吡格列酮对单钠尿酸盐诱导THP-1细胞炎症反应的影响及其机制

Effects of pioglitazone on inflammatory response of THP-1 cells induced by monosodium urate

目的探讨吡格列酮对单钠尿酸盐(MSU)诱导人单核细胞(THP-1细胞)炎症反应的影响及其机制。方法将THP-1细胞体外培养,分为PBS预处理组、T0070907预处理组、Piog预处理组,将上述三组细胞加佛波酯(PMA)100ng/mL预处理24h,分别以PBS、T0070907、Piog预处理1h,再各分两组,一组不予干预,一组以MSU(100μg/mL,1mL)刺激16h,继续在37℃、5%CO2环境中培养,得到PBS组、T0070907组、Piog组、MSU组、MSU+T0070907组、MSU+Piog组。用IL-1β抗体标记THP-1细胞。用流式细胞仪检测各组THP-1细胞构成比,ELISA法检测培养上清液IL-1β水平。再将体外培养的THP-1细胞分为四组,分别以PBS、MSU、Piog、LY294002干预,得到PBS组、PBS+MSU组,PBS+MSU+Piog组、PBS+MSU+Piog+LY294002组,Westernblotting法检测各组p-蛋白激酶B(AKT)、AKT、PPAR-γ蛋白表达。结果与其他组比较,MSU组、MSU+T0070907组IL-1β标记阳性THP-1细胞构成比升高(P均<0.05)。六组THP-1细胞上清液IL-1β水平差异有统计学意义(P<0.05)。MSU组、MSU+T0070907组IL-1β水平较PBS组、T0070907组、Piog组升高,而MSU+Piog组较MSU组、MSU+T0070907组下降(P均<0.05)。PBS组、PBS+MSU组、PBS+MSU+Piog组、PBS+MSU+Piog+LY294002组p-AKT、AKT、PPAR-γ蛋白相对表达量比较差异有统计学意义(P均<0.05)。结论吡格列酮可抑制MSU诱导THP-1细胞的炎症反应,其机制可能通过活化PPAR-γ/AKT通路实现。

Objective To investigate the effect of pioglitazone on inflammatory response of THP-1 cells induced by monosodium urate(MSU)and its mechanism.Methods The THP-1 cells were cultured in vitro.Pioglitazone and T0070907,an specific inhibitor of peroxisome proliferator-activated receptor-γ(PPAR-γ),were used to treat the MSU-induced THP-1 cells.The cells were divided into three groups and pre-treated with PMA for 24 h,and then PBS,T0070907,Piog were separately added to the three groups and treated for 1 h.After that,all the three groups were then divided into two groups.One group was not treated,and the other group was stimulated with MSU for 16 h in the same environment of 37℃and 5%CO2,and then this group were divided into six groups:PBS,T0070907,Piog,MSU,MSU+T0070907 and MSU+Piog groups.The above cells were labeled with IL-1βantibody,and then were detected by flow cytometry to research the composition ratio.IL-1βlevel in the THP-1 cells were assessed by ELISA.To further investigate the mechanism of Piog,LY294002 was used to block the expression of protein kinase B(AKT).The THP-1 cells were then divided into four groups:PBS,PBS+MSU,PBS+MSU+Piog,and PBS+MSU+Piog+LY294002 groups.Then the protein expression of p-AKT,AKT,and PPAR-γin THP-1 cells was detected by Western blotting.Results Compared with the other groups,the composition ratio of IL-1β-labelled positive THP-1 cells in the MSU group and MSU+T0070907 group increased(P<0.05).Statistically significant difference was found in the levels of IL-1βin the supernatants of THP-1 cells in the six groups(P<0.05).The levels of IL-1βin the MSU group and MSU+T0070907 group were higher than those in the PBS group,T0070907 group and Piog group,while MSU+The Piog group was lower than the MSU group and the MSU+T0070907 group(all P<0.05).Statistically significant differences were found in the relative protein expression levels of p-AKT,AKT and PPAR-γamong the PBS group,PBS+MSU group,PBS+MSU+Piog group,and PBS+MSU+Piog+LY294002 group(all P<0.05).Conclusion Pioglitazone inhibits the MSU-induced inflammation in THP-1 cells by activating the PPAR-γ/AKT pathway.