黄芪-莪术-重楼配伍降低血管内皮通透性抑制结肠癌转移作用的研究

刘甜甜; 卞勇; 关汉卿; 梁研; 郭文晖; 余倩慧; 唐德才

doi:10.14148/j.issn.1672-0482.2022.0115

黄芪-莪术-重楼配伍降低血管内皮通透性抑制结肠癌转移作用的研究

doi: 10.14148/j.issn.1672-0482.2022.0115

刘甜甜^1,,
卞勇^{1, 2},
关汉卿¹,
梁研¹,
郭文晖¹,
余倩慧¹,
唐德才^1, ,

1.
南京中医药大学中医学院 · 中西医结合学院, 江苏南京 210023
2.
南京中医药大学实验动物中心, 江苏南京 210023

基金项目:

国家自然科学基金面上项目 81873021

江苏省中医药科技发展专项( 2020ZX01

江苏省研究生科研与实践创新计划项目 KYCX21_1764

详细信息

作者简介:
刘甜甜，女，硕士研究生, E-mail: liutiantian1368@163.com

通讯作者:
唐德才，男，教授，主要从事中药药性、配伍机制及应用研究，E-mail: 290022@njucm.edu.cn

中图分类号: R285.5
计量
- 文章访问数: 360
- HTML全文浏览量: 242
- PDF下载量: 39
- 被引次数: 0
出版历程
- 收稿日期: 2021-10-29
- 网络出版日期: 2022-03-01
- 发布日期: 2022-02-10

Study on the Inhibitory Effect of Huangqi-Ezhu-Chonglou Combination on Metastasis of Colon Cancer by Reducing Vascular Endothelial Permeability

1.
School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Laboratory Animal Center, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的观察黄芪-莪术-重楼配伍对人脐静脉内皮细胞株(HUVEC)通透性与细胞紧密连接的影响及其抑制人结肠癌细胞系(HCT116)血行转移的作用机制。方法制备空白对照血清和黄芪-莪术-重楼配伍含药血清。构建HUVEC-HCT116共培养体系, 实验分为共培养模型组, 黄芪-莪术-重楼配伍低、中、高剂量(2.1、4.2、8.4 g · kg^-1)组。另设HUVEC单培养对照组。CCK-8法检测细胞增殖; FITC-dextran法、Transwell法检测HUVEC通透性与HCT116跨内皮迁移数量; 免疫荧光法检测ZO-1蛋白的分布; Western blot法检测RhoA、ROCK及ZO-1蛋白表达量。结果黄芪-莪术-重楼配伍不同剂量含药血清处理48 h后, HUVEC增殖均显著减少(P < 0.01)。与单培养对照组相比, 共培养模型组FITC-dextran渗透量、跨越内皮迁移的HCT116细胞数量明显增加(P < 0.01), ZO-1蛋白表达下调(P < 0.01), RhoA、ROCK蛋白表达上调(P < 0.01);与共培养模型组相比, 黄芪-莪术-重楼配伍各剂量组FITC-Dextran渗透量、跨越内皮迁移的HCT116细胞数量均减少(P < 0.05), ZO-1蛋白表达上调(P < 0.05), RhoA、ROCK蛋白表达下调(P < 0.05), 且呈现剂量依赖性。结论黄芪-莪术-重楼配伍可能通过抑制RhoA/ROCK通路调控HUVEC紧密连接相关蛋白ZO-1的表达, 降低血管内皮通透性, 进而抑制结肠癌血行转移。
- 黄芪-莪术-重楼配伍 /
- 结肠癌 /
- 血管通透 /
- 共培养 /
- 紧密连接
Abstract: OBJECTIVE To observe the effects of Huangqi-Ezhu-Chonglou combination (HEC) on the permeability and tight junction of human umbilical vein endothelial cells (HUVEC) and to explore the mechanism of HEC on inhibiting the hematogenous metastasis of colon cancer cells (HCT116). METHODS Control serum and HEC-medicated serum were prepared. The co-culture system of HUVEC-HCT116 was constructed and divided into single culture control group, co-culture model group and HEC low, medium and high dose (2.1, 4.2, 8.4 g ·kg^-1) groups. In addition, HUVEC single culture control group was set up. Cell proliferation was detected by CCK-8 method; FITC-dextran method and transwell method were used to detect the permeability of HUVEC and the number of cross endothelial migration of HCT116 cells; The distribution of ZO-1 protein in HUVEC was detected by immunofluorescence; The protein expressions of RhoA, ROCK and ZO-1 were detected by Western blot. RESULTS The proliferation of HUVEC was inhibited after 48 h of treatment with different doses of HEC medicated serum (P < 0.01). Compared with the single culture control group, FITC-dextran penetration and the number of transendothelial migration of HCT116 in the co-culture model group significantly increased (P < 0.01), the expression of ZO-1 was down-regulated (P < 0.01), and the expressions of RhoA and ROCK were up-regulated(P < 0.01); Compared with the co-culture model group, the FITC-dextran penetration and the number of transendothelial migration of HCT116 in the HEC group significantly decreased (P < 0.05), the expression of ZO-1 protein was up-regulated (P < 0.05), and the expressions of RhoA and ROCK were down-regulated in a dose-dependent manner (P < 0.05). CONCLUSION HEC may regulate the expression of tight junction associated proteins ZO-1 in HUVEC by inhibiting RhoA/ROCK pathway, reduce vascular endothelial permeability, and then inhibit colon cancer hematogenous metastasis.
- Huangqi-Ezhu-Chonglou combination /
- colon cancer /
- vascular permeability /
- co-culture /
- tight junction

