桑叶改善3T3-L1细胞胰岛素抵抗的作用及其机制分析
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摘要
目的:观察桑叶含药血清对脂肪细胞株3T3-L1胰岛素抵抗(IR)模型葡萄糖消耗量及细胞活力的影响,筛选出桑叶含药血清的最佳浓度,并检测其对炎症因子含量的影响,探讨可能的作用机制。方法:取对数生长期3T3-L1前脂肪细胞,10 mg·L~(-1)胰岛素(Ins),0. 25 mmol·L~(-1)地塞米松(DEX),0. 5 mmol·L~(-1)3-异丁基-1-甲基黄嘌呤(IBMX)诱导48 h后,10 mg·L~(-1)Ins再次诱导48 h,待其分化为成熟脂肪细胞后,1μmol·L~(-1)DEX诱导96 h以建立IR细胞模型。桑叶水提物含药血清培养12,24,36,72 h后,采用葡萄糖氧化酶法检测桑叶含药血清培养后细胞葡萄糖的消耗量,采用噻唑蓝(MTT)比色法检测桑叶含药血清培养后IR模型的细胞活力,酶联免疫吸附测定法(ELISA)检测桑叶含药血清对细胞肿瘤坏死因子-α(TNF-α)含量的影响,蛋白免疫印迹法(Western blot)测定桑叶含药血清对胰岛素信号通路胰岛素受体(InsR),胰岛素受体底物(IRS),磷酸化IRS1(p-IRS1),葡萄糖转运蛋白4(GLUT4)蛋白表达的影响。结果:桑叶含药血清可显著提高IR细胞葡萄糖消耗量(P <0. 01),增强细胞活力(P <0. 01),降低炎症因子TNF-α的含量(P <0. 01),调节胰岛素信号通路下游蛋白的表达量(P <0. 05,P <0. 01)。结论:桑叶可明显改善3T3-L1细胞IR状态,其作用机制可能与抑制TNF-α表达、促进胰岛素信号通路蛋白的表达有关。
Objective: To observe effect of Mori Folium-containing serum on glucose consumption and cell activity of fat cell line 3 T3-L1 insulin resistance(IR) model,in order to screen out the optimal concentration of drug-containing serum,detect effect of Mori Folium on the content of inflammatory factors,and explore the possible mechanism. Method: 3 T3-L1 preadipocytes in logarithmic growth phase were selected,and induced with10 mg·L~(-1) insulin(Ins),0. 25 mmol·L~(-1) dexamethasone(DEX) and 0. 5 mmol·L~(-1)3-isobutyl-methylxanthine(IBMX) for 48 h and then with 10 mg·L~(-1) Ins for 48 h. After the cells were differentiated into mature adipocytes,they were induced with 1 μmol·L~(-1) DEX for 96 h to establish IR model. Glucose content in the supernatant of cells was detected by glucose oxidase after serum containing Mori Folium cultured for 12,24,36,72 h. Methylthiazdyl-tetrazolium(MTT) was used to detect the effect of serum containing Mori Folium on IR cells activity. The content of tumor necrosis factor-α(TNF-α) was determined by enzyme-linked immunosorbent assay(ELISA).Meanwhile,the effects of inflammatory factors on the expressions of insulin signaling pathway proteins insulin receptor(InsR),insulin receptor substrate(IRS),p-IRS1 and glucose transporter 4(GLUT4) were determined by Western blot. Result: Serum containing Mori Folium could significantly increase the glucose consumption rate and cell activity of IR cells(P < 0. 01),reduce the content of TNF-α(P < 0. 01),and regulate the expression of insulin signaling pathway proteins(P < 0. 05,P < 0. 01). Conclusion: Mori Folium can significantly improve IR status of 3 T3-L1 cells,and its mechanism may be related to inhibiting TNF-α and promoting the expressions of insulin signaling pathway proteins.
Objective: To observe effect of Mori Folium-containing serum on glucose consumption and cell activity of fat cell line 3 T3-L1 insulin resistance(IR) model,in order to screen out the optimal concentration of drug-containing serum,detect effect of Mori Folium on the content of inflammatory factors,and explore the possible mechanism. Method: 3 T3-L1 preadipocytes in logarithmic growth phase were selected,and induced with10 mg·L~(-1) insulin(Ins),0. 25 mmol·L~(-1) dexamethasone(DEX) and 0. 5 mmol·L~(-1)3-isobutyl-methylxanthine(IBMX) for 48 h and then with 10 mg·L~(-1) Ins for 48 h. After the cells were differentiated into mature adipocytes,they were induced with 1 μmol·L~(-1) DEX for 96 h to establish IR model. Glucose content in the supernatant of cells was detected by glucose oxidase after serum containing Mori Folium cultured for 12,24,36,72 h. Methylthiazdyl-tetrazolium(MTT) was used to detect the effect of serum containing Mori Folium on IR cells activity. The content of tumor necrosis factor-α(TNF-α) was determined by enzyme-linked immunosorbent assay(ELISA).Meanwhile,the effects of inflammatory factors on the expressions of insulin signaling pathway proteins insulin receptor(InsR),insulin receptor substrate(IRS),p-IRS1 and glucose transporter 4(GLUT4) were determined by Western blot. Result: Serum containing Mori Folium could significantly increase the glucose consumption rate and cell activity of IR cells(P < 0. 01),reduce the content of TNF-α(P < 0. 01),and regulate the expression of insulin signaling pathway proteins(P < 0. 05,P < 0. 01). Conclusion: Mori Folium can significantly improve IR status of 3 T3-L1 cells,and its mechanism may be related to inhibiting TNF-α and promoting the expressions of insulin signaling pathway proteins.
