桂枝茯苓汤含药血清对子宫内膜癌细胞的影响

doi:10.13438/j.cnki.jdzk.2023.01.011

摘要/Abstract

摘要：为探究桂枝茯苓汤含药血清是否通过调控3-磷酸肌醇激酶（PI3K）、蛋白激酶B（AKT），参与调控子宫内膜癌细胞增殖、上皮-间质转化（EMT）与凋亡，将72只SD大鼠分为健康组、桂枝茯苓汤高剂量组、桂枝茯苓汤中剂量组、桂枝茯苓汤低剂量组和奥沙利铂组，对各组大鼠灌胃相应剂量药物，从大鼠的腹主动脉取血制备含药血清，培养子宫内膜癌HEC-B细胞，分为对照组、含药血清高剂量组、含药血清中剂量组、含药血清低剂量组、奥沙利铂血清组、含药血清高剂量+通路激活剂组，向各组细胞培养基中加入相应血清及试剂.采用四甲基偶氮唑蓝（MTT）法检测各组细胞增殖情况，流式细胞术检测各组细胞凋亡情况，Western Blot法检测EMT相关蛋白E-钙黏蛋白（E-cadherin）、细胞波形蛋白（Vimentin）、N-钙黏蛋白（N-cadherin）表达水平及PI3K/AKT通路相关蛋白磷酸化水平.结果表明：与对照组相比，含药血清高剂量组、含药血清中剂量组、含药血清低剂量组的细胞生存率、Vimentin和N-cadherin蛋白表达量、PI3K和AKT磷酸化程度均显著降低（P＜0.05），细胞凋亡率、E-cadherin蛋白表达量显著升高（P＜0.05），且呈剂量依赖性；与含药血清高剂量组相比，含药血清高剂量+通路激活剂组的细胞生存率、Vimentin和N-cadherin蛋白表达量、PI3K和AKT磷酸化程度均显著升高（P＜0.05），细胞凋亡率、E-cadherin蛋白表达量显著降低（P＜0.05），奥沙利铂血清组各项指标无显著差异（P＞0.05）.说明桂枝茯苓汤通过调控PI3K/AKT通路，抑制相关蛋白磷酸化，阻碍细胞生长，加剧细胞凋亡，缓解EMT，从而缓解子宫肌瘤病情.

关键词： 桂枝茯苓汤, 3-磷酸肌醇激酶, 蛋白激酶B, 子宫内膜癌, 细胞增殖, 细胞凋亡, 上皮-间质转化

Abstract: The purpose of this study was to investigate the effect of Guizhi Fuling Decoction-containing serum in regulation of endometrial cancer cell proliferation,epithelium-mesenchymal transformation (EMT) and apoptosis through the regulation of inositol 3-phosphate kinase (PI3K) and protein kinase B (AKT).Seventy-two SD rats were divided into healthy group,high-dose Guizhi Fuling Decoction group,middle-dose Guizhi Fuling Decoction group,low-dose Guizhi Fuling Decoction group and oxaliplatin group.Corresponding doses of drugs were given by gavage,and blood was drawn from the abdominal aorta to prepare drug-containing serum.Human endometrial cancer HEC-B cells were cultured and grouped into control group,high-dose drug-containing serum group,medium-dose drug-containing serum group,low-dose drug-containing serum group,oxaliplatin serum group,high-dose drug-containing serum + pathway activator group.Corresponding serum and reagents were added to the cell culture medium of each group.The proliferation of cells in each group was detected by tetramethylazolyl blue (MTT) method;cell apoptosis in each group was detected by flow cytometry;the expression levels of EMT-related proteins E-cadherin (E-cadherin),cellular vimentin (Vimentin),N-cadherin (N-cadherin) and the phosphorylation levels of PI3K/AKT pathway-related proteins were detected by Western Blot.Compared with the control group,the cell survival rate,Vimentin,N-cadherin protein expression,PI3K and AKT phosphorylation levels in the high-dose drug-containing serum group,the medium-dose drug-containing serum group,and the low-dose drug-containing serum group were obviously decreased (P＜0.05);while the apoptosis rate and E-cadherin protein expression were obviously increased (P＜0.05),in a dose-dependent manner.Compared with the high-dose drug-containing serum group,the cell survival rate,Vimentin,N-cadherin protein expression,PI3K and AKT phosphorylation levels in the high-dose drug-containing serum + pathway activator group were obviously increased (P＜0.05),and the apoptosis rate and E-cadherin protein expression were obviously decreased (P＜0.05),and there was no significant difference in each index between the oxaliplatin serum group (P＞0.05).Guizhi Fuling Decoction regulates the PI3K/AKT pathway to inhibit the phosphorylation of related proteins,hinder cell growth,aggravate cell apoptosis,relieve EMT,and thus alleviate the condition of uterine fibroids.

Key words: Guizhi Fuling Decoction, phosphatidylinositide 3-kinase, protein kinase B, endometrial cancer, cell proliferation, cell apoptosis, epithelial-mesenchymal transition

袁小波, 唐静, 阳帆. 桂枝茯苓汤含药血清对子宫内膜癌细胞的影响[J]. 吉首大学学报（自然科学版）, 2023, 44(1): 77-83.

YUAN Xiaobo, TANG Jing, YANG Fan. Effect of Guizhi Fuling Decoction-Containing Serum on Endometrial Cancer Cells[J]. Journal of Jishou University(Natural Sciences Edition), 2023, 44(1): 77-83.

