慢性高糖抑制小鼠RAW264.7巨噬细胞AKT磷酸化并促进其Ml型极化

Chronic high glucose inhibits AKT phosphorylation and promotes Ml polarization of mouse RAW264.7 macrophages

目的研究慢性高糖对小鼠巨噬细胞的影响及机制.方法以正常生长液和含(40、60)mmol/L葡萄糖的生长液培养小鼠RAW264.7巨噬细胞1、3、5、7 d,相差显微镜观察细胞形态,CCK-8法检测细胞增殖能力,Western blot法检测细胞白细胞介素1β(IL-1β)和IL-1O蛋白水平,ELISA检测培养细胞上清液IL-Iβ和IL,-10分泌量;实时定量PCR法检测细胞蛋白激酶B(AKT)mRNA水平,Western blot法检测细胞AKT、磷酸化的AKT(p-AKT)蛋白水平.结果60 mmol/L葡萄糖处理1 d即出现较多长梭形细胞,可见伸展的伪足,且随时间延长数量增加、体积变大;处理1 d即明显抑制细胞增殖,随时间延长抑制效果有所改善;处理3,5,7d后,IL-1β蛋白水平增加、IL-10蛋白水平降低;处理1 d后IL-1β分泌显著增加,但处理3d后IL-10分泌显著减少且随时间延长进一步减少;处理3,5,7 d后,AKT蛋白磷酸化被抑制,但AKT mRNA水平未见明显变化.40 mmol/L葡萄糖处理在一定程度上发挥作用,但不如60 mmol/L葡萄糖显著和稳定.结论长期高糖刺激诱导巨噬细胞向促炎性M1型转变,诱发巨噬细胞的持续慢性炎症反应,其机制可能与抑制AKT蛋白磷酸化激活有关.

Objective To explore the effect of chronic high glucose on mouse macrophages in vitro and its mechanism.Methods RAW264.7 macrophages were cultured in normal growth medium and glucose-containing 40 or 60 mmol/L growth medium for 1,3,5 and 7 days.Cell morphology was observed by phase contrast microscopy;cell proliferation was measured by CCK-8 assay;interleukin-1β(IL-1β) and IL-10 protein levels were detected by Western blot analysis;IL-1β and IL-10 secretion were detected by ELISA;the levels of AKT mRNA in the cells were tested by real-time quantitative PCR;the levels of AKT and phosphorylated AKT(p-AKT)were examined by Western blot analysis.Results After 60 mmol/L glucose treatment for 1 day,the longer spindle cells appeared,the extended pseudopod was visible,and the number and volume increased over time.After treatment for 1 day,cell proliferation was significantly inhibited,and the inhibitory effect was ameliorated with time.After treatment for 3,5,and 7 days,IL-1β protein level increased and IL-10 protein level decreased.After treatment for 1 day,the secretion of IL-1βincreased significantly,but after treatment for 3 days,the secretion of IL-10 decreased significantly and decreased further over time.AKT protein phosphorylation was inhibited after treatment for 3,5,and 7 days,but AKT mRNA level did not change obviously.Glucose(40 mmol/L)treatment played a role to some extent,but it was not as significant and stable as 60 mmol/L glucose.Conclusion Chronic high glucose induces macrophages to transform into pro-inflammatory Ml type and the continuous chronic inflammatory response,which may be related to the inhibition of AKT protein phosphorylation.