透骨草总生物碱对乳腺癌骨转移的抑制作用及其机制

doi:10.3969/j.issn.1004-616x.2018.06.004

摘要/Abstract

摘要： 目的：探讨透骨草总生物碱（TTAT）对乳腺癌骨转移的抑制作用及其机制。方法：提取透骨草中所含生物碱，通过碘化铋钾或改良碘化铋钾与碘化汞钾试剂显色反应进行鉴定。将42只裸鼠分为正常组（常规饲养）、模型组（仅造模）、TTAT低浓度组（100 mg/kg TTAT腹腔注射）、TTAT高浓度组（500 mg/kg TTAT腹腔注射）、盐酸多柔比星（DOX）组（5 mg/kg DOX腹腔注射）、唑来膦酸注射液（ZOL）组（0.4 mg/kg ZOL颈后皮下注射），每组7只，除正常组外其他组裸鼠右后肢胫骨内注射人乳腺癌MDA-MB-231细胞建立乳腺癌骨转移模型，除正常组和模型组外，其余组造模1周成瘤后开始给药，隔日1次，连续给药6周。给药期间，每周定期测量各组裸鼠体质量及肿瘤体积，密切观察各组裸鼠生活状态。实验结束时颈椎脱臼法处死各组裸鼠，以右侧髋关节为界切除右后肢，采用影像学技术、HE染色法和抗酒石酸酸性磷酸酶（TRAP）染色法评估TTAT对骨转移裸鼠的胫骨及肿瘤组织生长的影响；采用Western blot法检测肿瘤组织中核因子-κB配体（RANKL）、骨保护素（OPG）蛋白以及细胞外信号调节激酶（ERK）、p-ERK、c-Jun氨基末端激酶（JNK）、p-JNK蛋白的表达水平。结果：从500 g透骨草中提取得到3.28 g提取物，显色反应呈现阳性，表明提取物中含有生物碱；与模型组比较，100和500 mg/kg TTAT组瘤质量分别为（3.50±0.70）g和（2.63±0.59）g，较模型组瘤质量（5.01±1.03）g均明显下降（P < 0.01）；影像学评分均升高（P < 0.01）；HE染色结果显示100和500 mg/kg浓度组TTAT均能够显著抑制肿瘤癌巢形成，使核分裂像减少，并对乳腺癌胫骨骨转移裸鼠病变骨质具有保护作用；TRAP染色结果显示此2个TTAT组中破骨细胞数量明显减少（P < 0.05）；Western blot结果显示，TTAT显著降低肿瘤组织中RANKL/OPG比值（P < 0.05），同时下调p-ERK、p-JNK的表达（P < 0.05）。结论：透骨草总生物碱能显著抑制乳腺癌胫骨骨转移裸鼠肿瘤生长和骨质病变，其抑制乳腺癌骨转移作用可能是通过OPG/RANK/RANKL信号通路实现的。

关键词: 透骨草, 生物碱, 乳腺癌, 骨转移, NF-κB受体激活因子配体

Abstract: OBJECTIVE: To explore the inhibitory effect of the total alkaloids of Tougucao (TTAT) on bone metastasis of breast cancer cells in mice and to investigate the mechanisms. METHODS: Alkaloids contained in the extract was identified by chromogenic reaction. Female nude mice were randomly divided into 6 groups of 7 each:normal (routine feeding),model (only modeling),TTAT low concentration (100 mg/kg TTAT intraperitoneal injection),TTAT high concentration (500 mg/kg TTAT intraperitoneal injection),DOX (5 mg/kg DOX intraperitoneal injection),and ZOL group (0.4 mg/kg ZOL subcutaneous injection after neck). The human breast cancer MDA-MB-231 cells were injected into the tibia to establish the bone metastasis model of breast cancer. The other groups were administered the drugs once every other day for 6 weeks after tumor formation except the normal and the model groups. During the administration,the body weight and tumor volume of each group were measured regularly and the living activities of each group were observed closely. At the end of the experiment,all the nude mice were killed by cervical dislocations,and the right hind limbs were removed at the right hip joint. Imaging,HE staining and TRAP staining were used to observe and evaluate the effect of TTAT on the growth of tibia and tumor tissues of nude mice with tibial bone metastases from breast cancer. The effects of TTAT on the RANKL/OPG ratio and the expression of p-ERK,ERK,p-JNK and JNK in the tumor tissue of the tibial bone metastases of breast cancer were detected by Western blot. RESULTS: 3.28 g was extracted from Tougucao and three alkaloid chromogenic reagents showed positive results,indicating the presence of alkaloids. Compared with the model group,the quality of tumor in the TTAT group decreased significantly (P < 0.01) while imaging scores increased (P < 0.01). The HE staining showed that TTAT significantly inhibited the formation of tumor nests,reduced the nuclear division and protected metastases to the tibia. The TRAP staining results showed that the number of osteoclasts in the TTAT group decreased significantly (P < 0.05). Western blot results showed that TTAT significantly reduced the RANKL/OPG ratio in the tumor tissue of the tibial bone metastases (P < 0.05),and reduced the expression of p-ERK and p-JNK (P < 0.05). CONCLUSION: TTAT significantly inhibited the growth and tibial bone metastasis of breast cancer. The observed effect could be mediated via the OPG/RANK/RANKL signaling pathway.

Key words: Tougucao, alkaloids, breast cancer, bone metastases, NF-kappa B receptor activator ligand

中图分类号:

R73-3

杜娟, 胡向阳, 刘丹, 胡卫, 陈涛. 透骨草总生物碱对乳腺癌骨转移的抑制作用及其机制[J]. 癌变·畸变·突变, 2018, 30(6): 435-441.

DU Juan, HU Xiangyang, LIU Dan, HU Wei, CHEN Tao. Inhibitory effect of total alkaloids of Tougucao on bone metastasis of breast cancer cells in mice[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(6): 435-441.

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