Phosphoinositide 3-kinase/serine-threonine kinase (PI3K/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 PI3K/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 PI3K/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 (LPS 1 μg/mL),IGF-Ⅰ group (IGF-Ⅰ 100 ng/mL +LPS 1 μg/mL),wortmannin group (wortmannin 100 nmol/L +LPS 1 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 (MyD88),AKT,PI3K,p38 mitogen-activated protein kinase (p38MAPK)and nuclear factor-κB (NF-κB)were determined by real-time PCR.Results:After treated with LPS,IGF-Ⅰand wortmannin, respectively,no differences in cell viability of RAW264.7 cells were found between different concentrations groups (P>0.05).Protein expressions of TNF-αand 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-αand 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 PI3K as well as TLR4 and its downstream molecules MyD88,p38MAPK 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:PI3K/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.%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(MyD88)、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MyD88、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.
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