瑞香狼毒降低YAP1表达抑制肝癌细胞增殖的作用

doi:10.13560/j.cnki.biotech.bull.1985.2022-1418

摘要/Abstract

摘要：

探讨瑞香狼毒（Stellera chamaejasme L., SCL）抑制肝癌细胞增殖的效应和机制，为原发性肝细胞癌的病理机制研究和临床治疗提供依据。SCL含药血清处理HepG2215细胞24 h，CCK-8法分析肝癌HepG2215细胞增殖活力。Western blot分析肝癌HepG2215细胞中Yes关联蛋白1（yes-associated protein 1，YAP1）表达的情况；应用N-亚硝基二乙胺（diethylnirtosamine，DEN）/组成型雄甾烷受体激动剂（3,5-dichloro-2-[4-（3,5-dichloropyridin-2-yl）oxyphenoxy]pyridine，TCPOBOP）诱导肝细胞特异性Yap1敲除（Yap1^LKO）小鼠和Yap1^flox/flox小鼠构建肝原位癌模型；SCL水煎液灌胃7 d，取出肝脏，观察肝脏肿瘤大体形态；H&E染色观察肝脏肿瘤组织病理形态，Western blot分析肝脏肿瘤组织中YAP1的表达。细胞试验结果表明，SCL抑制HepG2215细胞的增殖，并降低了YAP1的表达水平。动物试验结果表明，SCL能减小Yap1^flox/flox和Yap1^LKO小鼠肝原位癌肿瘤体积和肝肿瘤中YAP1的表达水平。在NS组中，与Yap1^flox/flox小鼠相比，Yap1^LKO小鼠肝原位癌肿瘤体积减小了。在SCL组中，与Yap1^flox/flox小鼠相比，Yap1敲除减弱了SCL抑制肿瘤增长的作用。利用LC-MS检测SCL含药血清和水煎液的活性成分，并与YAP1蛋白进行分子对接。LC-MS结果显示，SCL的4种活性成分是3-硫酸咖啡酸、5-苄恶唑-2-酮、3,4,5-三羟基-6-[（2-氧代-2H-铬-5-基）氧]氧烷-2-羧酸和异东莨菪内酯。其中，3-硫酸咖啡酸、3,4,5-三羟基-6-[（2-氧代-2H-铬-5-基）氧]氧烷-2-羧酸和异东莨菪内酯这3种主要活性成分能直接结合YAP1。这些结果说明，SCL抑制肝癌细胞增殖与降低YAP1表达有关。

关键词: 瑞香狼毒, HepG2215细胞, 原发性肝细胞癌, YAP1

Abstract:

This study is to investigate the effect and mechanism of Stellera chamaejasme L.（SCL）on inhibiting proliferation of hepatocellular carcinoma cells, providing basis for the pathological mechanism study and clinical treatment of primary hepatocellular carcinoma. Firstly, CCK-8 assay was performed to illustrate the ability of tumor growth in the HepG2215 cells treated with SCL serum containing blood. Western blot was used to detect the expression of YAP1（yes-associated protein 1）protein in the HepG2215 cells. Secondly, Yap1^flox/flox and Yap1^LKO mice were induced to form liver tumors in situ by DEN（diethylnirtosamine）/TCPOBOP（3,5-dichloro-2-[4-（3,5-dichloropyridin-2-yl）oxyphenoxy]pyridine）. SCL decoction was given to the stomach of the mice for 7 d, the liver was taken out, and the general shape of the liver tumor was observed. H&E was performed to observe the pathological morphology in tumor tissue. Western blot was to detect the expressions of YAP1 in the liver tumor tissues. The results from cell experiment demonstrated that SCL inhibited the proliferation of HepG2215 cells and decreased YAP1 expression. Moreover, animal experiment revealed that SCL reduced tumor volume and YAP1 expression both in Yap1^flox/flox and Yap1^LKO mice with tumors in liver. The hepatocellular carcinoma tumor volume of Yap1^LKOmice in NS group reduced when compared with Yap1^flox/floxmice. Yap1 knockdown reduced the effect of SCL inhibiting tumor growing in SCL group when compared with Yap1^flox/floxmice. LC-MS was used to detect the active components of SCL drug-containing serum and water decoction, and molecular docking was carried out with YAP1 protein. LC-MS results showed that the active ingredients were caffeic acid 3-sulfate, 5-benzyloxolan-2-one, 3,4,5-trihydroxy-6-[（2-oxo-2h-chromen-5-yl）oxy]oxane-2-carboxylic acid and isoscopoletin in SCL. Among them, caffeic acid 3-sulfate, 3,4,5-trihydroxy-6-[（2-oxo-2h-chromen-5-yl）oxy]oxane-2-carboxylic acid and isoscopoletin directly formed stable docking with YAP1 protein. These results suggest that SCL inhibits liver tumor cell proliferation by reducingYAP1 expression.

