黄芪甲苷通过调控宿主核糖体翻译开关抑制HBV复制的机制

常凯; 王艳艳; 那琬琳; 刘晨霞; 叶雨笙; 江忠勇; 刘媛

doi:10.3969/j.issn.1001-5256.2022.07.009

黄芪甲苷通过调控宿主核糖体翻译开关抑制HBV复制的机制

DOI: 10.3969/j.issn.1001-5256.2022.07.009

常凯¹,
王艳艳¹,
那琬琳¹,
刘晨霞²,
叶雨笙³,
江忠勇⁴,
刘媛^1, , ,

1.
中国人民解放军西部战区总医院检验科，成都 610083
2.
四川省疾病预防控制中心微生物检验所，成都 610041
3.
成都市郫都区人民医院，成都医学院第三附属医院检验科，成都 611730
4.
成都市第七人民医院检验科，成都医学院附属肿瘤医院，成都 610041

基金项目:

四川省中医药专项课题 (2020JC0124);

西部战区总医院星火青年创新人才工程 ;

西部战区总医院院管课题 (2021-XZYG-C22);

国家自然科学基金 (81301445)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：常凯、王艳艳负责课题设计，资料分析，撰写论文；常凯、王艳艳、那琬琳、刘晨霞、叶雨笙、江忠勇参与实验实施与修改论文；常凯、刘媛负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
刘媛，liuyuan198231@163.com

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出版历程
- 收稿日期: 2021-11-11
- 录用日期: 2022-01-20
- 出版日期: 2022-07-20

Mechanism of astragaloside Ⅳ inhibits hepatitis B virus replication by regulating host ribosome translation process

1.
Department of Clinical Laboratory, The General Hospital of Western Theater Command, Chengdu 610083, China
2.
Institute of Microbiology, Sichuan Provincial Center for Disease Control and Prevention, Chengdu 610041, China
3.
Department of Clinical Laboratory, People's Hospital of Pidu District & The Third Affiliated Hospital of Chengdu Medical College, Chengdu 611730, China
4.
Department of Clinical Laboratory, Affiliated Cancer Hospital of Chengdu Medical College & Chengdu Seventh People's Hospital, Chengdu 610041, China

Research funding:

The Special Project of Sichuan Province Traditional Chinese Medicine Administration (2020JC0124);

The General Hospital of Western Theater Command Management Project of Science and The Spark Young Innovative Talent Project ;

The Applied Basic Research Program of The General Hospital of Western Theater Command (2021-XZYG-C22);

National Natural Science Foundation of China (81301445)

More Information

Corresponding author: LIU Yuan, liuyuan198231@163.com (ORCID: 0000-0002-4387-2250)

