PI3K/AKT激动剂和抑制剂对巨噬细胞炎症反应的影响
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摘要
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.
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.
引文
1 Xu P,Wang J,Yang ZW,et al.Regulatory roles of the PI3K/AKT signaling pathway in rats with severe acute pancreatitis[J].PLo S One,2013,8(11):e81767.
2 Zhao W,Ma G,Chen X.Lipopolysaccharide induced LOX-1 expression via TLR4/My D88/ROS activated p38MAPK-NF-κB pathway[J].Vascul Pharmacol,2014,63(3):162-172.
3 Cantley LC.The phosphoinositide 3-kinase pathway[J].Science,2002,296(5573):1655-1657.
4 康新,王立志,王屹刚,等.重症急性胰腺炎肺损伤时磷脂酰肌醇3激酶/蛋白激酶B信号转导通路的表达[J].中华医学杂志,2010,90(11):732-737.
5 Zhao M,Zhou A,Xu L,et al.The role of TLR4-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in neuroinflammation in hippocampal neurons[J].Neuroscience,2014,269:93-101.
6 Li Y,Zhou ZG,Zhang J,et al.Microcirculatory detection of Toll-like receptor 4 in rat pancreas and intestine[J].Clin Hemorheol Microcirc,2006,34(1-2):213-219.
7 Sharif R,Dawra R,Wasiluk K,et al.Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice[J].Gut,2009,58(6):813-819.
8 Sawa H,Ueda T,Takeyama Y,et al.Role of toll-like receptor 4 in the pathophysiology of severe acute pancreatitis in mice[J].Surg Today,2007,37(10):867-873.
9 Kim TH,Kim SJ,Lee SM.Stimulation of theα7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation[J].J Infect Dis,2014,209(10):1668-1677.
2 Zhao W,Ma G,Chen X.Lipopolysaccharide induced LOX-1 expression via TLR4/My D88/ROS activated p38MAPK-NF-κB pathway[J].Vascul Pharmacol,2014,63(3):162-172.
3 Cantley LC.The phosphoinositide 3-kinase pathway[J].Science,2002,296(5573):1655-1657.
4 康新,王立志,王屹刚,等.重症急性胰腺炎肺损伤时磷脂酰肌醇3激酶/蛋白激酶B信号转导通路的表达[J].中华医学杂志,2010,90(11):732-737.
5 Zhao M,Zhou A,Xu L,et al.The role of TLR4-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in neuroinflammation in hippocampal neurons[J].Neuroscience,2014,269:93-101.
6 Li Y,Zhou ZG,Zhang J,et al.Microcirculatory detection of Toll-like receptor 4 in rat pancreas and intestine[J].Clin Hemorheol Microcirc,2006,34(1-2):213-219.
7 Sharif R,Dawra R,Wasiluk K,et al.Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice[J].Gut,2009,58(6):813-819.
8 Sawa H,Ueda T,Takeyama Y,et al.Role of toll-like receptor 4 in the pathophysiology of severe acute pancreatitis in mice[J].Surg Today,2007,37(10):867-873.
9 Kim TH,Kim SJ,Lee SM.Stimulation of theα7 nicotinic acetylcholine receptor protects against sepsis by inhibiting Toll-like receptor via phosphoinositide 3-kinase activation[J].J Infect Dis,2014,209(10):1668-1677.