杨梅枝条醇提物对A375细胞增殖和凋亡的影响及其分子机制

doi:10.3969/j.issn.1004-1524.2022.05.12

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (5): 974-983.DOI: 10.3969/j.issn.1004-1524.2022.05.12

杨梅枝条醇提物对A375细胞增殖和凋亡的影响及其分子机制

郑园园¹^,²(), 俞浙萍², 张淑文², 李有贵³, 孙鹂², 郑锡良², 戚行江¹^,²^,^*()

1.浙江师范大学化学与生命科学学院,浙江金华 321004
2.浙江省农业科学院园艺研究所,浙江杭州 310021
3.浙江省农业科学院蚕桑与茶叶研究所,浙江杭州 310021

收稿日期:2021-11-03 出版日期:2022-05-25 发布日期:2022-06-06
作者简介:* 戚行江,E-mail: qixj@mail.zaas.ac.cn
郑园园(1995—),女,浙江杭州人,硕士研究生,植物学专业。E-mail: zhengyuanyuanky@163.com
通讯作者: 戚行江
基金资助:
浙江省农业(果品)新品种选育重大科技专项(2021C02066-2);浙江省重点研发计划(2021C02009)

Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism

ZHENG Yuanyua¹^,²(), YU Zheping², ZHANG Shuwen², LI Yougui³, SUN Li², ZHENG Xiliang², QI Xingjiang¹^,²^,^*()

1. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Institute of Sericultural and Tea, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2021-11-03 Online:2022-05-25 Published:2022-06-06
Contact: QI Xingjiang

摘要/Abstract

摘要：

杨梅(Myrica rubra)具有较高的药用价值,利用杨梅枝条醇提物(Myrica rubra branch extract, MRBE)开展其对人恶性黑色素瘤A375细胞的作用研究。采用MTT法、PI流式细胞术、Annexin V-PI双染流式细胞术、实时荧光定量PCR(qRT-PCR)等方法,解析MRBE诱导A375细胞阻滞与凋亡的潜在作用机制。结果表明:MRBE对A375细胞的抑制率呈剂量依赖效应关系,抑制率最高达49.46%;流式细胞术结果表明,MRBE作用48 h可使A375细胞阻滞于S期并发生凋亡;qRT-PCR结果进一步表明,MRBE可能通过上调P21和下调CyclinA、CyclinD、CDK2、CDK4、CDK6、E2F1等基因的表达,诱导A375细胞阻滞于S期,同时下调Bcl-w和Raf基因的表达促使A375细胞凋亡。初步解析了MRBE对人恶性黑色素瘤A375细胞增殖和凋亡的作用机制,为利用MRBE研发抗黑色素瘤潜在药物提供科学依据,同时有助于提高杨梅枝条的药用附加值,拓宽杨梅产业链多元化发展。

关键词: 杨梅, 枝条醇提物, 人黑色素瘤A375细胞, 细胞周期, 细胞凋亡

Abstract:

Chinese bayberry (Myrica rubra) has high medicinal values. This study was performed to evaluate the effect of alcohol extracts of Myrica rubra branch extract (MRBE) on human malignant melanoma cells A375. MTT assay, flow cytometry based on PI, Annexin V/PI double staining and quantitative reverse-transcription PCR (qRT-PCR) were used to dissect the potential mechanism of the arrest and apoptosis of A375 cells induced by MRBE. The results revealed that MRBE inhibited the proliferation of human A375 cells in a dose-dependent way, and the inhibition rate was up to 49.46%. Flow cytometry analyses suggested that 48 h treatment of MRBE induced both S-phase cell cycle arrest and apoptotic death in A375 cells. The results of qRT-PCR further showed that MRBE induced S phase arrest by promoting the upregulation of p21 and decreasing the expression of CyclinA, CyclinD, CDK2, CDK4, CDK6 and E2F1, and induced apoptosis by decreasing the expression of Bcl-w and Raf. This study revealed the action mechanism of MRBE on the induction of S-phase arrest and apoptosis of A375 cells, which could provide scientific evidence for the development of potential anti-melanom drugs by using MRBE, and help to improve the medicinal added value of Chinese bayberry branches and broaden the diversified development of its industry chain.

Key words: Chinese bayberry, alcohol extracts of branch extract, human melanoma A375 cells, cell cycle, cell apoptosis

中图分类号:

郑园园, 俞浙萍, 张淑文, 李有贵, 孙鹂, 郑锡良, 戚行江. 杨梅枝条醇提物对A375细胞增殖和凋亡的影响及其分子机制[J]. 浙江农业学报, 2022, 34(5): 974-983.

ZHENG Yuanyua, YU Zheping, ZHANG Shuwen, LI Yougui, SUN Li, ZHENG Xiliang, QI Xingjiang. Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism[J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 974-983.

图/表 5

图1 MRBE对A375细胞增殖的影响 A,不同浓度MRBE处理A375细胞48 h后的抑制率;B,不同浓度MRBE处理A375细胞48 h后的细胞形态情况。*表示0.01<P<0.05;**表示P<0.01。下同。

Fig.1 Effect of MRBE on proliferation of A375 cells A, Effects of different concentrations of MRBE on the inhibition rate of A375 cells after 48 h treatment. B, Effects of different concentrations of MRBE on cell morphology of A375 cells after 48 h treatment. * represented 0.01<P<0.05, ** represented P<0.01. The same as below.

图2 MRBE对A375细胞周期的影响 A,PI流式细胞术分析不同浓度MRBE对A375细胞周期的影响;B,不同细胞周期阶段的细胞数量百分比。

Fig.2 Effect of MRBE on A375 cell cycle A, Effects of different concentrations of MRBE on A375 cell cycle analysed by PI flow cytometry; B, Percentage of cells in different cell cycle phases.

图3 MRBE对A375细胞凋亡的影响 A,Annexin V-PI双染流式细胞术检测不同浓度MRBE对A375细胞凋亡的影响,左下为正常细胞,右下为早期调亡细胞,右上为晚期调亡细胞,左上为坏死细胞;B,不同类型细胞数量百分比。

Fig.3 Effect of MRBE on apoptosis of A375 cells A, Effects of different concentrations of MRBE on A375 cell apoptosis analysed by Annexin V-PI flow cytometry, Lower left quadrant showed normal cells, lower right quadrant showed early apoptotic cells, upper right quadrant showed late apoptotic cells, upper left quadrant showed necrotic cells. B, Percentages of cells in different death types.

图4 MRBE对A375细胞周期相关基因表达的影响

Fig.4 Effect of MRBE on expression of cycle related genes in A375 cells

图5 MRBE对A375细胞凋亡相关基因表达的影响

Fig.5 Effect of MRBE on expression of apoptosis related genes in A375 cells

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杨梅枝条醇提物对A375细胞增殖和凋亡的影响及其分子机制

Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism

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