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中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (03) : 129 -134. doi: 10.3877/cma.j.issn.2095-1221.2019.03.001

所属专题: 文献

论著

舒林酸调节IKK通路对3T3-L1细胞IRS-1酪氨酸磷酸化的影响
胡颖1, 丁晓颖1, 董维平1, 马宇航1, 徐浣白1, 王育璠1, 彭永德1, 张爱芳1,()   
  1. 1. 200080 上海交通大学附属第一人民医院内分泌代谢科
  • 收稿日期:2019-04-25 出版日期:2019-06-01
  • 通信作者: 张爱芳
  • 基金资助:
    国家自然科学基金(81870594); 上海交通大学医学院多中心临床研究项目(DLY201824); 上海交通大学医学院护理科研重中之重项目(Jyhz1802); 上海市第一人民医院临床研究创新团队(CTCCR-2018A02); 上海申康临床科技创新项目(16CR4025A); 松江卫计委第三周期疾病联合攻关合作项目(2018)

Effect of sulindac on tyrosine phosphorylations of IRS-1 in 3T3-L1 adipocytes by regulating IKK pathway

Ying Hu1, Xiaoying Ding1, Weiping Dong1, Yuhang Ma1, Huanbai Xu1, Yufan Wang1, Yongde Peng1, Aifang Zhang1,()   

  1. 1. Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated First People's Hospital , Shanghai 200080, China
  • Received:2019-04-25 Published:2019-06-01
  • Corresponding author: Aifang Zhang
  • About author:
    Corresponding author: Zhang Aifang, Email:
引用本文:

胡颖, 丁晓颖, 董维平, 马宇航, 徐浣白, 王育璠, 彭永德, 张爱芳. 舒林酸调节IKK通路对3T3-L1细胞IRS-1酪氨酸磷酸化的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2019, 09(03): 129-134.

Ying Hu, Xiaoying Ding, Weiping Dong, Yuhang Ma, Huanbai Xu, Yufan Wang, Yongde Peng, Aifang Zhang. Effect of sulindac on tyrosine phosphorylations of IRS-1 in 3T3-L1 adipocytes by regulating IKK pathway[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(03): 129-134.

目的

探讨舒林酸通过调节IKK通路对分化成熟3T3-L1细胞胰岛素受体后信号转导蛋白胰岛素受体底物1(IRS-1)蛋白酪氨酸/丝氨酸(Tyr/Ser)残基磷酸化表达的影响。

方法

用地塞米松、IBMX和胰岛素三联培养诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色观察脂肪细胞形态。诱导分化成熟的脂肪细胞如下分组干预,实时荧光定量PCR检测不同浓度炎症因子IL-1 β(0,1,10,100 ng/ml)和(或)不同浓度IKK特异阻断剂舒林酸(0,0.1,1,10 mmol/L)对诱导分化成熟的脂肪细胞IKK通路激活状态的影响。Western Blot检测IL-1β和(或)舒林酸对诱导分化成熟的脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化状态的影响。采用单因素方差分析进行统计学分析。

结果

实时荧光定量PCR和Western Blot结果显示,IL-1β 10 ng/ml组诱导成熟脂肪细胞IKKβ mRNA较对照组相对表达水平增加,分别为[(2.85±0.16)﹪,(1.00±0.12)﹪,P < 0.01];而IRS-1酪氨酸的磷酸化相对表达量较对照组下降,分别为[(0.72±0.26)﹪,(1.00±0.24)﹪,P < 0.01]。进一步予舒林酸(1?mmol/?L、10?mmol/L)干预后较对照组显著逆转IL-1β诱导脂肪细胞IRS-1酪氨酸磷酸化的表达水平,分别为[(1.72±0.16)﹪,(1.90±0.08)﹪,(1.00±0.13)﹪,P < 0.01],同时下调IRS-1丝氨酸磷酸化的表达水平[(0.79±0.16)﹪,(0.66±0.08)﹪,(1.00±0.10)﹪,P < 0.05]。

结?论

IL-1β通过促进诱导分化成熟脂肪细胞IKKβ的表达,激活脂肪细胞IKK炎症通路,抑制脂肪细胞IRS-1酪氨酸残基磷酸化的表达,舒林酸通过调节脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化的表达,改善脂肪细胞胰岛素受体后信号转导。

Objective

To investigate the effects of sulindac on tyrosine/serine phosphorylations of insulin postreceptor signal transducin IRS-1 in 3T3-L1 adipocytes by regulating the activity of IKK pathway in vitro.

Methods

3T3-L1 pre-adipocytes were incubated with isobuthyl-methylxanthine, dexamethasone, insulin and differentiated into mature adipocytes as determined by Oil Red O staining. The differentiated maturate adipocytes were divided into the following groups with or without various concentrations of IL-1β (0, 1, 10, 100 ng/m1) . Then the adipocytes of different groups were treated with different concentrations of sulindac (0, 0.1, 1, 10?mmol/l) at the different time points and subjected to real-time RT-PCR and Western Blot analysis for IKK as well as the IRS-1 Tyr/Ser phosphorylation expression. Statistical analysis was performed using one-way ANOVA procedure.

Results

Compared with control group, the relative expression of mRNA expression of IKKβ was significantly increased in IL-1β 10?ng/ml treatment 3T3L1 adipocytes[ (2.85±0.16) ﹪vs (1.00±0.12) ﹪, P < 0.01] while IRS-1 tyrosine phosphorylation obviously decreased, the difference was statistically significant [ (0.72±0.26) ﹪ vs (1.00±0.24) ﹪, P < 0.01]. Further Western Blot results showed that the sulindac treatment significantly up-regulated the relative expression of IRS-1impaired tyrosine phosphorylation induced by IL-1β induction [ (1.72±0.16) ﹪, (1.90±0.08) ﹪vs (1.00±0.13) ﹪, P < 0.01] but decreased IRS-1 serine phosphorylation[ (0.79±0.16) ﹪, (0.66±0.08) ﹪vs (1.00±0.10) ﹪, P < 0.05].

Conclusions

These data suggest that IL-1β can promote the expression of IKKβ in adipocytes, activate the inflammatory pathway of IKK in adipocytes and inhibit the phosphorylation of IRS-1 tyrosine residues which may be one of the causes of insuliresistance, while sulindac can improve the insulin postreceptor signal transduction pathway in adipocytes by regulating the tyrosine/serine residues phosphorylation of IRS-1 in adipocytes.

表1 内参GAPDH及各目的基因定量PCR引物序列
图1 倒置显微镜下观察3T3-L1诱导分化5 d细胞形态(×100)
图2 倒置显微镜下观察3T3-L1诱导分化10 d细胞形态(油红染色,×200)
表2 不同浓度IL-1β孵育分化成熟脂肪细胞IKK mRNA表达水平的变化(n = 3,±s
图3 IL-1β促进分化成熟脂肪细胞IKKβ mRNA的表达
图4 IL-1β抑制分化成熟脂肪细胞IRS-1酪氨酸残基磷酸化表达
表3 不同浓度舒林酸干预分化成熟脂肪细胞IRS-1酪氨酸?/丝氨酸残基磷酸化表达水平的变化(n = 3,±s
图5 舒林酸抑制分化成熟脂肪细胞IKKβ mRNA的表达
图6 舒林酸促进分化成熟脂肪细胞IRS-1酪氨酸残基磷酸化表达
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