IGF-1促进肺泡上皮细胞衰老致肺纤维化进展的分子机制研究

Molecular mechanism of IGF-1 promoting alveolar epithelial cells senescence for participating in pulmonary fibrosis progress

目的探讨胰岛素样生长因子-1(IGF-1)在肺泡上皮细胞衰老致肺纤维化中的作用。方法用博来霉素(BLM)构建小鼠肺纤维化模型,免疫组织化学观察IGF-1的表达变化。利用IGF-1刺激A549细胞株(72 h)建立细胞衰老模型,β-半乳糖苷酶染色观察衰老阳性细胞数量。免疫荧光观察IGF-1对A549细胞衰老关键蛋白P16、P21表达的影响。ELISA法观察IGF-1刺激A549细胞后转化生长因子-β1(TGF-β1)、基质金属蛋白酶-9(MMP-9)的表达变化。免疫印迹检测IGF-1对A549细胞转分化的影响。RT-PCR检测IGF-1对下游信号通路中磷酯酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)的影响。结果与正常小鼠肺脏相比,IGF-1在肺纤维化小鼠肺脏中广泛表达。与对照组相比,IGF-1刺激A549细胞72 h后的衰老阳性细胞数量明显增多(P<0.05),细胞衰老关键蛋白P16、P21表达也明显上调(P<0.05)。IGF-1刺激A549细胞72 h后,培养基中细胞衰老表型(SASP)主要成分TGF-β1、MMP-9的表达量要高于对照组(P<0.05)。与对照组相比,IGF-1使A549细胞发生了转分化现象,α-SMA、CollagenⅠ的表达升高(P<0.05)。与对照组相比,IGF-1明显增加A549细胞中PI3K、AKT的表达(P<0.05)。结论IGF-1是促进肺泡上皮细胞衰老致肺纤维化进展的重要因素,并且与激活IGF-1/PI3K/AKT信号通路相关。

Objective To investigate the effect of insulin-like growth factor-1(IGF-1)on alveolar epithelial cells senescence caused pulmonary fibrosis.Methods Bleomycin(BLM)was used to establish the mice pulmonary fibrosis model,and the expression change of IGF-1 was observed by immunohistochemistry.IGF-1 was used to stimulate A549 cell line(72 h)to establish the cell senescence model.The number of senescent positive cells was observed byβ-galactosidase staining.The effect of IGF-1 on the expression of P16 and P21 was observed by immunofluorescence.The expression changes of transforming growth factor-1(TGF-1)and matrix metalloproteinase-9(MMP-9)after IGF-1 stimulating A549 cells was observed by ELISA.The effect of IGF-1 on transdifferentiation of A549 cells was detected by Western blot.The effects of IGF-1 on PI3K and p-AKT in the downstream signaling pathway were detected by RT-PCR.Results Compared with the normal mice lung,IGF-1 was widely expressed in the lungs of BLM mice.Compared with the control group,the number of senescence positive cells after 72 h of IGF-1 stimulating A549 cells was significantly increased(P<0.05),and the expressions of cell senescence key proteins P16 and P21 were also significantly up-regulated(P<0.05).After IGF-1 stimulating A549 cells for 72 h,the expression levels of main components TGF-β1 and MMP-9 in the medium were higher than those in the control group(P<0.05).Compared with the control group,IGF-1 made the transdifferentiation phenomenon occurrence of A549 cells(EMT),and increase theα-SMA and CollagenⅠexpression(P<0.05).Compared with the control group,IGF-1 significantly increased the expressions of PI3K and p-AKT in A549 cells(P<0.05).Conclusion IGF-1 is an important factor in promoting the aging of alveolar epithelial cells and the progression of pulmonary fibrosis,moreover which is related to the activation of IGF-1/PI3K/AKT signaling pathway.