高糖对THP-1细胞TLR4介导的信号调节的影响

Effects of high glucose on TLR4-mediated signal regulation in THP-1 cells

目的探讨高糖状态下巨噬细胞株THP-1经TLR4不同配体刺激后信号通路激活的改变,明确巨噬细胞激活在糖尿病肾病中的炎症机制。方法用正常和高糖培养THP-1细胞,用TLR4的配体LPS和HSP60刺激,收集上清和细胞,采用ELISA和Real-time PCR检测IL-10、IL-1β、TNF-α的蛋白及m RNA的表达水平,用Western blot检测NF-κB(P65)、AKT(Ser473)介导的信号通路分子的表达水平。结果高糖刺激下IL-10 m RNA表达的高峰值晚于正常组,出现延迟现象,但蛋白表达无明显变化。而IL-1β和TNF-α无论m RNA和蛋白水平的表达均高于正常对照组。Western blot结果显示LPS刺激细胞0、15、30、60、120、180 min,正常组P-NF-κB p65的表达于60 min达到高峰,而高糖组15 min时达到高峰,P-AKT的表达于60 min时表达增高,高糖组在同一时间点总体高于正常组;高糖状态下LPS和HSP60刺激细胞0、30、60、180 min后,P-NF-κB p65、P-AKT(Ser473)均高于对照组。结论高糖状态能促进TLR4介导的促炎因子的表达,不同配体刺激的结果相似。

The study aimed to explore the change of signal pathway activation in macrophage cell line THP-1after stimulation with different TLR4 ligands under high glucose condition,and identify the mechanism of macrophage activation in inflammation of diabetic nephropathy.THP-1 cells were stimulated with LPS and HSP60 in normal or high glucose condition;ELISA and RT-PCR was used to detect the protein and mRNA levels of IL-10,IL-1β,and TNF-α;the expression level of NF-κB-and AKT-mediated signaling pathway molecules was detected by Western blotting.Data showed that the IL-10 mRNA expression peak in high glucose group appeared later than normal group;however,there were no obvious changes in IL-10 protein expression confirmed by Western blotting.The mRNA and protein expression levels of IL-1β and TNF-α in high glucose group were both higher than those in normal group.In normal group the expression level of P-NF-κB p65 reached the peak at 60 minutes post LPS stimulation,and the level in the high glucose group reached the peak at 15 minutes post LPS stimulation.While the expression of P-AKT increased at 60 min post LPS stimulation and was higher than the normal group at following time.The cells demonstrated higher levels of P-NF-κB p65 and P-AKT expression at 0,30,60,180 min post stimulation of LPS and HSP60,as compared with control group.In conclusion,high glucose can promote the expression of pro-inflammatory factor mediated by TLR4,and stimulation of different ligands come to the similar results.