Effect And Mechanism Of Fisetin On Alveolar Epithelial Cell Senescence And Pulmonary Fibrosis In Mice

Objective: Pulmonary fibrosis(PF)is a relentlessly progressive interstitial lung disease characterized by abnormal lung tissue repair and excessive collagen deposition,which often leads to lung function damage or respiratory failure.The pathogenesis of pulmonary fibrosis is not fully understood and satisfactory therapies are not yet available.Cell senescence is a state of cell cycle arrest in which cells shut down proliferation but retain metabolic activity.Senescent cells can secrete a variety of proinflammatory cytokines,growth factors,chemokines and matrix metalloproteinases,described as senescence associated secretory phenotype(SASP).Recent studies have shown that accelerated senescence of alveolar epithelial cells is involved in the pathogenesis of PF.Targeting senescent cells or blocking proinflammatory secretome is expected to be a new treatment for PF.Fisetin(FIS),a natural non-toxic flavonoid,has anti-inflammatory,anti-oxidant and anti-aging properties.However,the role of FIS in alveolar epithelial cell senescence and PF has not been explained.This study was designed to investigate the effect of FIS on type Ⅱ alveolar epithelial cell senescence,and to explore whether fisetin can partly alleviate PF by rescuing alveolar epithelial cells from senescence.Materials and Methods: 1.In vitro 1.1 Establish the cell model of type Ⅱ alveolar epithelial cell senescence: A549 cellswere stimulated with different doses of bleomycin(0,1,2,5,10 μg/ml)for 2 to 3 days respectively.Cell morphology was observed,the senescence associated β-glucosidase(SA-β-GAL)and p21 protein were detected to evaluate the model and screen the appropriate concentration and time.1.2 Screen the optimal concentration of fisetin: A549 cells were stimulated with different concentrations of fisetin for 3 days.The best concentration of fitetin was determined by detecting SA-β-GAL and protein expressions of p16 and p21.1.3 In order to investigate the role and mechanism of fisetin in type Ⅱ alveolar epithelial cell senescence,A549 cells were divided into five groups: the CON group,the FIS group,the BLM group,the BLM + FIS group and the BLM + FIS + CC group.Three days later,SA-β-GAL and the protein expressions of p16,p21,AMPK,pAMPK,NF-κB p65 and NF-κB p-p65 were measured in A549 cells.1.4 In order to observe the senescence associated secretory phenotype(SASP)of type Ⅱ alveolar epithelial cells and the effect of fisetin,the culture supernatants were discarded to eliminate the effect of bleomycin.The CON group,the BLM group and the BLM + FIS group were cultured in fresh mediums.Two days later,the supernatants were collected and the levels of pro-inflammatory and fibrogenic factors were tested by ELISA to reflect SASP.1.5 In order to explore the effect of SASP of alveolar epithelial cells on fibroblasts,conditioned mediums from the CON group(N-CM),the BLM group(B-CM)and the BLM + FIS group(BF-CM)were collected to culture HELF cells with one half of concentration for 3 days.The protein expressions of α-SMA and collagen Ⅰ in HELF cells were evaluated to reflect fibroblasts differentiation and collagen synthesis.2 In vivo70 mice were randomly divided into four groups: the CON group,the FIS group,the BLM group and the BLM + FIS group.Intratracheal instillation of bleomycin was used to induce pulmonary fibrosis in mice.On day 7 after BLM treatment,mice were orally administered with fisetin once every other day for 3 weeks.On day 28,mice were euthanized.2.1 Effect of fisetin on pulmonary fibrosis in mice: the weight of mice was monitored and the survival curves were plot.The wet/dry weight ratio and hydroxyproline content of lung tissue were measured.The lung tissue sections were stained with HE and Masson,and semi-quantitative pathological scores of inflammation and fibrosis were performed.The protein and m RMA expressions of collagen Ⅰ and α-SMA in lung tissue were detected.2.2 Effect of fisetin on alveolar epithelial cell senescence in fibrotic lung tissue: SA-β-GAL staining and immunohistochemical staining of p21 were performed in lung tissue sections.The protein and m RNA expressions of p21 and p16 in lung tissue were measured.2.3 Effects of fisetin on TGF-β/Smad and AMPK/NF-κB signaling pathway: the protein expressions of TGF-β,Smad3,p-Smad3,AMPK,p-AMPK,NF-κB p65 and NF-κB p-p65 in lung tissue were evaluated.2.4 Effect of fisetin on inflammatory response in lung tissue: the levels of proinflammatory factors and pro-fibrogenic factors in BALF were detected.Results: 1.In vitro 1.1 The cell model of type Ⅱ alveolar epithelial cell senescence was successfully constructed: with the increase of bleomycin