HTML全文

图 1 黄芪-莪术-重楼配伍对HUVEC细胞增殖的影响

注: 与空白对照组相比, ^**P < 0.01。

Figure 1. Effect of Huangqi-Ezhu-Chonglou combination on the proliferation of HUVECs

下载: 全尺寸图片幻灯片

图 2 黄芪-莪术-重楼配伍对单层HUVEC细胞通透性的影响

注: 与单培养对照组相比, ^**P < 0.01;与共培养模型组相比, ^##P < 0.01。

Figure 2. Effect of Huangqi-Ezhu-Chonglou combination on permeability of monolayer HUVECs

下载: 全尺寸图片幻灯片

图 3 黄芪-莪术-重楼配伍对HCT116细胞跨血管内皮迁移的影响

注: 绿色荧光为HUVEC致密单层；红色荧光为跨内皮迁移的HCT116；与单培养对照组相比, ^**P < 0.01;与共培养模型组相比, ^#P < 0.05, ^##P < 0.01。

Figure 3. Effect of Huangqi-Ezhu-Chonglou combination on transvascular endothelial migration of HCT116 cells

下载: 全尺寸图片幻灯片

图 4 免疫荧光法检测黄芪-莪术-重楼配伍对HUVEC细胞ZO-1表达的影响

注: 与单培养对照组相比, ^**P < 0.01;与共培养模型组相比, ^##P < 0.01。

Figure 4. Effect of Huangqi-Ezhu-Chonglou combination on the expression of ZO-1 in HUVECs by immunofluorescence assay

下载: 全尺寸图片幻灯片

图 5 各组HUVEC细胞中ZO-1蛋白表达的比较

注: 与单培养对照组相比, ^**P < 0.01;与共培养模型组相比, ^#P < 0.05, ^##P < 0.01。

Figure 5. Comparison of ZO-1 protein expression in HUVECs in each group

下载: 全尺寸图片幻灯片

图 6 各组HUVEC细胞中RhoA、ROCK蛋白表达的比较

注: 与单培养对照组相比, ^**P < 0.01;与共培养模型组相比, ^#P < 0.05, ^##P < 0.01。

Figure 6. Comparison of RhoA and ROCK protein expressions in HUVECs in each group

下载: 全尺寸图片幻灯片

参考文献(33)

