基于博来霉素诱导的A549细胞衰老探讨补阳还五汤改善特发性肺纤维化的作用机制OA

Objective:To investigate the effect of the Buyang Huanwutang(BHT)-containing serum on the bleomycin-induced senescence of A549 cells and explore the potential mechanism by which BHT ameliorates pulmonary fibrosis.Methods:A549 cells were cultured in vitro and modeled for senescence through the application of bleomycin.SD rats were administrated with BHT by gavage for the preparation of BHT-containing serum,and the effect of BHT-containing serum on the viability of the cell model was studied through a cell experiment designed with the following groups:blank group,model group,2.5％,5％,and 10％BHT-containing serumgroups.The effect of BHT-containing serum on the BLM-induced senescence of A549 cells was studied by the experiment designed with blank group,model group,positive group(PC,pirfenidone,600 mg·L-1),2.5％,5％,10％BHT-containing serum groups.SA-β-Gal staining was used to reveal the area of senescence-positive cells,and flow cytometry was employed to analyze the cell cycle distribution.Real-time PCR and the immunofluorescence assay were adopted to determine the mRNA and protein levels of cell senescence markers p16 and p21.Western blot was employed to quantify the protein levels of senescence-associated secretory phenotype(SASP)molecules interleukin(IL)-1β,IL-6,matrix metalloproteinase(MMP)-3,chemokine ligand(CCL)2,tumor necrosis factor(TNF)-α,and transforming growth factor-β(TGF-β)/Smad pathway molecules TGF-β1,Smad2,phosphorylated(p)-Smad2,Smad3,and p-Smad3.Results:Compared with the blank group,the model group showed a decrease in cell survival rate(P＜0.01),increases in the SA-β-Gal-positive area,the proportion of cells in G0/G,phase,the mRNA and protein levels of P16 and P21,and the protein levels of IL-1β,IL-6,TNF-α,MMP-3,CCL2,TGF-β1,p-Smad2,and p-Smad3(P＜0.05,P＜0.01).Compared with the model group,the BHT-containing serum at each dose increased the cell survival rate(P＜0.01)and decreased the SA-β-Gal-positive area,the proportion of cells in G0/G1 phase,the mRNA and protein levels of p16 and p21,and the protein levels of IL-1β,IL-6,TNF-α,MMP-3,and CCL2(P＜0.05,P＜0.01).Moreover,the inhibitory effect of BHT-containing serum on cell senescence showed a dose-dependent manner,with the 10％BHT-containing serum showing the most obvious effects and inhibiting the expression of TGF-β1,p-Smad2,and p-Smad3(P＜0.01).The effect of 10％BHT-containing serum was comparable to that of pirfenidone,and the serum even outperformed pirfenidone in inhibiting the expression of TNF-α and Smad2(P＜0.05).Conclusion:The BHT-containing serum can inhibit BLM-induced senescence of A549 cells in a dose-dependent manner by regulating the TGF-β/Smad signaling pathway.