替米沙坦对3T3－L1脂肪细胞过氧化物酶体增殖物激活受体γ信号通路的影响

Effect of telmisartan on peroxisome proliferator-activated receptor γ signaling pathway in 3T3-L1 adipocytes

目的观察替米沙坦对3T3-L1脂肪细胞过氧化物酶体增殖物激活受体γ（PPARγ）信号通路的影响，探讨替米沙坦延缓2型糖尿病病程的作用机制。方法诱导3T3-L1前脂肪细胞至80%成熟（对照组），加入50 ng/ml肿瘤坏死因子α（TNF-α）刺激1 h（TNF-α组），分别以0.1、5、10 μmol/L替米沙坦加入培养基干预24 h，即T0.1, T5和T10组。应用Western印迹检测PPARγ及其磷酸化水平、细胞周期依赖性蛋白激酶5（CDK5）表达变化；酶联免疫吸附测定（ELISA）检测培养基中脂联素含量。短发夹RNA（shRNA）沉默未分化3T3-L1的PPARγ，筛选PPARγ丝氨酸突变型3T3-L1细胞系即S273A、S112A、S186A，以进一步确定磷酸化位点。shRNA沉默CDK5，油红O染色、异丙醇萃取检测分化效率，以10 μmol/L替米沙坦干预成熟3T3-L1，Western印迹检测pPPARγ/PPARγ，ELISA检测培养基脂联素含量。结果TNF-α刺激及替米沙坦干预后各组CDK5表达差异均无统计学意义（均P〉0.05）。与TNF-α组相比，T5组、T10组pPPARγ/PPARγ降低，脂联素含量增加，差异均有统计学意义（均P〈0.05），T0.1组差异均无统计学意义（均P〉0.05）。与3T3-L1野生型（WT）相比，S186A、S112A组加入TNF-α后pPPARγ/PPARγ升高，脂联素降低，加入替米沙坦后pPPARγ/PPARγ降低，脂联素含量增加，差异均有统计学意义（均P〈0.05），S273A组pPPARγ/PPARγ、脂联素差异均无统计学意义（均P〉0.05）。异丙醇萃取显示沉默CDK5组（shCDK5）与随机对照（shCon）组相比3T3-L1分化差异无统计学意义（P〉0.05），Western印迹显示与shCDK5组相比，shCon组加入替米沙坦后pPPARγ/PPARγ降低，脂联素增加，差异均有统计学意义（均P〈0.05）。结论替米沙坦能够缓解因TNF-α刺激导致的PPARγ磷酸化水平升高，上调脂联素含量。CDK5介导了替米沙坦对3T3-L1脂肪细胞PPARγ信号通路的影响。替米沙坦的作用位点为PPARγ第273位丝氨酸，PPARγ第273位丝氨酸上游激酶为CDK5。

ObjectiveTo investigate the mechanisms of telmisartan on delaying the course of type 2 diabetes.Methods3T3-L1 preadipocytes were induced to 80% mature adipocytes （control group） and stimulated with 50 ng/ml tumor necrosis factor （TNF-α） for 1 hour （TNF-α group）. Then 0.1, 5, 10 μmol/L telmisartan was added to the culture medium for 24 h, respectively（T0.1, T5 and T10 group）. The cells from each group was collected to detect peroxisome proliferator-activated receptors γ （PPARγ） and its phosphorylation level, as well as upstream kinase cell cycle dependent kinase 5 （CDK5） by Western blot. Adiponectin in the culture supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. PPARγ or CDK5 of undifferentiated 3T3-L1 adipocytes were silenced by using targeted short hairpin RNA （shRNA）. Through infection of the cells with the retrovirus, stabled PPARγ or CDK5 knockdown cell lines were set up and screened by incubation with puromycin. 3T3-L1 adipocyte cell lines expressing serine mutant PPARγ （S273A, S112A, S186A） were obtained and thus their phosphorate sites were further determined. CDK5 knockdown cell lines were detected by oil red O staining to measure the lipid accumulation and differentiation efficiency. The 10 μmol/L telmisartan was used to treat mature CDK5 knockdown 3T3-L1 adipocytes, Western blot was used to detect PPARγ and its phosphorylation level, and ELISA was used to detect the release of adiponectin in the culture supernatant.ResultsThe TNF-α stimulation had no significant effect on the expression of PPARγ in each group （all P〉0.05）, but it could up-regulate the phosphorylation of PPARγ in the TNF-α group and down-regulate the release of adiponectin （all P〈0.05）. Compared with TNF-α group, telmisartan can reduce PPARγ phosphorylation levels and up-regulate adiponectin release in different degrees, among which T5 group and T10 group had statistically significant differences （all P〈0.05）, but for T0.1 group, the difference was not significant （P〉0.05）. Compared with the 3T3-L1 wild type （WT） adipocytes, adiponectin in cell line with only S273A mutant did not respond to TNF-α stimulation and telmisartan intervention. Oil red O staining showed that silencing of CDK5 did not affect the differentiation of 3T3-L1 preadipocytes. Western blot results showed that silencing of CDK5 （shCDK5 group） had no significant effect on PPARγ expression （P〉0.05）, but it could down-regulate the phosphorylation of PPARγ and up-regulate release of adiponectin, compared with the randomized control group （shCon group） and the differences were statistically significant （all P〈0.05）. ConclusionsTelmisartan can alleviate the increased PPARγ phosphorylation and up-regulation of adiponectin content due to TNF-α stimulation. CDK5 mediates the effect of telmisartan on PPARγ signaling pathway in 3T3-L1 adipocytes. Additonally, it also demonstrated the action site of telmisartan was PPARγ Ser 273, and CDK5 is upstream kinases of PPARγ.