目的 探讨柘木总黄酮对Lewis肺癌小鼠的抗肿瘤活性影响及其新组分配伍诱导小鼠肺癌细胞(LLC)细胞自噬的效应研究。方法 构建Lewis肺癌移植瘤小鼠模型,并检测服用柘木总黄酮后Lewis肺癌移植瘤小鼠的体重、瘤重、瘤体积、脏器指数和病理切片;ELISA法检测血清中肿瘤坏死因子α(TNF-α),白细胞介素2(IL-2),IL-6和IL-12细胞因子含量。采用UPLC测定柘木黄酮提取物中主要有效成分含量及其配比;Western blot检测其新组分配伍对LLC细胞自噬蛋白表达的影响,通过透射电镜观察LLC细胞超微结构的改变;流式细胞仪检测新组分配伍对LLC细胞自噬的影响。结果 柘木总黄酮高剂量组能显著抑制小鼠肿瘤的生长、提高荷瘤小鼠的脏器指数,且柘木总黄酮各组能较好地改善小鼠生存状态;各组荷瘤小鼠血清中TNF-α,IL-2,IL-6和IL-12含量均较模型组高,其中柘木总黄酮高剂量组具有显著差异(P<0.01)。UPLC结果表明,其主要有效成分为花旗松素、山柰酚、柚皮素,其中花旗松素与山柰酚含量比为60∶1;Western blot检测结果表明,新组分配伍使LLC细胞自噬蛋白表达显著升高(P<0.01);透射电镜可观察到细胞质中的自噬体;流式实验结果表明,新组分配伍平均荧光强度明显高于对照组。结论 柘木总黄酮可有效抑制肿瘤生长,且对机体免疫器官组织伤害较小;其机制可能与上调细胞因子TNF-α,IL-2,IL-6和IL-12含量、提高机体免疫功能和使LLC细胞自噬蛋白表达升高,诱导LLC细胞自噬有关。
Abstract
OBJECTIVE To explorethe effect of total flavonoids from Cudrania tricuspidata Bun on Lewis lung cancer mice and its new component compatibility on the autophagy of LLC cells. METHODS A mouse model of Lewis lung cancer xenografts was constructed. The body weight, tumor weight, tumor volume and organ index were measured before and after taking total flavonoids of Cudrania tricuspidata Bun. The HE staining of the xenograft pathological sections were also observed. The TNF-α, IL-2, IL-6 and IL-12 levels in the serum were calculated by ELISA. The content of the main active ingredient and its ratio in the flavonoid extract were measured by UPLC. Western blot was used to detect the effect of the new component compatibility on the expression of autophagy protein in LLC cells, and the ultra structural changes of LLC cells were observed by transmission electron microscopy. Flow cytometry was used to detect the effect of the new component compatibility on autophagy of LLC cells. RESULTS The high-dose group of total flavonoids from Cudrania tricuspidata Bun can significantly inhibit the growth of tumor in mice and enhance the organ index of tumor-bearing mice, and the survival rate of mice could be improved in all groups of total flavonoids from Cudrania tricuspidata Bun. The serum levels of TNF-α, IL-2, IL-6 and IL-12 in the tumor-bearing mice of each group were higher than those in the model group, and there was significant difference in the high dose group of total flavonoids (P<0.01). The main active ingredients were taxifolin, kaempferol and naringenin by UPLC. The ratio of taxifolin to kaempferol was 60∶1. Western blot assay showed that its new component compatibility significantly increased the expression of autophagic protein in LLC cells (P<0.01). The autophagosomes in the cytoplasm were observed by transmission electron microscopy. The results of flow experiments showed that the average fluorescence intensity of its new component compatibility was significantly higher than that of the control group. CONCLUSION Total flavonoids of Cudrania tricuspidata Bun can effectively inhibit tumor growth and have less harm to immune organs. Its mechanism may be related to up-regulation of cytokines TNF-α, IL-2, IL-6 and IL-12 levels and improve immune function in the body. Moreover, its main active ingredient promotes the increase of autophagy protein expression in LLC cells and induces autophagy in LLC cells.
关键词
柘木总黄酮 /
抗Lewis肺癌 /
细胞因子 /
组分配伍 /
细胞自噬
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Key words
Cudrania tricuspidata Bun total flavonid /
Lewis lung cancer inhibition /
cytokine /
component compatibility /
autophagy
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中图分类号：
R969.1
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脚注
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基金
国家自然科学基金项目资助(31370764);黑龙江省自然科学基金重点项目资助(ZD2015007)
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