范琦琦;折改梅;魏静;李芝奇;陈美琳;董英;于啊香;李建伟;秦文杰;王伟;林瑞超;赵崇军;游蓉丽;
FAN Qi-qi;SHE Gai-mei;WEI Jing;LI Zhi-qi;CHEN Mei-lin;DONG Ying;YU A-xiang;LI Jian-wei;QIN Wen-jie;WANG Wei;LIN Rui-chao;ZHAO Chong-jun;YOU Rong-li;Beijing Key Laboratory for Quality Evaluation of Chinese Materia Medica, School of Chinese Materia Medica, Beijing University of Chinese Medicine;Beijing Zhendong Guangming Pharmaceutical Research Institute Co., Ltd.;

• 1:北京中医药大学中药学院中药品质评价北京市重点实验室
• 2:北京振东光明药物研究院有限公司

摘要(Abstract)：

该研究基于体内斑马鱼模型评价复方苦参注射液(compound kushen injection, CKI)抗肿瘤及镇痛活性，并探究其抗肿瘤的作用机制。通过斑马鱼卵黄囊显微注射LPC H12肝癌细胞建立斑马鱼肿瘤异种移植模型，实验过程中设置复方苦参注射液高(H-CKI)、中(M-CKI)、低(L-CKI)剂量组和模型组，给药72 h后以肿瘤面积增长倍数、积分吸光度(integrated absorbance, IA)增长倍数为指标评价CKI对肿瘤的抑制作用；分别以冰乙酸(acetic acid, AA)和佛波酯(phorbol myristate acetate, PMA)建立斑马鱼外周疼痛和中枢疼痛模型，利用行为轨迹分析系统监测斑马鱼行为运动轨迹，以运动次数、运动时间、运动距离、运动速度等运动状态为指标评价CKI的镇痛活性；最后，通过实时定量聚合酶链反应(real-time quantitative polymerasechain reaction, RT-qPCR)检测B淋巴细胞瘤-2基因(B lymphocyte tumor-2, Bcl-2)与磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase, PI3K)/蛋白激酶B(protein kinase B,Akt或PKB)通路相关基因的表达，验证抗肿瘤作用机制。与模型组相比，M-CKI组和H-CKI组肿瘤面积及积分吸光度的增长倍数显著降低，对外周疼痛和中枢疼痛具有明显的缓解作用，机制研究发现CKI能够上调半胱氨酸天冬氨酸蛋白酶3(caspase-3, Casp3)、半胱氨酸天冬氨酸蛋白酶9(caspase-9, Casp9)表达，下调PI3K、Akt表达，且Bcl-2、缺氧诱导因子-1α(hypoxia-inducible factor 1α, HIF-1α)、血管内皮生长因子(vascular endothelial growth factor, VEGF)表达水平显著降低。综上，CKI具有显著的抑制肿瘤生长及镇痛的功效，其机制与PI3K/Akt信号通路有关，该通路介导细胞凋亡，抑制肿瘤生长，缓解肿瘤疼痛。
This study aims to evaluate the anti-tumor and analgesic activities of Compound Kushen Injection(CKI) based on zebrafish model in vivo and investigate the anti-tumor mechanism. To be specific, zebrafish tumor xenotransplantation model was established by microinjection of murine LPC H12 cells into yolk sac. Then the high-dose CKI(H-CKI), medium-dose CKI(M-CKI), low-dose CKI(L-CKI) groups, and the model group were set. The anti-tumor activity of CKI was evaluated with the tumor area growth fold and integral absorbance(IA) growth fold 72 h after administration. The peripheral pain and central pain in zebrafish were respectively induced with acetic acid(AA) and phorbol myristate acetate(PMA). Zebralab ViewPoint system was employed to monitor behavioral trajectory of zebrafish, and movement times, movement time, movement distance, and movement velocity were used to evaluate the analgesic activity of CKI. Finally, real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression levels of apoptosis-related B lymphocyte tumor-2(Bcl-2) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt or PKB) pathway-related genes, for the verification of the anti-tumor mechanism. Compared with the model group, M-CKI and H-CKI significantly reduced the growth folds of tumor area and IA, relief the peripheral pain and central pain. The mechanism was that CKI can up-regulate the expression of cysteine aspartic acid specific protease-3(caspase-3, Casp3) and caspase-9(Casp9), down-regulate the expression of phosphoinositide 3-kinase(PI3 K) and Akt, and significantly reduce the expression of Bcl-2, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF). In conclusion, CKI has significant inhibitory effect on tumor growth and pain, which is related to the PI3 K/Akt signaling pathway. The pathway mediates cell apoptosis, suppresses tumor growth, and alleviates tumor pain.

关键词(KeyWords)： 复方苦参注射液;活性;机制;抗肿瘤;镇痛;RT-qPCR
Compound Kushen Injection(CKI);activity;mechanism;anti-tumor;analgesic;RT-qPCR

Abstract:

Keywords:

基金项目(Foundation): 国家“重大新药创制”科技重大专项(2018ZX09735005);; 北京中医药大学新教师启动基金项目(2020-JYB-XJSJJ-009);北京中医药大学重点攻关项目(2020-JYB-ZDGG-035);; 国家中药标准化项目(ZYBZH-C-JIN-43);; 山西省重点研发计划重点项目(201603D3113011)

作者(Authors): 范琦琦;折改梅;魏静;李芝奇;陈美琳;董英;于啊香;李建伟;秦文杰;王伟;林瑞超;赵崇军;游蓉丽;
FAN Qi-qi;SHE Gai-mei;WEI Jing;LI Zhi-qi;CHEN Mei-lin;DONG Ying;YU A-xiang;LI Jian-wei;QIN Wen-jie;WANG Wei;LIN Rui-chao;ZHAO Chong-jun;YOU Rong-li;Beijing Key Laboratory for Quality Evaluation of Chinese Materia Medica, School of Chinese Materia Medica, Beijing University of Chinese Medicine;Beijing Zhendong Guangming Pharmaceutical Research Institute Co., Ltd.;

DOI: 10.19540/j.cnki.cjcmm.20211129.701

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