mTORC1/2双重抑制剂OSI-027抑制高氧诱导的肺成纤维细胞增殖和分化

mTORC1/2 dual inhibitor OSI-027 attenuates proliferation and differentiation of lung fibroblasts exposed to hyperoxia

目的分析哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)复合物1/2(mTOR complex 1/2,mTORC1/2)双重抑制剂OSI-027对高体积分数氧(高氧)所致人胚肺成纤维细胞增殖和分化的抑制作用。方法高氧(95%O_(2))处理人胚肺成纤维细胞MRC-5建立增殖分化模型,分为对照组、高氧组、高氧+OSI-027组和高氧+雷帕霉素组。Western blot检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、I型胶原蛋白(collagen type I,Col I)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白D1(cyclin D1)、RhoA、Rho相关含卷曲螺旋蛋白激酶1(Rho-associated coiled-coil-containing protein kinase 1,ROCK1)、蛋白激酶B(protein kinase B,PKB/AKT)、p-AKT和mTOR的表达;CCK-8实验检测细胞活力;流式细胞术检测细胞周期。结果与对照组相比,PCNA、cyclin D1、Col I和α-SMA表达随高氧处理时间增加而增加(P<0.05)。与高氧组相比,OSI-027及雷帕霉素干预后,细胞活力下降,细胞周期被抑制在G1期(P<0.05)。高氧+OSI-027组PCNA、cyclin D1、Col I及α-SMA蛋白表达明显低于高氧组和高氧+雷帕霉素组(P<0.05)。高氧组mTOR信号通路蛋白RhoA、ROCK1、AKT、p-AKT和mTOR表达显著增高(P<0.05),但经OSI-027处理后均显著下降(P<0.05)。结论高氧可促进MRC-5细胞以时间依赖的方式增殖和分化,同时伴有mTOR信号通路活化。OSI-027可通过抑制mTOR通路抑制高氧处理后MRC-5细胞的增殖和分化,有望为临床干预高氧肺纤维化提供新的靶点。

AIM To investigate the inhibitory effect of mammalian target of rapamycin(mTOR)complex 1/2(mTORC1/2)dual inhibitor OSI-027 on the proliferation and differentiation of lung fibroblasts induced by high volume fraction of oxygen(hyperoxia),so as to explore the occurrence and development of pulmonary fibrosis caused by hyperoxia.METHODS Human fetal lung fibroblast MRC-5 cells were induced by hyperoxia.The cells were divided into control group,hyperoxia group,hyperoxia+OSI-027 group,and hyperoxia+rapamycin(Rapa)group.Western blot was used to detected the expression levels of proliferating cell nuclear antigen(PCNA),cyclin D1,α-smooth muscle actin(α-SMA),collagen type I(Col I),RhoA,Rho-associated coiled-coil-containing protein kinase 1(ROCK1),protein kinase B(PKB/AKT),p-AKT,and mTOR.CCK-8 assay was used to detected the cell viability,and flow cytometry was used to detected the cell cycle.RESULTS Compared with control group,hyperoxia increased the expression levels of PCNA,cyclin D1,Col I andα-SMA protein(P<0.05).Compared with hyperoxia group,cell viability was decreased in hyperoxia+OSI-027 group and hyperoxia+Rapa group(P<0.05).The expression levels of PCNA,cyclin D1,Col I andα-SMA in hyperoxia+OSI-027 group was significantly lower than those in hyperoxia group and hyperoxia+Rapa group(P<0.05).The protein levels of RhoA,ROCK1,AKT,p-AKT and mTOR were increased in hyperoxia group,but decreased significantly after OSI-027 treatment(P<0.05).CONCLUSION Hyperoxia promotes the proliferation and differentiation of lung fibroblasts via mTOR signaling pathway.OSI-027 attenuates the proliferation and differentiation of lung fibroblasts under hyperoxia exposure by inhibiting mTOR signaling pathway,which is expected to provide a new target for clinical intervention in pulmonary fibrosis.