目的 考察汉防己甲素（Tet）对体内外肺纤维化模型中细胞周期蛋白依赖性激酶（CDK）-成视网膜细胞瘤蛋白（Rb）-E2F转录因子（E2F）信号通路的影响。方法 将60只Wistar大鼠随机分为对照组、模型组、吡非尼酮(阳性药，50 mg·kg^-1)组及Tet低、中、高剂量(13.5、27.0、54.0 mg·kg^-1)组，通过气管内注入博来霉素(BLM,5 mg·kg^-1)建立大鼠肺纤维化模型。造模24 h后，给药组给予相应药物，对照组与模型组给予等体积0.9%氯化钠溶液，连续干预28 d，检测Tet对大鼠肺组织中细胞周期蛋白D1（Cyclin D1）、E2F及α-平滑肌肌动蛋白（α-SMA）的蛋白表达影响；体外实验以转化生长因子-β(TGF-β,5 ng·mL^-1)刺激人胚肺成纤维细胞MRC-5构建肺纤维化模型，设对照组、模型组及Tet低、中、高剂量(5、10、50μmol·L^-1)组，Western blotting法检测Tet对MRC-5细胞增殖及Cyclin D1、Rb、磷酸化-成视网膜细胞瘤蛋白（p-Rb）、CDK4、CDK6的蛋白表达影响。结果 体内实验显示，与模型组相比，Tet高剂量组Cyclin D1蛋白表达显著降低（P<0.01），且α-SMA、E2F蛋白表达均下调；体外实验中，Tet高剂量组可显著抑制MRC-5细胞增殖（P<0.01）;Western blotting结果显示，Tet低剂量组CDK6蛋白表达显著下调（P<0.01），中剂量组E2F、CDK6、CDK4蛋白表达显著下调（P<0.05、0.001），高剂量组p-Rb、E2F、CDK6、CDK4、Cyclin D1蛋白表达均显著下调（P<0.01、0.001）；免疫荧光结果显示，Tet中、高剂量组CDK6相对荧光强度显著降低（P<0.01、0.001）。结论 Tet的抗肺纤维化作用可能通过调控CDK-Rb-E2F信号通路阻滞细胞周期，进而抑制成纤维细胞异常增殖实现。

Objective To investigate the effects of tetrandrine（Tet） on the cyclin-dependent kinase（CDK）-retinoblastoma protein（Rb）-E2F transcription factor（E2F） signaling pathway in vivo and in vitro pulmonary fibrosis models. Methods Sixty Wistar rats were randomly divided into the control group, the model group, the pirfenidone(positive drug, 50 mg·kg^-1) group, and the Tet low-, medium-, and high-dose(13.5, 27.0, and 54.0 mg·kg^-1) groups. A rat pulmonary fibrosis model was established by intratracheal injection of bleomycin(BLM, 5 mg·kg^-1). 24 hours after modeling, the drug groups were given the corresponding drugs, while the control and model groups were given the same volume of 0.9% sodium chloride solution. The intervention lasted for 28 days. The effects of Tet on the protein expression of cyclin D1（Cyclin D1）, E2F, and α-smooth muscle actin（α-SMA） in rat lung tissue were detected. In the in vitro experiment, a pulmonary fibrosis model was established by stimulating human embryonic lung fibroblasts MRC-5 with transforming growth factor-β(TGF-β, 5 ng·mL^-1). The control group, the model group, and the Tet low-, medium-, and high-dose(5, 10, and 50 μmol·L^-1) groups were set up. The effects of Tet on the proliferation of MRC-5 cells and the protein expression of Cyclin D1, Rb, phosphorylated retinoblastoma protein（p-Rb）, CDK4, and CDK6 were detected by Western blotting method. Results In the in vivo experiment, compared with the model group, the high-dose Tet group showed a significant decrease in Cyclin D1 protein expression（P ＜ 0.01）, and the protein expressions of α-SMA and E2F were also downregulated. In the in vitro experiment, the highdose Tet group significantly inhibited the proliferation of MRC-5 cells（P ＜ 0.01）. Western blotting results showed that the low-dose Tet group significantly downregulated the protein expression of CDK6（P ＜ 0.01）, the medium-dose group significantly downregulated the protein expressions of E2F, CDK6, and CDK4（P ＜ 0.05, 0.001）, and the high-dose group significantly downregulated the protein expressions of p-Rb, E2F, CDK6, CDK4, and Cyclin D1（P ＜ 0.01, 0.001）. Immunofluorescence results showed that the relative fluorescence intensity of CDK6 in the medium-and high-dose Tet groups was significantly decreased（P ＜ 0.01, 0.001）. Conclusion The anti-pulmonary fibrosis effect of Tet may be achieved by regulating the CDK-Rb-E2F signaling pathway to arrest the cell cycle and thereby inhibit the abnormal proliferation of fibroblasts.

R285.5;R563.9

国家自然科学基金青年基金资助项目(82204740); 解放军总医院第六医学中心创新培育基金资助项目(CXPY202406); 中央级公益性科研院所基本业务费专项资金资助(ZXKT22045); 中国中医科学院中央级公益性课题(ZXKT25006);中国中医科学院优秀青年科技人才培养专项(ZZ16-YQ-027)