Citrus comprising some of the
Citrus , comprising some of the most widely cultivated fruit crops worldwide, has been consumed as a food and dietary supplement. According to traditional Chinese medicine, dry pericarp of Citrus, as a Chinese herbal remedy, is capable of regulating Qi and expelling phlegm, and has been used to treat pulmonary diseases for centuries. Phytochemical studies demonstrated that Citrus reticulata (the mandarin orange) mainly contains flavonoids, volatile oils, and alkaloids . It has been reported that citrus and its active ingredients exerts antimicrobial, antioxidant, anticancer and anti-fibrosis activities, and have been used for the prevention and treatment of hypertension, PU-WS13 cancer, ulcerative colitis, and liver and lung fibrosis in mice [, , ]. Our previous studies demonstrated that citrus ethanol extracts contained the active constituents against pulmonary fibrosis in vitro and in vivo. While, the citrus alkaline extracts (CAE) exhibited anti-inflammation properties and suppressed collagen deposition through regulating matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tumor necrosis factor-α (TNF-α) [34,35]. Recently, we have identified the effects of CAE on inducing apoptosis in the primary fibroblasts isolated from mice with bleomycin-induced pulmonary fibrosis . However, the effects of CAE on senescent fibroblasts in terms of the inhibition of pulmonary fibrosis are still unclear.
Materials and methods
Discussion Cellular senescence was first termed by Hayflick and Moorhead to describe irreversible growth arrest in human diploid cells . It can also be induced by DNA damaging agents, which is attributed to telomere shorting . Numerous examples of senescence in aging pathology have been reported , 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  and fibroblast senescence  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 . 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 . 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 cell cycle 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.