摘要

摘要: 目的分析黄芪甲苷抑制HBV复制的宿主调控机制。方法使用不同浓度的黄芪甲苷处理人正常肝细胞(L-02)，根据黄芪甲苷浓度的不同，分为0、5、10和20 μg/mL 4组。应用CCK-8检测细胞活性，流式细胞法检测细胞凋亡，化学发光和生化方法检测AFP、ALT、AST和ALP外泌水平，评估黄芪甲苷对正常细胞的影响。用黄芪甲苷处理携带HBV的肝癌细胞Hep3B，应用qPCR方法检测HBV DNA、pgRNA、MTIF2、RPL10基因表达量，ELISA测法检测HBsAg和HBeAg，评估对HBV复制的影响。应用TCGA和GEO数据库结合R语言资料包对RPL10和MTIF2在临床样本中的预后影响进行分析验证。绘制Kaplan-Meier生存曲线，log-rank检验用于分析比较两组或多组之间的生存差异，进行了time ROC分析以比较RPL10和MTIF2基因的预测准确性和风险评分。计量资料多组间比较和同一组内不同时间段比较均采用单因素方差分析，进一步分析两两组间比较采用Bonferroni方法。结果 20 μg/mL组处理24 h和48 h相较未处理组细胞生长活性均显著提高(P值均＜0.05)，20 μg/mL组处理72 h相较10 μg/mL组生长活性提高(P＜0.05)，5 μg/mL组处理72 h相较未处理组生长活性提高(P＜0.05)。5、10和20 μg/mL 3个处理组AFP水平均较未处理组显著增高(P值均＜0.05)，10和20 μg/mL 2个处理组ALT水平分别均较未处理和5 μg/mL 2组显著降低(P值均＜0.05)，20 μg/mL组ALT水平较10 μg/mL组显著降低(P＜0.05)。5、10和20 μg/mL 3个处理组AST水平较未处理组均显著增高(P值均＜0.05)。5、10和20 μg/mL 3个处理组HBV DNA、pgRNA、HBsAg、HBeAg、RPL10和MTIF2水平与未处理组比较差异均有统计学意义(P值均＜0.05)。生物信息学分析结果显示，HBV感染的肝癌患者中RPL10和MTIF2基因较高水平表达的患者预后较差，而在无HBV感染的肝癌患者中不存在此现象。结论黄芪甲苷能够抑制翻译起始蛋白MTIF2和核糖体大亚基成分RPL10，通过调控宿主核糖体翻译开关降低HBV复制。
- 黄芪甲苷 /
- 乙型肝炎病毒 /
- 核糖体蛋白质类
Abstract: Objective To investigate the host regulatory mechanism of astragaloside Ⅳ in inhibiting hepatitis B virus (HBV) replication. Methods Normal human hepatocytes L-02 were treated with different concentrations of astragaloside Ⅳ, and according to the concentration of astragaloside Ⅳ, the cells were divided into 0, 5, 10, and 20 μg/mL groups. CCK-8 assay was used to measure cell viability, flow cytometry was used to measure cell apoptosis, and chemiluminescence and biochemical methods were used to measure the levels of alpha-fetoprotein (AFP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), so as to evaluate the influence of astragaloside Ⅳ on normal cells. Hepatoma cells Hep3B carrying HBV were treated with astragaloside Ⅳ; quantitative PCR was used to measure the mRNA expression levels of HBV DNA, pgRNA, MTIF2, and RPL10, and ELISA was used to measure the levels of HBsAg and HBeAg, so as to evaluate the effect of astragaloside Ⅳ on HBV replication. TCGA and GEO databases combined with R language package were used to analyze the prognostic effect of RPL10 and MTIF2 in clinical samples. The Kaplan-Meier method was used for survival analysis, and the log-rank test was used for comparison of survival between two or multiple groups; the time-dependent ROC curve analysis was performed to compare the predictive accuracy and risk score of RPL10 and MTIF2 genes. A one-way analysis of variance was used for comparison of continuous data between multiple groups and within each group at different time points, and the Bonferroni method was used for further comparison between two groups. Results Compared with the untreated group, the 20 μg/mL group had a significant increase in cell growth activity at 24 and 48 hours of treatment (both P < 0.05); compared with the 10 μg/mL group, the 20 μg/mL group had a significant increase in cell growth activity at 72 hours of treatment (P < 0.05); compared with the untreated group, the 5 μg/mL group had a significant increase in cell growth activity at 72 hours of treatment (P < 0.05). Compared with the untreated group, the 5, 10, and 20 μg/mL groups had a significant increase in AFP (all P < 0.05); compared with the untreated group and the 5 μg/mL group, the 10 and 20 μg/mL groups had a significant reduction in ALT (all P < 0.05), and compared with the 10 μg/mL group, the 20 μg/mL group had a significant reduction in ALT (P < 0.05). Compared with the untreated group, the 5, 10, and 20 μg/mL groups had a significant increase in AST (all P < 0.05). There were significant differences in the levels of HBV DNA, pgRNA, HBsAg, HBeAg, RPL10, and MTIF2 between the 5/10/20 μg/mL groups and the untreated group (all P < 0.05). The bioinformatics analysis showed that among the liver cancer patients with HBV infection, the patients with high mRNA expression levels of RPL10 and MTIF2 genes tended to have a poor prognosis, while this phenomenon was not observed in liver cancer patients without HBV infection. Conclusion Astragaloside Ⅳ can inhibit the translation initiation factor MTIF2 and the large ribosomal subunit RPL10 and reduce HBV replication by regulating the initiation of host ribosome translation.
- Astragaloside Ⅳ /
- Hepatitis B virus /
- Ribosomal Proteins

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图 1 不同浓度的黄芪甲苷对L-02外泌蛋白的影响

注：与0 μg/mL组比较，* P＜0.05；与5 μg/mL组比较，# P＜0.05。

Figure 1. Effect of Astragaloside Ⅳ on L-02 exocrine protein

下载: 全尺寸图片幻灯片

图 2 RPL10 & MTIF2蛋白互作分析和黄芪甲苷参与HBV复制的分子机制示意图

注：a, RPL10 & MTIF2蛋白互作分析图；b, 黄芪甲苷参与HBV复制的分子机制示意图。

Figure 2. RPL10 and MTIF2 protein interaction analysis with GeneMANIA and molecular mechanism of Astragaloside Ⅳ on hepatitis B virus replication

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图 3 RPL10 & MTIF2调控HBV复制相关基因的富集分析及临床生存曲线分析

注：a, 一致性聚类结果热图；b, 相关基因表达聚类热图；c, RPL10基因生存曲线分析；d, MTIF2基因生存曲线分析；e, RPL10和MTIF2基因联合生存曲线分析；f, 不同样本Risk score对应的生存时间和生存状态散点分布图。

Figure 3. RPL10 & MTIF2 regulated gene enrichment and clinical survival curve analysis in HBV replication

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图 4 黄芪甲苷对HBV复制装配中相关基因及蛋白表达的影响