[1]	TOMITA T, KATO M, HIRATSUKA S. Regulation of vascular permeability in cancer metastasis[J]. Cancer Sci, 2021, 112(8): 2966-2974. doi: 10.1111/cas.14942
[2]	单云龙, 汪思亮, 祝娉婷, 等. 抑制肿瘤转移的新策略: 靶向肿瘤细胞的外渗过程[J]. 肿瘤, 2014, 34(8): 754-757. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZLL201408013.htm SHAN YL, WANG SL, ZHU PT, et al. The new therapeutic strategy for inhibition of cancer metastasis: Targeting extravasation of cancer cells[J]. Tumor, 2014, 34(8): 754-757. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZLL201408013.htm
[3]	CONG X, KONG W. Endothelial tight junctions and their regulatory signaling pathways in vascular homeostasis and disease[J]. Cell Signal, 2020, 66: 109485. doi: 10.1016/j.cellsig.2019.109485
[4]	ZIHNI C, MILLS C, MATTER K, et al. Tight junctions: From simple barriers to multifunctional molecular gates[J]. Nat Rev Mol Cell Biol, 2016, 17(9): 564-580. doi: 10.1038/nrm.2016.80
[5]	LIN YN, ZHANG C, XIANG PP, et al. Exosomes derived from HeLa cells break down vascular integrity by triggering endoplasmic Reticulum stress in endothelial cells[J]. J Extracell Vesicles, 2020, 9(1): 1722385. doi: 10.1080/20013078.2020.1722385
[6]	LI RR, QI YN, JIANG M, et al. Primary tumor-secreted VEGF induces vascular hyperpermeability in premetastatic lung via the occludin phosphorylation/ubiquitination pathway[J]. Mol Carcinog, 2019, 58(12): 2316-2326. doi: 10.1002/mc.23120
[7]	HARATI R, HAFEZI S, MABONDZO A, et al. Silencing miR-202-3p increases MMP-1 and promotes a brain invasive phenotype in metastatic breast cancer cells[J]. PLoS ONE, 2020, 15(10): e0239292. doi: 10.1371/journal.pone.0239292
[8]	张荣嘎. 芪术蚤复方对乳腺癌模型小鼠的抑制作用及机制研究[J]. 四川中医, 2018, 36(12): 53-56. https://www.cnki.com.cn/Article/CJFDTOTAL-SCZY201812020.htm ZHANG RG. Effect and mechanism of Qizhuzao formulation on breast cancer model[J]. J Sichuan Tradit Chin Med, 2018, 36(12): 53-56. https://www.cnki.com.cn/Article/CJFDTOTAL-SCZY201812020.htm
[9]	曾普华, 郜文辉, 潘敏求, 等. 益气化瘀解毒方对人肝癌裸鼠HepG2移植瘤MVD、HIF1α、VEGF/KDR表达的影响[J]. 中华中医药学刊, 2014, 32(7): 1563-1565. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYHS201407010.htm ZENG PH, GAO WH, PAN MQ, et al. Effect of prescription of strengthening qi and clearing poison and toxin on expressions of MVD, HIF1α and VEGF/KDR from HepG2 hepatocellular carcinoma nude mice transplantation tumor[J]. Chin Arch Tradit Chin Med, 2014, 32(7): 1563-1565. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYHS201407010.htm
[10]	彭巍, 唐建清, 龚辉, 等. 健脾消癌方治疗对大肠癌肝转移患者临床疗效、血管生成因子及QLQ-c30评分的影响[J]. 四川中医, 2021, 39(8): 76-79. https://www.cnki.com.cn/Article/CJFDTOTAL-SCZY202108024.htm PENG W, TANG JQ, GONG H, et al. Effect of Jianpi Xiaoai Recipe on clinical efficacy, angiogenesis factors and QLQ-c30 score for patients with liver metastases of colorectal cancer[J]. J Sichuan Tradit Chin Med, 2021, 39(8): 76-79. https://www.cnki.com.cn/Article/CJFDTOTAL-SCZY202108024.htm
[11]	关汉卿, 刘甜甜, 梁研, 等. 黄芪-莪术-重楼配伍对结肠癌原位移植瘤模型裸鼠肿瘤及癌旁组织中侵袭性伪足相关蛋白表达的影响[J]. 中医杂志, 2021, 62(16): 1427-1433. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ202116013.htm GUAN HQ, LIU TT, LIANG Y, et al. Effect of Huangqi-Ezhu-Chonglou combination on the expression of invadopodia-related proteins in the tumors and paracancerous tissues of nude mice with colon cancer-derived orthotopic transplant model[J]. J Tradit Chin Med, 2021, 62(16): 1427-1433. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ202116013.htm
