王瓜葫芦素D的提取及其对大鼠乳腺癌细胞增殖、凋亡和体内成瘤作用的影响

Extraction of cucurbitacin D from Trichosanthes cucumeroides and its effects on proliferation,apoptosis and tumorigenesis of rat breast cancer cells

目的从王瓜中提取葫芦素D,并观察其对大鼠乳腺癌细胞增殖、凋亡和体内成瘤作用的影响。方法采用硅胶柱层析、葡聚糖凝胶柱层析,结合硅胶柱色谱、高效液相色谱法(HPLC)对王瓜果实进行分离纯化,运用现代光谱技术(~1H-NMR)对提取化合物(葫芦素D)的结构进行鉴定。培养大鼠乳腺癌细胞株Walker256,将细胞分为实验1、2、3组和对照组,分别加入2、4、8μmol/L的葫芦素D和等量培养液。采用Ed U法观察细胞增殖情况,TUNEL法观察凋亡情况。将80只雌性SPF大鼠随机分为Con组、Sham组、IP-L组和IP-H组,各20只。Sham组、IP-L组和IP-H组取Walker256细胞制作大鼠乳腺癌骨转移瘤模型,Con组不制作模型;IP-L组经腹腔注射0.75mg/(kg·d)的葫芦素D,IP-H组经腹腔注射1.5 mg/(kg·d)的葫芦素D,Sham组腹腔注射等量生理盐水,每天1次,连续21 d。观察各组大鼠左侧胫骨组织病理变化,测量胫骨肿瘤瘤体大小,观察毒性反应发生情况。结果从王瓜提取物中分离出1个化合物,鉴定为葫芦素D。对照组及实验1、2、3组细胞增殖抑制率分别为5.32%±4.54%、46.17%±4.87%、57.37%±5.15%、64.61%±5.33%,细胞凋亡率分别为6.24%±4.31%、35.43%±5.09%、44.79%±4.92%、73.19%±4.86%,组间两两相比,P均<0.05。Con组、Sham组、IP-L组、IP-H组大鼠转移瘤代谢体积平均值分别为0、2.1、1.7、1.2 cm^3;IP-L组、IP-H组与Sham组相比,P均<0.05;IP-L组与IP-H组相比,P<0.05。Sham组大鼠胫骨骨组织结构紊乱,多处骨皮质缺损,骨髓腔内可见大量肿瘤细胞,骨小梁破坏明显;IP-L组与IP-H组骨质破坏较Sham组轻,IP-H组可见部分正常骨髓组织。Con组、Sham组、IP-L组、IP-H组分别有0、0、6、13只大鼠死亡;IP-L组、IP-H组死亡率与Con组、Sham组相比,P均<0.05;IP-L组死亡率与IP-H组相比,P<0.05。结论在王瓜中分离得到葫芦素D,其可抑制大鼠乳腺癌细胞株Walker256增殖,诱导其凋亡;葫芦素D对大鼠乳腺癌骨转移瘤有生长抑制作用,但可导致大鼠死亡率增加。

Objective To extract cucurbitacin D from Trichosanthes cucumeroides and to observe its effects on proliferation,apoptosis and tumorigenesis of rat breast cancer cells. Methods The fruit of Trichosanthes cucumeroides was extracted with ethanol. The solution was isolated and purified by silica and Sephadex LH-20 column chromatography,as well as silica gel column chromatography and high performance liquid chromatography（ HPLC）. The structures of the compounds were identified by the modern spectrum technology（~1H-NMR）. The rat breast cancer cell line Walker256 was divided into the experiment groups 1,2,3 and the control group,which was respectively treated with 2,4,8 μmol / L cucurbitacin D and nutrient solution for 24 h. Ed U and TUNEL methods were performed to detect the proliferation and antitumor activities. Mouse models of bone metastasis were established in SPF rats. The 80 rats were randomly divided into four groups,20 in each group： the blank control group（ Con group without any treatment）,the bone metastasis in the left leg group（ Sham group,treated with normal saline）,the bone metastasis and low-dose injection of cucurbitacin D in the left leg group〔IP-L group,0. 75 mg /（ kg·d） 〕and high-dose injection of cucurbitacin D in the left leg group〔IP-H,1. 5 mg /（ kg·d） 〕. They were all treated once a day and for 21 d in these three groups. Histopathological changes on the rat left side of the tibia were observed in each group,and we measured the tibia tumor size and observed toxicity reaction. Results One tetracyclic triterpenoid was isolated and identified as cucurbitacin D from Trichosanthes cucumeroides. The proliferation inhibition rates of the control group and experimental groups 1,2 and 3 were 5. 32% ± 4. 54%,46. 17% ± 4. 87%,57. 37% ± 5. 15% and 64. 61% ± 5. 33%,respectively. The apoptotic rates were 6. 24% ± 4. 31%,35. 43% ± 5. 09%,44. 79% ± 4. 92% and 73. 19% ± 4. 86%,respectively. Significant differences were found between every two groups,all P〈0. 05. The average metabolic tumour volume of the Con group,Sham group,IP-L group and IP-H group was 0,2. 1,1. 7 and 1. 2 cm^3. There were significant differences between IP-L,IP-H groups and Sham group,between IP-L group and IP-H group（ all P〈0. 05）. The tibia of Sham group showed the disorder structure and incomplete bony cortex. Tumor cells assembled in the marrow cavity with the obviously destroyed of bone trabecula. The degree of Sham group was higher than IP-L group and IP-H group. Part of the normal bone marrow tissues were found in the IP-H group. The mortality of IP-L and IP-H groups were 6 and 13,and significant difference was found in the mortality between IP-L,IP-H groups and Con,Sham groups,between IP-L group and IP-H groups（ P〈0. 05）. Conclusions Cucurbitacin D is isolated from Trichosanthes cucumeroides. It can inhibit the proliferation of rat breast cancer cell line Walker256 and induce its apoptosis. Cucurbitacin D can inhibit the growth of metastatic tumor of bone,but can also lead to death in rats.