注：与0 μg/mL组比较，*P＜0.05。

Figure 4. The effect of Astragaloside Ⅳ on the expression of related genes and proteins in HBV replication assembly

下载: 全尺寸图片幻灯片

表 1 RT-PCR引物列表

Table 1. List of RT-PCR primers

基因名称		引物序列(5′-3′)	Tm值(℃)
MTIF2	F-MTIF2	CTATGATGCTTCACACGAGTG	53
	R-MTIF2	TGCTCTTCCACAGCACAGG	53
RPL10	F-RPL10	CGGTATTGTAAGAACAAGCCGT	60
	R-RPL10	GCCACAAAGCGGAAACTCAT	60
pgRNA	F-pgRNA	CTCAATCTCGGGAATCTCAATGT	53
	R-pgRNA	TGGATAAAACCTAGCAGGCATAAT	53
GAPDH	F-GAPDH	CAGGAGGCATTGCTGATGAT	52
	R-GAPDH	GAAGGCTGGGGCTCATTT	52

下载: 导出CSV

表 2 黄芪甲苷对人正常肝细胞L-02细胞生长活性的影响

Table 2. Effect of Astragaloside Ⅳ on L-02 cell growth activity

组别	细胞活性(%)
组别	24 h	48 h	72 h
0 μg/mL组	95.11±0.88	91.69±0.52	90.85±1.41
5 μg/mL组	94.40±6.05	92.35±3.81	91.13±3.37¹⁾
10 μg/mL组	94.31±7.44	94.47±4.60	91.64±1.84
20 μg/mL组	100.23±1.40¹⁾	97.35±0.67¹⁾	99.56±0.44²⁾
F值	0.682	1.433	8.301
P值	0.617	0.362	0.030
注：与0 μg/mL组比较，1)P＜0.05；与10 μg/mL组，2)P＜0.05。

下载: 导出CSV

表 3 黄芪甲苷对L-02细胞凋亡的影响

Table 3. Effect of Astragaloside Ⅳ on L-02 cell apoptosis

组别	死亡率(%)	凋亡率(%)	死亡+凋亡率(%)
0 μg/mL组	2.74±0.45	2.72±0.27	5.46±0.72
5 μg/mL组	3.07±0.68	3.27±0.43	6.34±0.92
10 μg/mL组	3.16±0.56	3.10±0.34	6.26±0.47
20 μg/mL组	3.35±0.69	2.96±0.76	6.31±0.96
F值	0.533	0.689	0.853
P值	0.672	0.584	0.503

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表 4 不同浓度的黄芪甲苷处理对L-02外泌蛋白的影响

Table 4. Effect of different concentrations of Astragaloside Ⅳ treatment on L-02 cell exoprotein

组别	AFP(ng/mL)	ALT(U/L)	AST(U/L)	ALP(U/L)
0 μg/mL组	13.25±0.20	6.46±0.67	5.68±0.21	40.68±0.61
5 μg/mL组	14.75±0.20¹⁾	6.27±0.16	7.63±0.22¹⁾	38.68±0.92¹⁾
10 μg/mL组	14.48±0.13¹⁾	3.09±0.08¹⁾²⁾	6.57±0.46¹⁾	40.37±0.37
20 μg/mL组	14.42±0.48¹⁾	1.80±0.13¹⁾²⁾³⁾	7.61±0.17¹⁾	43.77±0.34¹⁾
F值	16.180	129.523	31.670	36.847
P值	0.001	0.001	0.001	0.001
注：与0 μg/mL组比较，1)P＜0.05；与5 μg/mL组，2)P＜0.05；与10 μg/mL组，3)P＜0.05。

下载: 导出CSV

表 5 黄芪甲苷对HBV复制装配中相关基因及蛋白表达的影响

Table 5. The effect of Astragaloside Ⅳ on the expression of related genes and proteins in HBV replication assembly

组别	HBV DNA(IU/mL)	pgRNA	HBsAg	HBeAg	RPL10	MTIF2
0 μg/mL组	43 300.00±23 356.25	1.07±0.09	0.70±0.03	0.62±0.06	1.30±0.51	1.02±0.05
5 μg/mL组	7 420.00±955.98¹⁾	0.46±0.19¹⁾	0.58±0.04¹⁾	0.41±0.04¹⁾	0.79±0.04¹⁾	0.51±0.18¹⁾
10 μg/mL组	6 730.00±141.07¹⁾	0.63±0.13¹⁾	0.32±0.04¹⁾	0.46±0.01¹⁾	0.32±0.04¹⁾	0.63±0.08¹⁾
20 μg/mL组	4900.00±400.37¹⁾	0.74±0.03¹⁾	0.56±0.05¹⁾	0.34±0.02¹⁾	0.56±0.05¹⁾	0.56±0.22¹⁾
F值	7.532	12.159	33.026	17.251	4.633	4.712
P值	0.010	0.002	0.003	0.009	0.037	0.035
注：与0 μg/mL组比较，1)P＜0.05。

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