[12]	吴幸冬, 唐德才. 黄芪配伍莪术对小鼠结肠癌细胞CT26黏附和迁移能力的影响[J]. 中医杂志, 2020, 61(13): 1176-1183. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ202013017.htm WU XD, TANG DC. Effects of compatibility of Radix astragali seu hedysa and Rhizoma curcumae on adhesion and migration ability of colon cancer cell CT26 in mice[J]. J Tradit Chin Med, 2020, 61(13): 1176-1183. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ202013017.htm
[13]	张硕, 唐德才, 谭喜莹, 等. 黄芪、莪术配伍对斑马鱼血管生成的影响[J]. 中华中医药学刊, 2020, 38(8): 179-182, 281-283. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYHS202008043.htm ZHANG S, TANG DC, TAN XY, et al. Effect of angiogenesis by compatibility of Huangqi (Radix astragali) and ezhu (Curcuma) in zebrafsh model[J]. Chin Arch Tradit Chin Med, 2020, 38(8): 179-182, 281-283. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYHS202008043.htm
[14]	骆殊, 尹刚, 唐德才. 黄芪甲苷与姜黄素配伍对人肝癌裸鼠原位移植瘤新生血管生成的影响[J]. 中华肿瘤防治杂志, 2016, 23(S1): 12-13. https://www.cnki.com.cn/Article/CJFDTOTAL-QLZL2016S1006.htm LUO S, YIN G, TANG DC. Effect of astragaloside Ⅳ combined with curcumin on angiogenesis of orthotopic transplanted human hepatocellular carcinoma in nude mice[J]. Chin J Cancer Prev Treat, 2016, 23(S1): 12-13. https://www.cnki.com.cn/Article/CJFDTOTAL-QLZL2016S1006.htm
[15]	AGRAWAL V, MAHARJAN S, KIM K, et al. Direct endothelial junction restoration results in significant tumor vascular normalization and metastasis inhibition in mice[J]. Oncotarget, 2014, 5(9): 2761-2777. doi: 10.18632/oncotarget.1942
[16]	刘珂, 邱炳勋, 邹利, 等. 黄芪及其有效成分对内皮细胞及其连接的保护作用及机制研究进展[J]. 中草药, 2016, 47(21): 3912-3917. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO201621031.htm LIU K, QIU BX, ZOU L, et al. Research progress in mechanism of Astragali Radix and its active components on endothelial cells and their junction[J]. Chin Tradit Herb Drugs, 2016, 47(21): 3912-3917. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO201621031.htm
[17]	许成勇, 徐冉, 王毓国, 等. 黄芪-莪术配伍对Lewis肺癌生长转移及血管生成的抑制作用及机制研究[J]. 世界中西医结合杂志, 2018, 13(5): 596-598, 602. https://www.cnki.com.cn/Article/CJFDTOTAL-SJZX201805002.htm XU CY, XU R, WANG YG, et al. Inhibitive effects and mechanism of the compatibility of astragali radix and curcumae rhizome on the growth, metastasis and angiogenesis in Lewis lung carcinoma[J]. World J Integr Tradit West Med, 2018, 13(5): 596-598, 602. https://www.cnki.com.cn/Article/CJFDTOTAL-SJZX201805002.htm
[18]	冒慧敏. 莪术成分Zedoarondiol对血管平滑肌细胞增殖和内皮细胞损伤的影响[D]. 北京: 北京中医药大学, 2017. MAO HM. Effects of Zedoarondiol on proliferation of vascular smooth muscle cells and injury of endothelial cells[D]. Beijing: Beijing University of Chinese Medicine, 2017.
[19]	赵保胜, 朱寅荻, 马勇, 等. 中药重楼研究进展[J]. 中国实验方剂学杂志, 2011, 17(11): 267-270. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201111083.htm ZHAO BS, ZHU YD, MA Y, et al. Advances in studies on paridis rhizoma[J]. Chin J Exp Tradit Med Formulae, 2011, 17(11): 267-270. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201111083.htm
[20]	胡静, 钱晓萍, 刘宝瑞, 等. 重楼醇提物抗鼠H22移植瘤血管生成的体内实验研究[J]. 现代肿瘤医学, 2010, 18(10): 1886-1889. https://www.cnki.com.cn/Article/CJFDTOTAL-SXZL201010003.htm HU J, QIAN XP, LIU BR, et al. Experimental study on inhibiting angiogenesis in H22 hepatoma transplantation tumor mice by alcohol extract of paridis[J]. J Mod Oncol, 2010, 18(10): 1886-1889. https://www.cnki.com.cn/Article/CJFDTOTAL-SXZL201010003.htm