Key words: Stellaria chamaejasme L., HepG2215 cells, hepatocellular carcinoma, YAP1

周文汉, 郑康宁, 李永民. 瑞香狼毒降低YAP1表达抑制肝癌细胞增殖的作用[J]. 生物技术通报, 2023, 39(7): 316-324.

ZHOU Wen-han, ZHENG Kang-ning, LI Yong-min. Stellera chamaejasme L. Inhibiting Cell Proliferation by Reducing YAP1 Expression in Hepatocellular Carcinoma[J]. Biotechnology Bulletin, 2023, 39(7): 316-324.

图/表 8

图1 低浓度瑞香狼毒含药血清对人正常肝细胞L02增殖的影响 A：瑞香狼毒水煎液对肝脏外观形态的影响；B：低浓度瑞香狼毒含药血清对L02细胞增殖作用（*P<0.05）。NS serum：生理盐水血清；SCL serum：瑞香狼毒含药血清

Fig. 1 Effects of low concentration of SCL serum on the proliferation of normal liver cells L02 A: The effect of SCL decoction on the morphological appearance of liver.B: Proliferative effect of low concentration of SCL containing serum on L02 cells（*P<0.05）. NS serum: Normal saline serum group; SCL serum : Stellera chamaejasme L. serum group

图2 不同浓度瑞香狼毒含药血清抑制HepG2215细胞的增殖 A：不同浓度瑞香狼毒含药血清对HepG2215细胞形态的影响，标尺 =50 μm；B：不同浓度瑞香狼毒含药血清对HepG2215细胞增殖活力的影响；C：瑞香狼毒抑制HepG2215细胞的IC50值。*P<0.05，NS：生理盐水组；SCL：瑞香狼毒含药血清组

Fig. 2 Proliferation of HepG2215 cells inhibited by different concentrations of SCL serum A: The effects of different concentrations of SCL serum on the morphologyies of HepG2215 cells. Bar = 50 μm. B: The effects of different concentrations of SCL serum on the survival rates of HepG2215 cells. C: The IC50 values of HepG2215 cells treated with SCL serum for 24 h. *P<0.05, NS serum: Normal saline serum group. SCL serum: Stellera chamaejasme L. serum group

图3 瑞香狼毒含药血清降低HepG2215细胞YAP1蛋白表达水平 YAP1在HepG2215细胞中的表达（*P<0.05）。NS serum：生理盐水血清组；SCL serum：瑞香狼毒含药血清组

Fig. 3 Expression of YAP1 decreased by SCL serum in HepG2215 cells Expression of YAP1 in HepG2215 cells（*P<0.05）. NS serum: Normal saline serum group. SCL serum: Stellera chamaejasme L. serum group

图4 瑞香狼毒抑制肝原位癌小鼠肿瘤的增长 A：肝脏的形态学外观和肝脏肿瘤的体积（*P<0.05）；B：肝脏肿瘤的H&E染色，标尺 =100 μm。NS：生理盐水组；SCL：瑞香狼毒含药血清组

Fig. 4 SCL inhibiting tumor growth in the mice with hepatic carcinoma in situ A: Morphological appearance of the liver and the volume of liver tumors（*P<0.05）. B: H&E staining of liver tumors. Bar = 100 μm. NS: Normal saline group, SCL: Stellera chamaejasme L. decoction group

图5 瑞香狼毒降低肝原位癌小鼠肝癌细胞YAP1的表达 YAP1在肝原位肿瘤小鼠中的表达（*P<0.05）； NS：生理盐水组；SCL：瑞香狼毒水煎液组

Fig. 5 Expression of YAP1 decreased by SCL in the mice with tumors in liver Expression of YAP1 in the mice with tumors in liver（*P<0.05）. NS: normal saline group. SCL: Stellera chamaejasme L. decoction group

