消癌解毒方联合顺铂通过调控组蛋白去甲基化酶抑制4T1荷瘤小鼠肿瘤生长

Inhibitory Effect of Xiaoai Jiedu Decoction Combined with Cisplatin on Breast Cancer Cell Activity Through PI3K/Akt Pathway

目的：观察消癌解毒方联合顺铂抑制4T1荷瘤小鼠肿瘤生长的作用及机制。方法：BALB/c小鼠皮下接种4T1乳腺癌细胞形成荷瘤模型,分别单独给予顺铂（2 mg·kg-1）、消癌解毒方（1×104mg·kg-1）及两者联合用药进行治疗,给药期间称量并记录小鼠体质量和瘤体积,15 d后脱颈处死,剥离并称量瘤块质量,计算抑瘤率,苏木素-伊红（HE）染色观察心、肝、脾、肾和瘤块的病理情况,原位末端转移酶标记技术（TUNEL）观察肿瘤细胞凋亡情况并统计凋亡率,蛋白免疫印迹法（Western blot）检测肿瘤组织中B淋巴细胞瘤-2（Bcl-2）,Bcl-2相关X蛋白（Bax）,组蛋白去甲基化酶1（LSD1）,组蛋白H3（Histone H3）,组蛋白H3第4位赖氨酸二甲基化（Histone H3K4me2）,组蛋白H3第9位赖氨酸二甲基化（Histone H3K9me2）的表达水平。结果：与模型组相比,顺铂给药组小鼠体质量均有下降,其中消癌解毒方＋顺铂组较单用顺铂组体质量增加（P〈0.01）;消癌解毒方＋顺铂较单用顺铂更能缩小移植瘤体积,提高抑瘤率（P〈0.01）;更明显提高了肿瘤细胞凋亡率（P〈0.05,P〈0.01）;更明显下调肿瘤组织Bcl-2表达水平,上调Bax表达水平（P〈0.05,P〈0.01）;各给药组均能下调LSD1蛋白的表达,增加组蛋白H3K4,H3K9的二甲基化,消癌解毒方＋顺铂组作用最为明显（P〈0.05,P〈0.01）。结论：消癌解毒方＋顺铂用药对于4T1荷瘤小鼠抑瘤作用明显,其机制可能与下调组蛋白去甲基化酶水平有关。

Objective： To study the effect and mechanism of Xiaoai Jiedu decoction combined with cisplatin in inhibiting tumor growth of 4 T1 tumor bearing mice. Method： BALB/c mice were subcutaneously inoculated with 4 T1 breast cancer cells to establish the tumor bearing model. They were treated with cisplatin（ CDDP,2 mg·kg-1） and Xiaoai Jiedu decoction（ XJF,1 × 104 mg·kg-1） respectively,and the combination therapy. Body weight and tumor volume were quantified and recorded during drug treatment. The mice were put to death after 15 days, and tumor masses were stripped and weighed. The inhibition rate was calculated.Hematoxylin-eosin（ HE） staining was used to observe the pathological changes of heart,liver,spleen,kidney and tumor. The apoptosis of tumor cells was observed by Td T-mediated-d UTP nick end labeling（ TUNEL） staining,and the apoptosis rate was counted. Western blot was used to detect the expressions of B-cell lymphoma-2（ Bcl-2）,Bcl-2 related X protein（ Bax）,lysine-specific demethylase 1（ LSD1）,Histone H3,Histone H3 K4 me2 and Histone H3 K9 me2 in tumor tissues. Result： Compared with model group,the body weight of mice in the cisplatin group decreased,and the mice in the combined group had a higher body weight than those in cisplatin group（ P 0. 01）. The combination therapy can further reduce the volume of transplanted tumor,improve the tumor inhibition rate,and promote the apoptosis rate of tumor cells significantly compared with cisplatin therapy（ P〈 0. 05,P 0. 01）. The expression of Bcl-2 in tumor tissues was up-regulated,and Bax was up-regulated（ P〈 0. 05,P 0. 01）. Each drug group could down regulate the protein expression of LSD1,and increase the methylation of histone H3 K4 and H3 K9,and the combination group showed the most significant effect on the protein expression（ P〈 0. 05,P〈 0. 01）. Conclusion： Xiaoai Jiedu prescription combined with cisplatin can obviously inhibit the growth of 4 T1 tumor in tumor bearing mice. Its mechanism may be related to the down-regulation of histone demethylation enzyme level.