PI3K/Akt/mTOR通路介导巨噬细胞自噬影响矽尘致肺成纤维细胞表型转化

Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure

目的:磷脂酰肌醇-3-激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin,PI3K/Akt/mTOR)信号通路是自噬相关主要信号通路之一。自噬在硅沉着病纤维化形成过程中发挥关键作用。肺成纤维细胞向肌成纤维细胞表型转化是硅沉着病由炎症期进入纤维化期的标志之一。本研究旨在探讨PI3K/Akt/mTOR通路是否通过介导巨噬细胞自噬影响矽尘诱导的肺成纤维细胞向肌成纤维细胞表型转化。方法:采用100 ng/mL佛波酯诱导人单核细胞白血病细胞系THP-1细胞24 h获得巨噬细胞。以不同浓度(0、25、50、100、200、400μg/mL)二氧化硅(silicon dioxide,SiO_(2))粉尘悬浮液染毒巨噬细胞不同的时间(0、6、12、24、48 h)。采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测巨噬细胞存活率,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)法检测巨噬细胞上清液中转化生长因子β1(transforming growth factor-β1,TGF-β1)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的含量。采用Transwell技术建立巨噬细胞和HFL-1细胞共培养体系,设空白对照组、SiO_(2)组、LY294002组、SC79组、LY294002+SiO_(2)组、SC79+SiO_(2)组。LY294002+SiO_(2)组、SC79+SiO_(2)组分别用LY294002(PI3K抑制剂)、SC79(Akt激活剂)预处理巨噬细胞18 h、24 h,再用SiO_(2)(100μg/mL)粉尘悬浮液染毒巨噬细胞12 h。采用免疫荧光法检测巨噬细胞中微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达情况;蛋白印迹法检测巨噬细胞PI3K、Akt、mTOR、Beclin-1、LC3的蛋白质表达水平及HFL-1细胞中Ⅲ型胶原蛋白(collagen Ⅲ,Col Ⅲ)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)、基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)、金属蛋白酶组织抑制因子-1(tissue matrix metalloproteinase inhibitor-1,TIMP-1)的蛋白质表达水平。结果:用不同浓度的SiO_(2)粉尘悬浮液染毒巨噬细胞12 h,巨噬细胞的存活率随SiO_(2)浓度的增加逐渐降低,与0μg/mL组比较,100、200、400μg/mL组细胞存活率均明显降低,细胞上清液中TGF-β1和TNF-α含量均明显增加(均P<0.05);用100μg/mL SiO_(2)粉尘悬浮液染毒巨噬细胞,巨噬细胞的存活率随染毒时间的延长逐渐降低,与0 h组比较,6、12、24、48 h组细胞存活率明显降低(均P<0.05),细胞上清液中TGF-β1和TNF-α含量均明显增加,Beclin-1和LC3Ⅱ的蛋白质表达水平均明显上调(均P<0.05)。免疫荧光法结果显示:100μg/mL的SiO_(2)粉尘处理巨噬细胞12 h后LC3的荧光呈点状聚集,且荧光强度明显高于空白对照组(P<0.05)。与空白对照组相比,SiO_(2)组HFL-1细胞中Col Ⅲ、FN、α-SMA、MMP-1、TIMP-1的蛋白质表达均上调(均P<0.05);与SiO_(2)组相比,LY294002+SiO_(2)组中巨噬细胞PI3K、Akt、mTOR的蛋白质表达均下调(均P<0.05),LC3Ⅱ和Beclin-1的蛋白质表达均上调(均P<0.05),细胞上清液中TGF-β1和TNF-α含量均降低(均P<0.01),HFL-1细胞中Col Ⅲ、FN、α-SMA、MMP-1、TIMP-1的蛋白质表达均下调(均P<0.05);SC79+SiO_(2)组巨噬细胞PI3K、Akt、mTOR的蛋白质表达均上调(均P<0.05),LC3Ⅱ和Beclin-1的蛋白质表达均下调(均P<0.05),细胞上清液中TGF-β1和TNF-α含量均增加(均P<0.01),HFL-1细胞中Col Ⅲ、FN、α-SMA、MMP-1、TIMP-1的蛋白质表达均上调(均P<0.05)。结论:矽尘染毒引起巨噬细胞的PI3K/Akt/mTOR通路抑制、自噬发生及炎症因子分泌增加,进而促进HFL-1细胞向肌成纤维细胞表型转化;调控PI3K/Akt/mTOR通路可通过影响矽尘对巨噬细胞的自噬诱导及炎症因子分泌,从而调节HFL-1细胞向肌成纤维细胞表型转化。