引文
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[2]年雪,王小丽,于东升,等.小檗碱通过抑制Traf2-MEKK1-MEK-ERK通路改善TNF-α诱导的胰岛素抵抗[J].现代生物医学进展,2017,17(36):7001-7007.
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[18]郭锡熔,陈荣华,费莉,等.神经肽YY5受体反义寡核苷酸对肥胖大鼠血清胰岛素与C肽水平及脂肪组织TNF-α基因mRNA表达的影响[J].中华儿科杂志,2002(11):30-34.
[19] Fujishiro M,Gotoh Y,Katagiri H,et al. Three mitogenactivated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes[J]. Mol Endocrinol,2003,17(3):487-497.
[20] Uysal K T,Wiesbrock S M,Marino M W,et al.Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function[J]. Nature,1997,389(6651):610-614.
[21]唐锋,梁少瑜,陈飞龙,等.血清药物化学和血清药理学相结合的方法探讨麻黄附子细辛汤抗炎和免疫抑制的物质基础[J].中国中药杂志,2015,40(10):1971-1976.
[22]唐明敏,田恒康,杨文宁,等.桑叶水提物及其含药血清化学成分分析[J].中国实验方剂学杂志,2016,22(9):25-29.
[23]江正菊,宁林玲,胡霞敏,等.桑叶总黄酮对高脂诱导大鼠高血脂及高血糖的影响[J].中药材,2011,34(1):108-111.
[2]年雪,王小丽,于东升,等.小檗碱通过抑制Traf2-MEKK1-MEK-ERK通路改善TNF-α诱导的胰岛素抵抗[J].现代生物医学进展,2017,17(36):7001-7007.
[3]季涛,宿树兰,郭盛,等.基于α-葡萄糖苷酶抑制活性评价桑叶多组分药效相互作用研究[J].中国中药杂志,2016,41(11):1999-2006.
[4]陈群,苏傲蕾,黄正团.糖脉康颗粒对2型糖尿病气阴两虚夹瘀证大鼠CRP及脏器指数的影响[J].光明中医,2017,32(23):3393-3396.
[5]朱群.降糖饮治疗2型糖尿病60例[J].中国中医药现代远程教育,2011,9(14):36-37.
[6]刘洪凤,韩智学,聂影,等.桑叶多糖对糖尿病肾病大鼠肾脏纤维化的影响[J].中国老年学杂志,2014,34(14):3959-3960.
[7]杨桂枝,高小平,晏菊芳,等.地塞米松和胰岛素诱导3T3-L1脂肪细胞胰岛素抵抗的分子机理[J].西南师范大学学报:自然科学版,2003(3):460-464.
[8]张敬升,王晓婉,张广霞,等.小檗碱对3T3-L1前脂肪细胞及其胰岛素抵抗模型的影响[J].中国实验方剂学杂志,2015,21(22):145-149.
[9]王丽静,张尉,刘小莺,等.地塞米松诱导3T3-L1脂肪细胞胰岛素抵抗模型的建立[J].福建医科大学学报,2007(3):282-284.
[10]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.
[11]姜廷良,霍海如,李兰芳,等. 15种中西药物含药血清对大鼠成骨细胞增殖成熟的影响[J].中国骨质疏松杂志,2002(4):65-68.
[12]陆菊明.胰岛素抵抗与2型糖尿病的发生发展[J].药品评价,2014,11(23):12-15,40.
[13]袁群,俞璐,邵致格,等.糖脉康颗粒对2型糖尿病患者胰岛素敏感性的临床疗效观察[J].中国临床药理学杂志,2015,31(12):1099-1102.
[14]陆施婷,陈清光,徐佩英,等.基于中医传承辅助平台探讨丁学屏诊治糖尿病的临证经验及用药规律[J].中国实验方剂学杂志,2017,23(7):198-205.
[15] Oh D Y,Morinaga H,Talukdar S,et al. Increased macrophage migration into adipose tissue in obese mice[J]. Diabetes,2012,61(2):346-354.
[16] Olefsky J M,Glass C K. Macrophages,inflammation,and insulin resistance[J]. Annu Rev Physiol,2010,72(72):219-246.
[17]王凤华. 2型糖尿病患者ANGPTL3、TNF-α、FFA水平变化及其与胰岛素抵抗相关性研究[D].济南:山东大学,2008.
[18]郭锡熔,陈荣华,费莉,等.神经肽YY5受体反义寡核苷酸对肥胖大鼠血清胰岛素与C肽水平及脂肪组织TNF-α基因mRNA表达的影响[J].中华儿科杂志,2002(11):30-34.
[19] Fujishiro M,Gotoh Y,Katagiri H,et al. Three mitogenactivated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes[J]. Mol Endocrinol,2003,17(3):487-497.
[20] Uysal K T,Wiesbrock S M,Marino M W,et al.Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function[J]. Nature,1997,389(6651):610-614.
[21]唐锋,梁少瑜,陈飞龙,等.血清药物化学和血清药理学相结合的方法探讨麻黄附子细辛汤抗炎和免疫抑制的物质基础[J].中国中药杂志,2015,40(10):1971-1976.
[22]唐明敏,田恒康,杨文宁,等.桑叶水提物及其含药血清化学成分分析[J].中国实验方剂学杂志,2016,22(9):25-29.
[23]江正菊,宁林玲,胡霞敏,等.桑叶总黄酮对高脂诱导大鼠高血脂及高血糖的影响[J].中药材,2011,34(1):108-111.