图6 LC-MS检测不同组别化合物数量Venn图 A：不同组别LC-MS负极检测化合物数量；B：不同组别LC-MS正极检测化合物数量。NS serum：生理盐水血清；SCL serum：瑞香狼毒含药血清；SCL decoction：瑞香狼毒水煎液

Fig. 6 Venn diagram of the number of compounds of different groups detected by LC-MS A: Number of compounds detected by LC-MS negative electrode in different groups. B: Number of compounds detected by LC-MS positive electrode in different groups. NS serum: Normal saline serum group. SCL serum: Stellera chamaejasme L. serum group. SCL decoction: Stellera chamaejasme L. decoction group

表1 瑞香狼毒发挥作用的活性成分

Table 1 Active ingredients of SCL

化合物 Compound	峰ID ID	代谢物Metabolite	离子模式 Mode	加合离子模式 Adducts	分子式 Formula	Metlin数据库得分 Fragmentation score	HMDB 数据库得分 Theoretical fragmentation score	瑞香狼毒组 M_5	瑞香狼毒水煎液组W_6
2.89_240.9800m/z	neg_1438	3-硫酸咖啡酸Caffeic acid 3-sulfate	neg	M-H₂O-H	C₉H₈O₇S	-	87.8	42 837.95	4 520 349
6.01_213.0311m/z	neg_2060	5-苄恶唑-2-酮5-Benzyloxolan-2-one	neg	M+K-2H	C₁₁H₁₂O₂	-	73.3	7 321.147	7 124.684
2.33_337.0552m/z	neg_5643	3,4,5-三羟基-6-[（2-氧代-2H-铬-5-基）氧]氧烷-2-羧酸 3,4,5-Trihydroxy-6-[（2-oxo-2H-chromen-5-yl）oxy]oxane-2-carboxylic acid	neg	M-H	C₁₅H₁₄O₉	-	54.7	56 091.29	34 451.94
2.51_193.0492m/z	pos_1816	异东莨菪内酯Isoscopoletin	pos	M+H	C₁₀H₈O₄	89.9	-	227.2022	5 515 259

图7 瑞香狼毒活性成分与YAP1分子对接示意图 A：3-硫酸咖啡酸和YAP1；B：3,4,5-三羟基-6-[（2-氧代-2H-铬-5-基）氧]氧烷-2-羧酸和YAP1；C：异东莨菪内酯和YAP1；D: 维替泊芬和YAP1

Fig. 7 Schematic diagram of molecular docking between the active ingredients of Stellera chamaejasme L. and YAP1 A: Caffeic acid 3-sulfate and YAP1. B: 3,4,5-trihydroxy-6-[（2-oxo-2H-chromen-5-yl）oxy]oxane-2-carboxylic acid and YAP1. C: Isoscopoletin and YAP1.D：Verteporfin and YAP1

