目的 探讨血根碱与5-氟尿嘧啶联合使用对鼻咽癌裸鼠移植瘤细胞自噬与凋亡的影响及机制。方法 建立移植瘤模型;Western blot法、TUNEL法、免疫双荧光法检测各组别中自噬和凋亡相关蛋白以及PI3K/AKT/mTOR信号通路关键蛋白的表达变化。结果 相较于模型组,血根碱组、5-氟尿嘧啶组以及联用药物组移植瘤质量明显下降(P<0.05),且联用药物组较之单药组效果更优(P<0.05);Western blot结果显示,与模型组比较,自噬蛋白Beclin1、LC3表达均上调,P62蛋白表达下调,凋亡蛋白cleaved caspase3、cleaved PARP的表达均上调,且联用药物组自噬和凋亡蛋白上调较单药组上调更明显(P<0.05)。联用药物组的p-PI3K、p-AKT和p-mTOR蛋白的表达明显低于模型组和各单药组(P<0.05);TUNEL检测发现,各药物组的凋亡细胞较模型组明显增多,联合用药组增多最为明显(P<0.05);免疫双荧光结果显示,LC3、caspase3在各药物组中表达有不同程度的升高,其中在联合用药组中表达升高得最为明显(P<0.05)。结论 血根碱联用5-氟尿嘧啶可协同抑制体内鼻咽癌移植瘤的生长,并诱导移植瘤发生自噬和凋亡,与PI3K/AKT/mTOR信号通路有着密切联系。
Abstract
OBJECTIVE To investigate the effect and possible mechanism of sanguinarine(Sang) combined with 5-fluorouracil(5-FU) on autophagy and apoptosis of nasopharyngeal carcinoma xenograft cells in nude mice. METHODS The transplanted tumor model was established; Western blot. TUNEL and immunofluorescence methods were used to detect the expression changes of autophagy and apoptosis-related proteins and key proteins of PI3K/AKT/mTOR signaling pathway in each group. RESULTS Compared with the model group, the masses of the transplanted tumor in the Sang group, 5-FU group and the combined drug group were decreased significantly (P<0.05), while that in the combined drug group were significantly different from those in the single drug groups (P<0.05). Western blot results showed that compared with the model group, the expressions of autophagy proteins Beclin1 and LC3 and the expressions of apoptotic proteins cleaved caspase3 and cleaved PARP were up-regulated, and the expression of P62 protein was down-regulated in each drug groups. These changes were more significant in the combined drug group than those in the single-drug group (P<0.05). The expressions of p-PI3K, p-AKT and p-mTOR proteins in the combined drug group were significantly lower than those in the model group and each single drug group (P<0.05). The combination group had the most significant increase (P<0.05). The results of immunofluorescence showed that the expressions of LC3 and Caspase3 increased to varying degrees in each drug group, among which the expression increased most significantly in the combination group (P<0.05). CONCLUSION Sang combined with 5-FU can synergistically inhibit the growth of nasopharyngeal carcinoma xenografts in vivo, and induce autophagy and apoptosis in the xenografts, which is closely related to the PI3K/AKT/mTOR signaling pathway.
关键词
鼻咽癌 /
5-氟尿嘧啶 /
血根碱 /
自噬 /
凋亡
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Key words
sanguinarine /
5-fluorouracil /
nasopharyngeal carcinoma /
autophagy /
apoptosis
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金项目资助(81874408);湖南省自然科学基金项目资助(2020JJ5419);湖南省教育厅科研基金项目资助(21C0234);湖南省卫计委科研基金项目资助(202207015049,202207015643);湖南省大学生创新创业训练计划项目资助(S202110541043);2022年度湖南中医药大学中医学开放基金项目资助(2022ZYX03,2022ZYX22);2022年湖南中医药大学大学生创新课题资助项目资助
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