concentration and stimulation time,A549 cells became wider and flatter,SA-β-GAL staining and the expression of p21 protein increased in a time and dose-dependent manner.A549 cells showed a senescent phenotype.5 μg/ml bleomycin and 3 days were the optimal concentration and time point for modeling.1.2 Bleomycin induced type Ⅱ alveolar epithelial cell senescence: compared with the CON group,expression of SA-β-GAL and protein levels of p16 and p21 were significantly raised in the BLM group(p < 0.01).The levels of IL-1β,IL-6,TGF-β and TNF-α were significantly raised in the supernatant of BLM group(p < 0.01).1.3 Decreased AMPK activity and increased NF-κB activity were found in senescent alveolar epithelial cells: compared with the CON group,the protein expression of pAMPK and the ratio of p-AMPK/AMPK were significantly decreased in the BLM group(p < 0.01),however,the protein level of NF-κB p-p65 and the ratio of p-p65/p65 were significantly up-regulated in the BLM group(p < 0.01).1.4 Fisetin alleviated type Ⅱ alveolar epithelial cell senescence and inflammatory phenotype: 10 μM fisetin can significantly inhibit the expression of p16,p21 and SA-β-GAL,therefore,this concentration was used for subsequent experiments.Compared with the BLM group,the positive rate of β-galactosidase(p < 0.01)and the protein levels of p16 and p21 were significantly decreased in the BLM+FIS group(p < 0.05).The levels of IL-1β,IL-6 and TGF-β were significantly decreased in the supernatant of BLM+FIS group(p < 0.01).1.5 Fisetin regulated the AMPK/NF-κB signaling pathway: compared with the BLM group,the protein expression of p-AMPK and the ratio of p-AMPK/AMPK were significantly raised in the BLM+FIS group(p < 0.05),while the protein level of NF-κB p-p65 and the ratio of p-p65/p65 were significantly decreased in the BLM+FIS group(p < 0.01).Compared with the BLM+FIS group,the protein expression of pAMPK and the ratio of p-AMPK/AMPK were significantly down-regulated in the BLM+FIS+CC group(p < 0.05),while the protein level of NF-κB p-p65 and the ratio of p-p65/p65 were significantly up-regulated in the BLM+FIS+CC group(p < 0.01).2 In vivo 2.1 The mouse model of pulmonary fibrosis was established successfully: a large number of inflammatory cells infiltration and obvious collagen deposition were observed in the BLM group.The content of hydroxyproline,the protein and m RNA expressions of collagen Ⅰ and α-SMA were significantly raised in the BLM group(p < 0.01).2.2 Increased senescent alveolar epithelial cells were found in fibrotic lung tissue: compared with the CON group,the protein and m RNA expressions of p21 and the level of p16 m RNA in lung tissue were significantly up-regulated in the BLM group(p < 0.01).Strongly positive SA-β-Gal staining and positive expression of p21 primarily located in alveolar epithelial cells.2.3 Fisetin alleviated pulmonary fibrosis in mice: compared with the BLM group,the inflammatory reaction and fibrosis degree of the lung tissue were alleviated in the BLM+FIS group.The content of hydroxyproline and the expressions of collagen I and α-SMA were significantly reduced in the BLM+FIS group(p < 0.05).2.4 Fisetin attenuated alveolar epithelial cell senescence in mice: compared with the BLM group,decreased alveolar epithelial cells with positive expression of p21 and β-glucosidase were found in the BLM+FIS group.The expressions of p16 and p21 in lung tissue were significantly down-regulated in the BLM+FIS group(p < 0.05).2.5 Fisetin inhibited the TGF-β/Smad3 signaling pathway: compared with the BLM group,the protein level of TGF-β and the ratio of p-Smad3/Smad3 in the lung tissue were significantly decreased in the BLM+FIS group(p < 0.01).2.6 Fisetin regulated the AMPK/NF-κB signaling pathway: compared with the BLM group,the protein expression of p-AMPK and the ratio of p-AMPK/AMPK in lung tissue were significantly raised in the BLM+FIS group,while the protein level of NF-κB p-p65 and the ratio of p-p65/p65 were significantly decreased in the BLM+FIS group(p < 0.05).2.7 Fisetin inhibited inflammatory response in lung tissue: compared with the BLM group,except for MMP-9,the levels of TNF-α,IL-1β,IL-6 and TGF-β in BALF were significantly decreased in the BLM+FIS group(p < 0.05).Conclusion: Fisetin can partly relieve alveolar epithelial cell senescence and associated inflammatory secretome by regulating AMPK/NF-κB signaling pathway,and therefore reduce the transdifferentiation of fibroblasts to myofibroblasts as well as collagen synthesis.Fisetin can alleviate the development of BLM-induced PF partly by rescuing alveolar epithelial cells from senescence,which is related to the regulation of AMPK/NF-κB signaling pathway and inhibition of TGF-β/Smad3 signaling pathway.