参考文献

[1] ZHOU Lanyun,WANG Wei,WANG Fenfen,et al.Plasma-Derived Exosomal miR-15a-5p as a Promising Diagnostic Biomarker for Early Detection of Endometrial Carcinoma[J].Molecular Cancer,2021,20(1):1-6.
[2] PENDLEBURY ADAM,RADEVA MILENA,ROSE PETER G.Surgical Lymph Node Assessment Influences Adjuvant Therapy in Clinically Apparent Stage I Endometrioid Endometrial Carcinoma,Meeting Mayo Criteria for LymphadEnectomy[J].Journal of Surgical Oncology,2021,123(5):1292-1298.
[3] KWAKYE AKOSUA KOTAA,KAMPO SYLVANUS,LV Jiaxin,et al.Levobupivacaine Inhibits Proliferation and Promotes Apoptosis of Breast Cancer Cells by Suppressing the PI3K/Akt/mTOR Signalling Pathway[J].BMC research notes,2020,13(1):386-389.
[4] PATEL PUJA,UMAPATHY DEVAN,MANIVANNAN SELVAMBIGAI,et al.A Doxorubicin-Platinum Conjugate System:Impacts on PI3K/AKT Actuation and Apoptosis in Breast Cancer Cells[J].RSC Advances,2021,11(8):4818-4828.
[5] MEI Yan,LIAO Xiaoming,ZHU Lingyu,et al.Overexpression of RSK4 Reverses Doxorubicin Resistance in Human Breast Cancer Cells via PI3K/AKT Signalling Pathway[J].The Journal of Biochemistry,2020,167(6):603-611.
[6] ZHU Mingliang,ZHANG Peimin,JIANG Min,et al.Myricetin Induces Apoptosis and Autophagy by Inhibiting PI3K/Akt/mTOR Signalling in Human Colon Cancer Cells[J].BMC Complementary Medicine and Therapies,2020,20(1):209-213.
[7] SUN Xiao,NG TONY T H,SHAM KATHY W Y,et al.Bufalin,a Traditional Chinese Medicine Compound,Prevents Tumor Formation in Two Murine Models of Colorectal Cancer[J].Cancer Prevention Research,2019,12(10):653-666.
[8] ZHANG Siqin,LAI Xinxing,WANG Xin,et al.Deciphering the Pharmacological Mechanisms of Guizhi-Fuling Capsule on Primary Dysmenorrhea Through Network Pharmacology[J].Frontiers in Pharmacology,2021,12(1):1-7.
[9] 黄秋艳.桂枝茯苓汤加减治疗卵巢囊肿的效果及其对卵巢功能的影响[J].医学食疗与健康,2020,18(9):1-2.
[10] 唐喜云.桂枝茯苓汤加减、维生素E结合艾灸治疗子宫肌瘤的临床观察[J].中国民间疗法,2021,29(19):63-65.
[11] RABE KIMMIE M,KLEIN MOLLY E,GHATAK SAYAK,et al.Sentinel Nodal Metastasis Detection in Endometrial Carcinoma with Microcystic,Elongated,and Fragmented (MELF) Pattern by Cytokeratin Immunostaining:A Recommendation for Routine Processing Protocols[J].American Journal of Clinical Pathology,2021,156(5):846-852.
[12] WANG Anna,GUO Hongyan,LONG Zaiqiu.Integrative Analysis of Differently Expressed Genes Reveals a 17-Gene Prognosis Signature for Endometrial Carcinoma[J].BioMed Research International,2021,14(5):1-18.
[13] 宋亭亭,刘国燕,王言研,等.桂枝茯苓汤对卵巢癌术后化疗的增敏效果及对炎症因子和免疫指标的影响[J].中华中医药学刊,2021,39(5):251-254.
[14] 何凤仪,王弋,罗艳华,等.桂枝茯苓汤对子宫内膜癌的药效及其机制初步探讨[J].中国比较医学杂志,2019,29(3):43-47.
[15] 明松林,刘燕,杨铁柱,等.桂枝茯苓汤对乳腺癌细胞株线粒体凋亡途径相关蛋白和细胞周期的影响[J].中医药导报,2015,21(17):23-26.
[16] 周凌,孙慧婷,杨思慧,等.shRNA靶向血管生成素2基因沉默联合奥沙利铂对子宫内膜癌生长抑制作用的研究[J].东南大学学报,2021,40(2):148-157.
[17] 董安琦,李东宝,周进.间皮-间充质转化在恶性肿瘤中的研究进展[J].中华实验外科杂志,2022,39(1):178-182.
[18] 闫飞,刘颖,冯年平.调控EMT的中药有效成分纳米递送系统抑制肿瘤转移的研究进展[J].中国实验方剂学杂志,2020,26(4):235-241.
[19] 代洪波,王梅,张艳,等.顺铂热化疗对卵巢癌PI3K/AKT及凋亡相关调控因子表达的影响[J].现代生物医学进展,2016,16(18):3515-3518.
[20] 代丽丽,陈丽娜,蔡丽娜,等.miR-19通过靶向PTEN调节PI3K-AKT通路促进子宫内膜癌KLE细胞的侵袭和迁移[J].中国肿瘤生物治疗杂志,2022,28(11):1107-1112.
[21] DAI Yifei,QIANG Weijie,YU Xiankuo,et al.Guizhi Fuling Decoction Inhibiting the PI3K and MAPK Pathways in Breast Cancer Cells Revealed by HTS2 Technology and Systems Pharmacology[J].Computational and Structural Biotechnology Journal,2020,18:1121-1136.