参考文献 19

[1]	Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA A Cancer J Clin, 2018, 68(6): 394-424. doi: 10.3322/caac.v68.6 URL
[2]	Anwanwan D, Singh SK, Singh S, et al. Challenges in liver cancer and possible treatment approaches[J]. Biochim Biophys Acta BBA Rev Cancer, 2020, 1873(1): 188314.
[3]	Imajo M, Ebisuya M, Nishida E. Dual role of YAP and TAZ in renewal of the intestinal epithelium[J]. Nat Cell Biol, 2015, 17(1): 7-19. doi: 10.1038/ncb3084
[4]	Thompson BJ. YAP/TAZ: drivers of tumor growth, metastasis, and resistance to therapy[J]. Bioessays, 2020, 42(5): e1900162.
[5]	王琪, 邓志华. 短发夹RNA沉默YAP表达对肝癌细胞SMMC-7721增殖和凋亡的影响[J]. 中国药物与临床, 2013, 13(9): 1117-1120, 1245.
	Wang Q, Deng ZH. Effects of shRNA on human hepatocellular carcinoma cell line SMMC-7721 apoptosis and proliferation via Yes-associated protein expression silencing[J]. Chin Remedies & Clin, 2013, 13(9): 1117-1120, 1245.
[6]	Yang ZY, Kan JT, Cheng ZY, et al. Daphnoretin-induced apoptosis in HeLa cells: a possible mitochondria-dependent pathway[J]. Cytotechnology, 2014, 66(1): 51-61. doi: 10.1007/s10616-013-9536-8 URL
[7]	刘阳, 卢年华, 冯亭亭, 等. HPLC结合响应面分析法优化提取瑞香狼毒中西瑞香素的含量研究[J]. 河北中医药学报, 2019, 34(5): 48-52.
	Liu Y, Lu NH, Feng TT, et al. Optimization of Daphne content extraction in Stellera chamaejasme by HPLC combined with response surface methodology[J]. J Hebei Tradit Chin Med Pharmacol, 2019, 34(5): 48-52.
[8]	Guo YL, Peng Q, Hao LY, et al. Dihydroartemisinin promoted FXR expression independent of YAP1 in hepatocellular carcinoma[J]. FASEB J, 2022, 36(6): e22361.
[9]	Li SH, Ji JM, Zhang ZQ, et al. Cisplatin promotes the expression level of PD-L1 in the microenvironment of hepatocellular carcinoma through YAP1[J]. Mol Cell Biochem, 2020, 475(1): 79-91. doi: 10.1007/s11010-020-03861-0
[10]	潘国凤, 杨庆, 翁小刚, 等. 瑞香狼毒抗肿瘤作用机制研究进展[J]. 中华中医药杂志, 2007, 22(12): 873-875.
	Pan GF, Yang Q, Weng XG, et al. Research progress on anti-tumor mechanism of stellera chamaejasme[J]. China J Tradit Chin Med Pharm, 2007, 22(12): 873-875.
[11]	周芳月, 李佳, 潘泽政, 等. Hippo-YAP通路在多种干细胞尤其是卵巢生殖干细胞自我更新中作用的研究[J]. 中国细胞生物学学报, 2015, 37(7): 1053-1058.
	Zhou FY, Li J, Pan ZZ, et al. Research progress on the role of hippo-YAP pathway in regulating various stem cells especially ovarian germ stem cell self-renewal[J]. Chin J Cell Biol, 2015, 37(7): 1053-1058.
[12]	Zhang X, Qiao YX, Wu Q, et al. The essential role of YAP O-GlcNAcylation in high-glucose-stimulated liver tumorigenesis[J]. Nat Commun, 2017, 8: 15280. doi: 10.1038/ncomms15280 pmid: 28474680
[13]	Hao LY, Guo YL, Peng Q, et al. Dihydroartemisinin reduced lipid droplet deposition by YAP1 to promote the anti-PD-1 effect in hepatocellular carcinoma[J]. Phytomedicine, 2022, 96: 153913. doi: 10.1016/j.phymed.2021.153913 URL
[14]	Xu ZH, Qin GW, Li XY, et al. New biflavanones and bioactive compounds from Stellera chamaejasme L[J]. Yao Xue Xue Bao, 2001, 36(9): 669-671.
[15]	Liu XN, Wang S, Yang Q, et al. ESC reverses epithelial mesenchymal transition induced by transforming growth factor-β via inhibition of Smad signal pathway in HepG2 liver cancer cells[J]. Cancer Cell Int, 2015, 15: 114. doi: 10.1186/s12935-015-0265-2 URL
[16]	Kan XX, Li Q, Chen X, et al. A novel cell cycle blocker extracted from Stellera chamaejasme L. inhibits the proliferation of hepatocarcinoma cells[J]. Oncol Rep, 2016, 35(6): 3480-3488. doi: 10.3892/or.2016.4742 URL
[17]	Zhao DD, Zhao QS, Liu L, et al. Compounds from Dryopteris fragrans(L.) schott with cytotoxic activity[J]. Molecules, 2014, 19(3): 3345-3355. doi: 10.3390/molecules19033345 URL
[18]	McGovern PE, Christofidou-Solomidou M, Wang W, et al. Anticancer activity of botanical compounds in ancient fermented beverages(review)[J]. Int J Oncol, 2010, 37(1): 5-14. pmid: 20514391
[19]	杨喜花, 王俊田, 陈丽霞, 等. 瑞香狼毒提取物对肝癌原位移植瘤小鼠的生命延长作用[J]. 中国药物与临床, 2017, 17(4): 501-503.
	Yang XH, Wang JT, Chen LX, et al. Effect of stellera chamaejasme extract on prolonging life of mice with orthotopic liver cancer transplantation[J]. Chin Remedies & Clin, 2017, 17(4): 501-503.