虫草素靶向HDAC7介导上皮细胞−间充质转化改善肺纤维化

Cordycepin targets HDAC7 to mediate epithelial-mesenchymal transition to ameliorate pulmonary fibrosis

虫草素(cordycepin,Cpn)是来源于传统中药冬虫夏草的天然活性化合物,具有抗纤维化、抗氧化、抗炎作用,其对肺纤维化的作用和机制尚不明确。本研究以人肺上皮A549细胞为实验细胞系,采用转化生长因子β1(transforming growth factorβ1,TGF-β1)诱导细胞模型,利用CCK-8法、划痕实验及Transwell实验检测虫草素对细胞活力、迁移能力及侵袭能力的影响,通过分子对接和分子动力学模拟对组蛋白去乙酰化酶7(histone deacetylase 7,HDAC7)与波形蛋白(vimentin)的相互作用及虫草素与HDAC7的关联进行预测。采用博来霉素(bleomycin,BLM)构建小鼠肺纤维化模型,考察虫草素对小鼠肺组织病理变化的作用效果。利用免疫印迹法(Western blot)、细胞转染实验、免疫共沉淀实验、免疫荧光实验、免疫组化及实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)实验等研究其对肺纤维化的作用及其分子机制。动物福利和实验过程均遵循上海儿童医学中心实验动物伦理委员会的规定(批准号:SCMC-LAWEC-2022-017)。实验结果显示,虫草素在100μmol·L-1浓度内对细胞无毒,可抑制A549细胞的迁移及侵袭;虫草素可显著减少胶原沉积,降低小鼠肺组织炎症和纤维化程度。在体内外模型中,虫草素显著逆转上皮细胞−间充质转化(epithelial-mesenchymal transition,EMT)及肺纤维化标志物I型胶原α1(collagen type Iα1 chain,collagen I)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、N-钙黏蛋白(N-cadherin)、vimentin及E-钙黏蛋白(E-cadherin)及HDAC7的mRNA和蛋白表达;HDAC7敲低可抑制EMT及collagen I的表达,且虫草素可靶向HDAC7抑制EMT及collagen I的表达;vimentin与HDAC7存在相互作用,且分子对接实验发现虫草素与HDAC7存在相互关联。综上所述,虫草素可靶向HDAC7抑制EMT发挥抗肺纤维化作用,为开发抗肺纤维化药物提供新的思路与选择。

Cordycepin(Cpn),a natural active compound derived from the traditional Chinese medicine Cordyceps sinensis,has antifibrotic,antioxidant,and anti-inflammatory effects,but the impact of Cpn on pulmonary fibrosis and the downstream molecular mechanism remain unclear.In this study,A549 cells were induced by transforming growth factorβ1(TGFβ1)in vitro,the viability of A549 cells was evaluated by CCK-8 assay;and migration of A549 cells were detected by wound healing assay,invasion of A549 cells were detected by transwell assay.Molecular docking and molecular dynamics simulations were used to predict the interaction of histone deacetylase 7(HDAC7)with vimentin and the association of Cpn with HDAC7.The pulmonary fibrosis model of mice was established by bleomycin in vivo to investigate the effect of Cpn on pathological changes of lung tissue.The impact of Cpn and molecular mechanism on pulmonary fibrosis were studied by Western blot assay,cell transfection assay,immunoprecipitation assay,immunofluorescence assay,immunohistochemistry and real-time quantitative PCR(RT-qPCR).All animal experiments were approved by the Shanghai Children's Medical Center Experimental Animal Ethics Committee(grant No.SCMC-LAWEC-2022-017).Results showed that Cpn had no toxic effect on A549 cells even at the concentration of 100μmol·L-1.Cpn inhibited migration and invasion of A549 cells and reduced deposition of collagen,the degree of lung inflammation and fibrosis in mice;in vivo and in vitro models,Cpn significantly reversed mRNA and protein expressions of epithelial-mesenchymal transition(EMT)and lung fibrosis markers collagen I,α-smooth muscle actin(α-SMA),N-cadherin,vimentin and E-cadherin and HDAC7.Deficiency of HDAC7 suppressed TGFβ1-induced EMT and expression of collagen I;vimentin interacted with HDAC7;and molecular docking experiment revealed that Cpn was interrelated with HDAC7.In conclusion,Cpn can target HDAC7 to mediate EMT and exert its anti-fibrotic effect,the inhibition of EMT and the improvement of pulmonary fibrosis provide a new idea and choice for the development of anti-pulmonary fibrosis drug.