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  • br Materials and methods br Results br


    Materials and methods
    Discussion Cellular senescence was first termed by Hayflick and Moorhead to describe irreversible growth arrest in human diploid cells [45]. It can also be induced by DNA damaging agents, which is attributed to telomere shorting [8]. Numerous examples of senescence in aging pathology have been reported [46], and mounting evidence has shown that cellular senescence is also linked to IPF. However, recent reports regarding the role of this process in the pathogenesis of IPF are still controversial. Both the alveolar epithelial cell senescence [11] and fibroblast senescence [12] impede fibrosis resolution and contribute to the lung fibrosis development. On the other hand, miR-34a restrains fibrotic response in the lungs by promoting fibroblast senescence [47]. In this study, we tested the effects of CAE on both senescent mouse primary lung fibroblasts and human lung fibroblasts, and demonstrated that it could attenuate fibroblast senescence and trans-differentiation into myofibroblasts. These findings suggested that cellular senescence contributes to the development of pulmonary fibrosis and treatment with CAE via anti-senescence might be beneficial and ameliorate the fibrotic burden. The characteristic phenotype of senescent cells is expression of SA-β-gal and, induction of P16INK4a and P21. These senescent cells can secrete lots of SASP, including cytokines, chemokines, matrix remodelling proteases and growth factors, which can paracrinely promote proliferation and tissue deterioration [8]. Our results illustrated that CAE could decrease the number of senescent fibroblasts and regulate senescence. First, we used P16INK4a and P21 as markers to detect cellular senescence, as they function as furin regulators and are up-regulated in senescent cells. Our present animal experiments demonstrated that P16INK4a and P21 were co-localized with the myofibroblasts marker α-SMA in the murine fibrotic lung. While, treatment with CAE down-regulated the expression of P16INK4a and p21, together with α-SMA in a dose-dependent manner. Next, we further measured the inhibitory effect of CAE on cellular senescence in vitro. After exposure to etoposide, fibroblasts showed a senescent state, as indicated by morphological enlargement, increased SA-β-gal activity, and enhanced expression of P16INK4a and P21, and CAE treatment significantly inhibited fibroblasts senescence. In addition, we determined that senescent fibroblasts were a potent source of the pro-fibrotic components of SASP, particularly capable of secreting MCP-1, CTGF, PDGF-a, PDGF-b, α-SMA, and collagen I, which were all profibrotic factors involved in the pathogenesis of IPF, however, they were down-regulated by CAE treatment. Together, these findings revealed that CAE can alleviate pulmonary fibrosis by regulating fibroblast senescence. It is well known that the tumor suppressor p53 plays an important role in regulating the cell cycle and cellular senescence [43,46,48]. For example, FOXO4 could interact with P53, which selectively induced apoptosis in senescent cells [48]. In addition, loss of MECP2 was found to lead to the induction of P53 and senescence [49]. Cell cycle arrest is a typical feature of cellular senescence, which is a consequence of cross-talk between cyclins and CDKs, and especially, Cyclin D1 and CDK4 are required for the G1/S transition. Our data showed that CAE could dose-dependently down-regulate P53, and its inhibitor PTF-α exhibited an anti-senescence effect based on decreasing SA-β-gal activity, inhibiting P16INK4a and P21, as well as promoting the expression of Cyclin D1 and CDK4. Furthermore, the anti-senescence effect was enhanced when PTF-α was added with CAE together. Therefore, our results indicated that the inhibition of fibroblast senescence by P53 might be a therapeutic target for the inactivation of fibroblasts by CAE.