舒林酸调节IKK通路对3T3-L1细胞IRS-1酪氨酸磷酸化的影响

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。

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

ObjectiveTo 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. Methods3T3-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. ResultsCompared 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]. ConclusionsThese 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.