土鳖虫醇提蛋白的分离纯化及其体外抗肝癌与抑制肝纤维化活性研究 |
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作者姓名: | 谢佳雨 戴隆超 王娜 刘成 王令充 |
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作者单位: | 1.南京中医药大学药学院, 江苏 南京 210023 |
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基金项目: | 国家自然科学基金面上项目81673788国家自然科学基金面上项目81873136 |
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摘 要: | 目的 采用体外细胞实验评价土鳖虫醇提物中大分子蛋白各分离产物的抗肝癌与抑制肝纤维化活性, 明确土鳖虫的有效部位与成分。 方法 采用盐析法从土鳖虫醇提物中回收蛋白类成分, 将土鳖虫粗蛋白产物按分子量差异进行超滤, 将其分为EEPA、EEPB及EEPC三个不同的蛋白部位, 以抑制肝异常细胞增殖活性为导向, 筛选最佳活性蛋白部位; 接着经过DEAE阴离子层析及Sephadex G-100凝胶层析对活性粗蛋白部位进一步的纯化, 从EEPC中分离出一种纯化蛋白PC3-Ⅲ, 并对其进行LC-MS/MS分析; 最后利用细胞实验评价EEPC及PC3-Ⅲ体外抗肝癌及抑制肝纤维化的活性。 结果 在EEPA、EEPB及EEPC中, 分子量最大的蛋白部位EEPC表现出最高的抑制肝异常细胞增殖活性; 纯化蛋白PC3-Ⅲ分子量为10 kDa左右, 经LC-MS/MS鉴定表明,PC3-Ⅲ对同源蛋白A0A0M3STY1的覆盖率达到35%, 查库得到的肽段序列为QYSINFISAR、CNGDSCVCTFR和SNNFR; 体外细胞实验证明PC3-Ⅲ不仅通过抑制肿瘤细胞生长、诱导肿瘤细胞凋亡、抑制肿瘤细胞迁移来共同发挥抗肝癌药效, 还能抑制活化的肝星状细胞增殖从而延缓肝纤维化的进程, 疗效与EEPC相当。 结论 研究证实了土鳖虫大分子蛋白的抗肿瘤与抗肝纤维化的活性, 并分离得到均一蛋白产物PC3-Ⅲ, 为推进土鳖虫功效物质基础的认识与相关产品研发奠定了基础。
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关 键 词: | 土鳖虫 分离纯化 活性蛋白 肝癌 肝纤维化 |
收稿时间: | 2022-07-01 |
Isolation and Purification of Alcohol-Extracted Protein from Eupolyphaga Sinensis Walker and Its in vitro Anti-Hepatocellular Carcinoma and Anti-Hepatic Fibrosis Activities |
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Institution: | 1.School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China2.Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China |
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Abstract: | OBJECTIVE In vitro cell experiments were used to evaluate the anti-hepatocarcinoma and anti-hepatic fibrosis activities of the macromolecular proteins isolated from the alcohol extract of Eupolyphaga sinensis Walker, and gradually focused on the effective parts and components. METHODS The protein fractions were recovered from the alcohol extracts of Eupolyphaga sinensis Walker by salting out, and the crude protein products of Eupolyphaga sinensis Walker were divided into three different protein sites EEPA, EEPB and EEPC, according to their molecular weight by ultrafiltration. Based on the activity of inhibiting the proliferation of abnormal liver cells, the best part was screened by cell experiments. Then, a purified protein (PC3-Ⅲ) was isolated from EEPC by DEAE anion chromatography and Sephadex G-100 gel chromatography, and identified by LC-MS/MS. Finally, cell experiments were performed to evaluate the in vitro anti-hepatocarcinoma and anti-hepatic fibrosis activities of EEPC and PC3-Ⅲ. RESULTS Among EEPA, EEPB, and EEPC, the protein site EEPC with the largest molecular weight showed the most potent inhibitory activity against abnormal liver cell proliferation. The molecular weight of PC3-Ⅲ was about 10 kDa. LC-MS/MS identification showed that the coverage rate of PC3-Ⅲ to the homologous protein A0A0M3STY1 reached 35%. The peptide sequences obtained by checking the reference were QYSINFISAR, CNGDSCVCTFR and SNNFR. In vitro experiments suggested that PC3-Ⅲ not only exerted anti-hepatoma by inhibiting tumor cell growth, inducing tumor cell apoptosis, and inhibiting tumor cell migration, but also inhibited the proliferation of activated hepatic stellate cells and delayed the process of liver fibrosis. The efficacy was comparable to that of EEPC. CONCLUSION This study confirms the anti-tumor and anti-liver fibrosis activities of the macromolecular protein of Eupolyphaga sinensis Walker and isolates the homogeneous protein product PC3-Ⅲ, laying a foundation for the advanced understanding of the material basis for the efficacy of Eupolyphaga sinensis Walker and the research and development of related products. |
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