川芎增强替莫唑胺对小鼠脑转移黑色素瘤脑的抑制作用

doi:10.12122/j.issn.1673-4254.2024.06.09

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1088-1097.doi: 10.12122/j.issn.1673-4254.2024.06.09

• • 上一篇

川芎增强替莫唑胺对小鼠脑转移黑色素瘤脑的抑制作用

赵倩¹(), 张振¹, 周星琦¹, 荣翔宇¹, 刘雪柔¹, 赵新丽¹, 王豪杰¹, 庞金龙¹, 李姗姗¹(), 李娴¹^,²^,³()

^1.蚌埠医科大学药学院，安徽蚌埠 233030
^2.中药饮片制造新技术安徽省重点实验室，安徽亳州 236800
^3.安徽协和成药业饮片有限公司博士后工作站，安徽亳州 236800

收稿日期:2024-02-22 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 李姗姗,李娴 E-mail:1340251163@qq.com;lishanshan@bbmc.edu.cn;Lixian202101@163.com
作者简介:赵倩，在读硕士研究生，E-mail: 1340251163@qq.com
基金资助:
安徽省科技重大专项(201903a07020029);蚌埠医科大学横向课题(2019-BYHX-03);安徽省教育厅高校科研项目(KJ2021a0702);安徽省自然科学基金(1908085QH373);安徽省教育厅自然科学研究项目(KJ2020a0565);安徽省重点研究与开发计划资助项目(202104g01020017);蚌埠医科大学研究生科研创新计划项目(Byycx22059)

Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice

Qian ZHAO¹(), Zhen ZHANG¹, Xingqi ZHOU¹, Xiangyu RONG¹, Xuerou LIU¹, Xinli ZHAO¹, Haojie WANG¹, Jinlong PANG¹, Shanshan LI¹(), Xian LI¹^,²^,³()

^1.School of Pharmacy, Bengbu Medical University, Bengbu 233030, China
^2.Anhui Provincial Key Laboratory of New Technology for Chinese Herbal Pieces Manufacturing, Bozhou 236800, China
^3.Postdoctoral Workstation at Anhui Xiehecheng Pharmaceutical Yinpian Co. Ltd, Bozhou 236800, China

Received:2024-02-22 Online:2024-06-20 Published:2024-07-01
Contact: Shanshan LI, Xian LI E-mail:1340251163@qq.com;lishanshan@bbmc.edu.cn;Lixian202101@163.com

摘要/Abstract

摘要：

目的探讨引经药川芎对黑色素瘤B16F10脑转移的影响。方法超声下左心室内注射黑色素瘤B16F10建立C57BL/6J鼠脑转移瘤动物模型，通过小动物活体成像系统监测颅内荧光信号大小，当荧光信号强于5×10⁶光子/s时，将小鼠按照体质量随机分为2组。对照组：每日灌胃生理盐水；川芎组：每日灌胃给药，川芎饮片水提物以生理盐水溶解，浓度为1 mg/g。与对照组（生理盐水组）相比，考察川芎（1 mg/g）对黑色素瘤B16F10脑转移的影响。通过动物行为学立柱和粘签实验观察川芎对荷瘤小鼠的神经保护作用，以免疫荧光法观察川芎对血脑屏障完整性相关蛋白（ZO-1、Claudin-5、Occludin、P-gp、 TNF-α、AQP4和PDGFRβ）、对神经元细胞增殖和凋亡的作用以及对小胶质细胞凋亡和活化的作用。通过小动物活体成像监测小鼠脑部荧光信号强度，考察川芎联合化疗药替莫唑胺对B16F10脑转移瘤的疗效。结果与对照组比较，川芎组小鼠脑转移瘤无明显进展，体质量下降减慢，血脑屏障相关指标ZO-1、Claudin-5、Occludin、P-gp、TNF-α、AQP4和PDGFRβ受体蛋白水平下降（P<0.05）。动物行为学实验表明，川芎组小鼠木棍停留时间更长（P<0.05），取下粘签时间更短（P<0.05），免疫荧光结果显示脑部神经元细胞增殖增多（P<0.05），凋亡减少（P<0.05），小胶质细胞凋亡减少（P<0.05）。与对照组相比，川芎组小鼠脑转移瘤微环境中CD86、CD206、IL-4、IL-10水平升高（P<0.05），CD163、IL-1β水平降低（P<0.05）。川芎（1 mg/g）联合替莫唑胺（25 mg/kg）组脑部荧光信号显著低于替莫唑胺单药组。结论川芎作为引经药在开放血脑屏障的同时并未促进黑色素瘤脑转移，且与替莫唑胺联用能增强替莫唑胺对脑转移黑色素瘤B16F10的治疗效果。

关键词: 川芎, 替莫唑胺, 黑色素瘤, 血脑屏障, 脑转移

Abstract:

