鲜广地龙纯化蛋白的制备及其体外抗肺纤维化活性评价

目的 寻找一种方便快捷的鲜广地龙蛋白纯化方法，并探究其是否具有抗肺纤维化作用。方法 采用冷冻干燥法对鲜广地龙进行粗提取，再用分子排阻色谱分离纯化得到纯化蛋白（P2），并质谱分析P2蛋白组成。细胞实验分组：C57BL/6小鼠24只，随机分3组（8只/组）。对照组、模型组（5 ng/mL TGF-β1处理）、SB431542组（2 μmol/L）和鲜广地龙纯化蛋白P2组（13.125 μg/mL）。动物实验分组：对照组（不处理）、模型组（博来霉素处理）、博来霉素+鲜广地龙纯化蛋白P2（5 mg/mL）。造模后给药21 d，在第22天进行取材。采用CCK8-法检测P2对MRC-5细胞的细胞毒性和肌成纤维细胞异常增殖的抑制；流式细胞术检验其对肌成纤维细胞的凋亡；HE 和 Massson 染色观察其对肺组织的病理学变化；RT-PCR、Western blot 检测其对α-SMA、Vimentin、E-cadherin和Collagen I的调节；机制方面通过检验P2对Smad2/3及P-Smad2/3的调节作用，进而证明其可通过TGF-β/Smad通路起到抗肺纤维化作用。结果 通过分子排阻色谱法可方便、稳定得到鲜广地龙纯化蛋白P2。体外实验显示P2具有抑制肌成纤维细胞的作用（P<0.01），降低纤维化模型中α-SMA、Vimentin和Collagen I的表达（P<0.001或P<0.01），升高E-cadherin的表达（P<0.01），可抑制Smad2/3和P-Smad2/3的表达（P<0.001或P<0.01）。体内实验显示，P2可降低HE染色中炎症细胞的数量以及Masson染色中蓝色的胶原纤维区域。结论 运用分子排阻色谱可较为理想的获得鲜广地龙纯化蛋白P2，且其可通过调节TGF-β/Smad通路抑制肺纤维化进程。

Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice

Objective To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis. Methods The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice. Results We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P<0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P< 0.001 or P<0.01), increased the expression of E-cadherin (P<0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P<0.001 or P<0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs. Conclusion The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/Smad pathway.