学报,2014,30(2):202-206.
[19]李芬.小檗碱改善胰岛素抵抗作用分子机制的研究[D].武汉:华中科技大学,2016.