[21]	TANG DC, ZHANG S, SHI XX, et al. Combination of astragali polysaccharide and curcumin improves the morphological structure of tumor vessels and induces tumor vascular normalization to inhibit the growth of hepatocellular carcinoma[J]. Integr Cancer Ther, 2019, 18: 1534735418824408.
[22]	TORNAVACA O, CHIA MH, DUFTON N, et al. ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation[J]. J Cell Biol, 2015, 208(6): 821-838.
[23]	武勰, 晏馥霞. 紧密连接对肺通透性影响的研究进展[J]. 基础医学与临床, 2021, 41(2): 272-276. https://www.cnki.com.cn/Article/CJFDTOTAL-JCYL202102025.htm WU X, YAN FX. Research progress on the effects of tight junction on lung permeability[J]. Basic Clin Med, 2021, 41(2): 272-276. https://www.cnki.com.cn/Article/CJFDTOTAL-JCYL202102025.htm
[24]	FENG SR, CEN JN, HUANG YH, et al. Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins[J]. PLoS ONE, 2011, 6(8): e20599.
[25]	JIANG M, QIN CY, HAN MY. Primary breast cancer induces pulmonary vascular hyperpermeability and promotes metastasis via the VEGF-PKC pathway[J]. Mol Carcinog, 2016, 55(6): 1087-1095.
[26]	ZHANG YY, ZHAO YZ, WU Y, et al. Ophiopogon saponin C1 inhibits lung tumors by stabilizing endothelium permeability via inhibition of PKCδ[J]. Int J Biol Sci, 2020, 16(3): 396-407.
[27]	魏殿芳. Aspirin减少肺癌细胞释放TNF-α对体外肺癌脑转移的影响及其机制[D]. 唐山: 华北理工大学, 2020. WEI DF. Effect and mechanism of Aspirin reducing TNF-α release from lung cancer cells on lung cancer brain metastasis in vitro[D]. Tangshan: North China University of Science and Technology, 2020.
[28]	杜相宇. 以β-catenin为中心研究芪术抗癌方联合五氟尿嘧啶对CT26. WT原位移植瘤小鼠肝转移的影响[D]. 南京: 南京中医药大学, 2020. DU XY. Effect of Qizhu anticancer formula combined with pentafluorouracil on liver metastasis of CT26. WT orthotopic transplanted tumor mice based on β-catenin[D]. Nanjing: Nanjing University of Chinese Medicine, 2020.
[29]	GONZáLEZ-MARISCAL L, TAPIA R, CHAMORRO D. Crosstalk of tight junction components with signaling pathways[J]. Biochim Biophys Acta, 2008, 1778(3): 729-756.
[30]	赵晓云. Flt-1在小细胞肺癌细胞穿过人脑微血管内皮细胞单层中的作用及机制研究[D]. 沈阳: 中国医科大学, 2008. ZHAO XY. Involvement of flt-1 in small cell lung cancer cells migration through human brain microvascular endothelial cells[D]. Shenyang: China Medical University, 2008.
[31]	蔡倩, 郭慕真, 朱晨笛, 等. p115RhoGEF/RhoA信号通路在高糖致脑微血管内皮细胞通透性异常中的作用[J]. 营养学报, 2019, 41(2): 154-162. https://www.cnki.com.cn/Article/CJFDTOTAL-YYXX201902013.htm CAI Q, GUO MZ, ZHU CD, et al. Role of p115RhoGEF/RhoA signaling pathway in permeability of brain microvascular endothelial cells exposed to high glucose[J]. Acta Nutr Sin, 2019, 41(2): 154-162. https://www.cnki.com.cn/Article/CJFDTOTAL-YYXX201902013.htm
[32]	LI B, ZHAO WD, TAN ZM, et al. Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells[J]. FEBS Lett, 2006, 580(17): 4252-4260.
[33]	赵晓云, 李波, 曹立业, 等. 小细胞肺癌细胞诱发人脑微血管内皮细胞紧密连接的开放[J]. 解剖科学进展, 2009, 15(2): 183-187, 191. https://www.cnki.com.cn/Article/CJFDTOTAL-JPKX200902016.htm ZHAO XY, LI B, CAO LY, et al. Opening of tight junction of human brain microvascular endothelial cells induced by small cell lung cancer cells[J]. Prog Anat Sci, 2009, 15(2): 183-187, 191. https://www.cnki.com.cn/Article/CJFDTOTAL-JPKX200902016.htm