Objective:The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway is one of the main signaling pathways related to autophagy.Autophagy plays a key role in the formation of silicosis fibrosis.The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis.This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.Methods:The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h.Macrophages were exposed to different concentrations(0,25,50,100,200,400μg/mL)and different times(0,6,12,24,48 h)of SiO_(2) dust suspension.The survival rate of macrophages was measured by cell counting kit-8(CCK-8)method.Enzyme linked immunosorbent assay(ELISA)was used to measure the contents of transforming growth factor-β1(TGF-β1)and tumor necrosis factor-α(TNF-α)in the cell supernatant.The co-culture system of macrophages and HFL-1 cells was established by transwell.A blank control group,a SiO_(2) group,a LY294002 group,a SC79 group,a LY294002+SiO_(2) group,and a SC79+SiO_(2) group were set up in this experiment.Macrophages in the LY294002+SiO_(2) group were pretreated with LY294002(PI3K inhibitor)for 18 hours,and macrophages in the SC79+SiO_(2) group were pretreated with SC79(Akt activator)for 24 hours,and then exposed to SiO_(2)(100μg/mL)dust suspension for 12 hours.The expression of microtubule-associated protein 1 light chain 3(LC3)protein in macrophages was detected by the immunofluorescence method.The protein expressions of PI3K,Akt,mTOR,Beclin-1,LC3 in macrophages,and collagen III(Col III),α-smooth muscle actin(α-SMA),fibronectin(FN),matrix metalloproteinase-1(MMP-1),tissue metalloproteinase inhibitor-1(TIMP-1)in HFL-1 cells were measured by Western blotting.Results:After the macrophages were exposed to SiO_(2) dust suspension of different concentrations for 12 h,the survival rates of macrophages were gradually decreased with the increase of SiO_(2) concentration.Compared with the 0μg/mL group,the survival rates of macrophages in the 100,200,and 400μg/mL groups were significantly decreased,and the concentrations of TGF-β1 and TNF-αin the cell supernatant were obviously increased(all P<0.05).When 100μg/mL SiO_(2) dust suspension was applied to macrophages,the survival rates of macrophages were decreased with the prolonged exposure time.Compared with the 0 h group,the survival rates of macrophages were significantly decreased(all P<0.05),the concentrations of TGF-β1 and TNF-αin the cell supernatant were significantly increased,and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6,12,24,and 48 h groups(all P<0.05).Immunofluorescence results demonstrated that after exposure to SiO_(2)(100μg/mL)dust for 12 h,LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group(P<0.05).Compared with the blank control group,the protein expressions of Col III,FN,α-SMA,MMP-1,and TIMP-1 in HFL-1 cells were up-regulated in the SiO_(2) group(all P<0.05).Compared with the SiO_(2) group,the protein expressions of PI3K,Akt,and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages(all P<0.05),the contents of TNF-αand TGF-β1 in the cell supernatant were decreased(both P<0.01),and the protein expressions of Col III,FN,α-SMA,MMP-1,and TIMP-1 in HFL-1 cells were down-regulated(all P<0.05)in the LY294002+SiO_(2) group.Compared with the SiO_(2) group,the protein expressions of PI3K,Akt,and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages(all P<0.05),the contents of TNF-αand TGF-β1 in the cell supernatant were increased(both P<0.01),and the protein expressions of Col III,FN,α-SMA,MMP-1,and TIMP-1 in HFL-1 cells were up-regulated(all P<0.05)in the SC79+SiO_(2) group.Conclusion:Silica dust exposure inhibits the PI3K/Akt/mTOR pathway,increases autophagy and concentration of inflammatory factors in macrophages,and promotes the phenotype transformation of HFL-1 cells into myofibroblasts.The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure,and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.