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

谢佳雨; 戴隆超; 王娜; 刘成; 王令充

doi:10.14148/j.issn.1672-0482.2022.1116

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

doi: 10.14148/j.issn.1672-0482.2022.1116

谢佳雨^1,,
戴隆超¹,
王娜¹,
刘成²,
王令充^1, ,

1.
南京中医药大学药学院, 江苏南京 210023
2.
上海中医药大学附属普陀医院, 上海 200062

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

详细信息

作者简介:
谢佳雨, 女, 硕士研究生, E-mail: 20190441@njucm.edu.cn

通讯作者:
王令充, 男, 副研究员, 主要从事天然高分子成分及中药多糖的研究，E-mail: 993wlc@njucm.edu.cn

中图分类号: R285.5
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- 文章访问数: 52
- HTML全文浏览量: 2
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- 被引次数: 0
出版历程
- 收稿日期: 2022-07-01
- 网络出版日期: 2022-12-15
- 发布日期: 2022-12-10

Isolation and Purification of Alcohol-Extracted Protein from Eupolyphaga Sinensis Walker and Its in vitro Anti-Hepatocellular Carcinoma and Anti-Hepatic Fibrosis Activities

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

摘要

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。
- 土鳖虫 /
- 分离纯化 /
- 活性蛋白 /
- 肝癌 /
- 肝纤维化
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.
- Eupolyphaga sinensis Walker /
- separation and purification /
- active protein /
- hepatoma /
- hepatic fibrosis

HTML全文

图 1 土鳖虫分离产物EE、EEP、EEPA、EEPB、EEPC对人正常肝细胞株L-02和肝肿瘤细胞株SMMC-7721增殖的影响

注: 与空白对照组比较, ^*P＜0.05, ^{* *}P＜0.01。

Figure 1. Effects of EE, EEP, EEPA, EEPB and EEPC on the proliferation of human normal stem cell line L-02 and liver tumor cell line SMMC-7721

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图 2 土鳖虫分离产物EE、EEP、EEPA、EEPB、EEPC对正常肝星状细胞株HSC-T6和活化后的肝星状细胞HSC-T6增殖的影响

注: 与模型对照组比较, ^*P＜0.05, ^{* *}P＜0.01。

Figure 2. Effects of EE, EEP, EEPA, EEPB and EEPC on the proliferation of hepatic stellate cell line HSC-T6 and activated HSC-T6

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图 3 EEPC经DEAE和Sephadex G-100纯化

注: A.EEPC阴离子交换色谱洗脱曲线; B.EEPC经DEAE洗脱所得6组分峰面积定量分析; C.PC3凝胶过滤色谱洗脱曲线; D.PC3经Sephadex G-100洗脱所得3组分峰面积定量分析

Figure 3. Purification of EEPC using DEAE and Sephadex G-100 chromatography

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图 4 PC3-Ⅲ对人正常肝细胞株L-02和肝肿瘤细胞株SMMC-7721增殖的影响

注: 与空白对照组比较, ^{* *}P＜0.01。

Figure 4. Effects of PC3-Ⅲ on the proliferation of human normal stem cell line L-02 and liver tumor cell line SMMC-7721

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图 5 EEPC及PC3-Ⅲ诱导SMMC-7721细胞凋亡的形态学变化

Figure 5. Morphological changes during the apoptosis induced by EEPC and PC3-Ⅲ in SMMC-7721 cells

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图 6 流式细胞术检测样品处理后SMMC-7721细胞凋亡情况

注：与空白对照组比较, ^*P＜0.05, ^{* *}P＜0.01。

Figure 6. Detection of apoptosis of SMMC-7721 cells by flow cytometry

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图 7 EEPC及PC3-Ⅲ处理不同时间后SMMC-7721细胞迁移情况

Figure 7. Migration of SMMC-7721 cells treated with EEPC and PC3-Ⅲ at different time point

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图 8 PC3-Ⅲ对正常肝星状细胞株HSC-T6和活化后的肝星状细胞HSC-T6增殖的影响

注: 与模型对照组比较, ^{* *}P＜0.01。

Figure 8. Effects of PC3-Ⅲ on the proliferation of hepatic stellate cell line HSC-T6 and activated HSC-T6

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图 9 PC3-Ⅲ蛋白的表征

注: A.RP-HPLC分析; B.SDS-PAGE分析; C.QE序列分析二级质谱图

Figure 9. Characterization of PC3-Ⅲ protein

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表 1 由Q-Exactive质谱仪鉴定的PC3-Ⅲ肽片段的信息

Table 1. Information on PC3-Ⅲ peptide fragments identified by Q-Exactive mass spectrometer

编号	序列	分子量/kDa	查库结果	PI	A0A0M3STY1的对应位置
1	QYSINFISAR	1.20	tr\|A0A0M3STY1\|A0A0M3STY1_9NEOP	8.75	39~48
2	CNGDSCVCTFR	1.38	tr\|A0A0M3STY1\|A0A0M3STY1_9NEOP	5.82	49~59
3	SNNFR	0.64	tr\|A0A5K7VJ13\|A0A5K7VJ13_9NEOP	9.47	-

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