PI3K/AKT激动剂和抑制剂对巨噬细胞炎症反应的影响

Effect of Agonist and Inhibitor of PI3K/AKT on Inflammatory Response in Macrophages

背景:近年发现磷脂酰肌醇3激酶/丝氨酸-苏氨酸激酶(PI3K/AKT)在重度急性胰腺炎(SAP)的发病中发挥重要作用,但机制尚未明确。目的:探讨PI3K/AKT激动剂胰岛素样生长因子-Ⅰ(IGF-Ⅰ)和抑制剂wortmannin对巨噬细胞株RAW264.7 Toll样受体4(TLR4)信号通路的影响,阐明PI3K/AKT参与调节SAP炎症反应的作用机制。方法:分别以不同浓度脂多糖(LPS)、IGF-Ⅰ、wortmannin处理RAW264.7细胞,采用CCK-8实验检测细胞活性。RAW264.7细胞分为空白对照组(不予处理)、LPS组(LPS 1μg/mL)、IGF-Ⅰ组(IGF-Ⅰ100 ng/mL+LPS 1μg/mL)、wortmannin组(wortmannin 100 nmol/L+LPS 1μg/mL)和IGF-Ⅰ+wortmannin组(wortmannin 100 nmol/L+IGF-Ⅰ100 ng/mL+LPS 1μg/mL),采用ELISA法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)蛋白表达,采用real-time PCR检测TLR4、髓样分化因子88(MyD 88)、AKT、PI3K、p38丝裂原活化蛋白激酶(p38MAPK)、核因子-κB(NF-κB)mRNA表达。结果:RAW264.7细胞经不同浓度LPS、IGF-Ⅰ、wortmannin处理后,各浓度组间细胞活性无明显差异(P>0.05)。LPS组、IGF-Ⅰ组、wortmannin组、IGF-Ⅰ+wortmannin组TNF-α、IL-6表达水平均较空白对照组显著升高(P<0.05);wortmannin组TNF-α、IL-6表达水平较LPS组和IGF-Ⅰ组显著降低(P<0.05);IGF-Ⅰ+wortmannin组TNF-α、IL-6表达水平较IGF-Ⅰ组显著降低(P<0.05)。LPS组AKT、PI3K、TLR4及其下游分子MyD 88、p38MAPK、NF-κB mRNA表达均显著高于空白对照组(P<0.05);IGF-Ⅰ组上述指标较LPS组进一步升高,差异有统计学意义(P<0.05);wortmannin组上述指标较LPS组和IGF-Ⅰ组显著降低(P<0.05);IGF-Ⅰ+wortmannin组上述指标显著高于wortmannin组(P<0.05),但较IGF-Ⅰ组显著降低(P<0.05)。结论:PI3K/AKT可能通过调节巨噬细胞中的TLR4及其下游分子影响促炎细胞因子表达,从而参与SAP炎症反应的发生。

Background： Phosphoinositide 3-kinase / serine-threonine kinase（ PI3 K / AKT） has been found playing an important role in the pathogenesis of severe acute pancreatitis（ SAP） in recent years,but the underlying mechanism has not been clarified. Aims： To investigate the role of PI3 K / AKT in regulating the inflammatory response in SAP by evaluating the effect of insulin-like growth factor-Ⅰ（ IGF-Ⅰ） and wortmannin,the agonist and inhibitor of PI3 K / AKT on Toll-like receptor 4（ TLR4） signaling pathway in macrophage cell line RAW264. 7. Methods： RAW264. 7 cells were treated with different concentrations of lipopolysaccharide（ LPS）, IGF-Ⅰ and wortmannin, respectively, and cell viability was determined by CCK-8 assay. RAW264. 7 cells were divided into blank control group（ no treatment）,LPS group（ LPS1 μg / mL）,IGF-Ⅰ group（ IGF-Ⅰ 100 ng / mL ＋ LPS 1 μg / mL）,wortmannin group（ wortmannin 100 nmol / L ＋ LPS1 μg / mL） and IGF-Ⅰ ＋ wortmannin group（ wortmannin 100 nmol / L ＋ IGF-Ⅰ 100 ng / mL ＋ LPS 1 μg / mL）. Protein expressions of tumor necrosis factor-α（ TNF-α） and interleukin-6（ IL-6） were detected by ELISA; mRNA expressions of TLR4,myeloid differentiation factor 88（ MyD 88）,AKT,PI3 K,p38 mitogen-activated protein kinase（ p38MAPK） and nuclear factor-κB（ NF-κB） were determined by real-time PCR. Results： After treated with LPS,IGF-Ⅰ and wortmannin,respectively,no differences in cell viability of RAW264. 7 cells were found between different concentrations groups（ P〈 0. 05）. Protein expressions of TNF-α and IL-6 in LPS,IGF-Ⅰ,wortmannin and IGF-Ⅰ ＋ wortmannin groups were significantly higher than those in blank control group（ P〈 0. 05）. Protein expressions of TNF-α and IL-6 in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups（ P〈 0. 05）,and those in IGF-Ⅰ ＋ wortmannin group were significantly lower than those in IGF-Ⅰ group（ P〈 0. 05）. In LPS group,mRNA expressions of AKT and PI3 K as well as TLR4 and its downstream molecules MyD 88,p38 MAPK and NF-κB were significantly higher than those in blank control group（ P〈 0. 05）. Expressions of all above-mentioned mRNAs in IGF-Ⅰ group were further increased and significantly higher than those in LPS group（ P〈 0. 05）. Expressions of all above-mentioned mRNAs in wortmannin group were significantly lower than those in LPS and IGF-Ⅰ groups（ P〈 0. 05）,and those in IGF-Ⅰ ＋ wortmannin group were significantly higher than those in wortmannin group（ P〈 0. 05）,but significantly lower than those in IGF-Ⅰ group（ P〈 0. 05）. Conclusions： PI3 K / AKT might regulate TLR4 signaling pathway and its downstream molecules in macrophages,thereby affects the expressions of inflammatory cytokines and being involved in the pathogenesis of inflammatory response in SAP.