Objective To investigate the effect of the aqueous extract of Chuan Xiong Rhizoma (CR) on brain metastasis of melanoma B16F10 cells in mice. Methods C57BL/6J mouse models of brain metastasis of melanoma were established by ultrasound-guided intraventricular injection of Luc-labeled B16F10 cells, and brain tumor growth was monitored by in vivo imaging. The mouse models were then randomized for daily gavage of saline or aqueous extract of CR (equivalent crude drug concentration of 1 mg/g). Behavioral tests were used to evaluate the neuroprotective effects of CR in the tumor-bearing mice, and the changes in proteins associated with blood-brain barrier integrity, neuronal cell proliferation and apoptosis, and microglial cell apoptosis and activation were observed using immunofluorescence assay. The efficacy of CR combined with temozolomide (25 mg/kg) against brain metastases of B16F10 cells was observed by in vivo imaging. Results CR-treated mouse models did not show obvious progression of brain metastases and had a reduced rate of body weight loss and lowered protein expressions of ZO-1, claudin-5, occludin, P-gp, TNF‑α, AQP4 and PDGFRβ. In the behavioral tests, the CR-treated mice showed prolonged stay on the wooden stick with a shortened time of sticky stick removal. Immunofluorescence assay showed increased proliferation and decreased apoptosis of neuronal cells and microglia in CR-treated mice. CR treatment significantly increased the levels of CD86, CD206, IL-4 and IL-10 and decreased the levels of CD163 and IL-1β in the microenvironment of brain metastases. The mice receiving combined treatments with CR and temozolomide showed significantly lower intensity of fluorescent signals in the brain than those treated with temozolomide alone. Conclusion CR does not promote brain metastasis of melanoma while inducing opening of the blood-brain barrier, and its combined use with TMZ results in enhanced inhibition against brain metastasis of melanoma B16F10 cells in mice.

Key words: Chuan Xiong Rhizoma, Melanoma, temozolomide, blood-brain barrier, brain metastases

赵倩, 张振, 周星琦, 荣翔宇, 刘雪柔, 赵新丽, 王豪杰, 庞金龙, 李姗姗, 李娴. 川芎增强替莫唑胺对小鼠脑转移黑色素瘤脑的抑制作用[J]. 南方医科大学学报, 2024, 44(6): 1088-1097.

Qian ZHAO, Zhen ZHANG, Xingqi ZHOU, Xiangyu RONG, Xuerou LIU, Xinli ZHAO, Haojie WANG, Jinlong PANG, Shanshan LI, Xian LI. Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice[J]. Journal of Southern Medical University, 2024, 44(6): 1088-1097.

图/表 4

图1 川芎不影响C57BL/6J鼠生存、体质量与颅内肿瘤生长

Fig.1 The aqueous extract of Chuan Xiong Rhizom (CR) does not affect survival, body weight or intracranial tumor growth in C57BL/6J mice. A: Bioluminescence imaging scan 6 days after intracardiac injection of the tumor cells (n=6). B, C: Bioluminescence imaging of intracranial tumors in C57BL/6J mice on day 6 and day 12, respectively (n=8). D: Quantitative analysis of intracranial tumor signals of bioluminescence imaging BLI (n=8). E: Quantification of intracranial tumor signals in control and CR-treated mice on day 12 (n=8). F: Kaplan-Meier survival curves of control and CR-treated mice (n=8). G: Body weight changes of control and CR-treated mice (n=8).

图3 川芎对B16F10脑转移C57BL/6J鼠具有神经保护作用

Fig.3 Neuroprotective effect of CR in mice with brain metastasis of B16-F10-Luc cells (×200). A: Vertical pole test for assessing forelimb grasping ability (n=10). B: Sticky dot test for assessing sensory abilities (n=10). C: Immunofluorescence staining of neuronal cells (red) and quantification of mean fluorescence intensity. D: Immunofluorescence staining of Tunnel (green) and quantification of mean fluorescence intensity. E: Immunofluorescence staining of TMEM119 (red) and cleaved caspase3 (green) and quantification of mean fluorescence intensity. *P<0.05, **P<0.01 vs control or Normal.

图4 川芎调节荷瘤C57BL/6J鼠脑部CD68、CD86、CD163、CD206、IL-1β、IL-4、IL-6和IL-10的表达

Fig.4 CR regulates the expression of CD68, CD86, CD163, CD206, IL-1β, IL-4, IL-6 and IL-10 (A-H,reepectively) in the brain of the tumor-bearing C57BL/6J mice （×200）. n=6, *P<0.05, **P<0.01 vs Control.

图5 川芎增强替莫唑胺抑制脑转移黑色素瘤B16F10生长的作用

Fig.5 CR enhances the inhibitory of temozolomide against brain metastasis of melanoma B16F10 cells in mice. A: Bioluminescence imaging of intracranial tumors in C57BL/6J mice on day 13. B: Quantitative detection of intracranial tumor signals. C: Kaplan-Meier survival curves in both groups. D: Body weight changes in both groups. E: Vertical pole test for assessing forelimb grasping ability of the mice. F: Sticky dot test for assessing sensory abilities of the mice. n=17, *P<0.05, **P<0.01 vs Control.

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川芎增强替莫唑胺对小鼠脑转移黑色素瘤脑的抑制作用

Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice

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