Page 52 - 2019年10月第30卷第20期

Effects of Benzoyl Aconitine on Autophagy and Apoptosis of Human Lung Cancer Cell Line A549
SHAO Xin ，HAN Bin ，JIANG Xianhong ，LI Feng ，HE Mei ，LIU Fu（1. School of Pharmacy，North Sichuan
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1，2
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3
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Medical College，Sichuan Nanchong 637000，China；2. Dept. of Pharmacy，the Affiliated Hospital of North
Sichuan Medical College，Sichuan Nanchong 637000，China；3. Dept. of Clinical Medicine，North Sichuan
Medical College，Sichuan Nanchong 637000，China）
ABSTRACT OBJECTIVE：To study the effects of benzoyl aconitine（BAC）on autophagy and apoptosis of human lung cancer
A549 cells，and to investigate its mechanism in anti-non-small cell lung cancer. METHODS：A549 cells were treated with different
doses of BAC（10，50，100，200，400 μmol/L），and then cell morphology was obtained；the proliferation inhibition rate of the
cell was determined by CCK-8 assay. The cells were divided into control group（without drug），BAC low-dose and high-dose
groups（200，400 μmol/L）. After treated with relevant drugs，the apoptosis rate of cells was determined by flow cytometry. The
gene and protein expression of apoptosis-related factors Bcl-2，Bax，Caspase-3 as well as autophagy-related factors Beclin1，LC3，
P62 were determined by RT-PCR and Western blotting assay. RESULTS：After treated with different doses of BAC，the cells
were shrunken and sparsely arranged；inhibitory rate of cell proliferation was increased significantly in BAC 100，200，400 μmol/L
groups（P＜0.05 or P＜0.01）. Results of flow cytometry showed that the apoptotic rates of cells were increased to different extents
in BAC low-dose and high-dose groups after treated for 24 and 48 h，in a concentration and time-dependent manner. Compared
with control group，mRNA and protein expression of Bcl-2 and P62 were decreased to different extents in BAC groups；mRNA
expression of Bax，Caspase-3，Beclin1 and LC3 as well as protein expression of Bax，Active caspase-3，P62，Beclin1，LC3Ⅱ/Ⅰ
were increased to different extent；there was statistical significance in mRNA expression of Caspase-3，and protein expression of
Bcl-2，Active Caspase-3，Beclin1，LC3Ⅱ/Ⅰ and P62 in BAC low-dose group as well as all target mRNA and protein expression
in BAC high-dose group（P＜0.05 or P＜0.01），in dose-dependent manner. CONCLUSIONS：BAC can inhibit the proliferation
and promote the apoptosis of A549 cells，promote Beclin1，LC3（LC3Ⅱ/Ⅰ），Bax and Caspase-3（Active Caspase-3）gene and
their protein expression，but inhibit P62 and Bcl-2 gene and their protein expression. The mechanism may be related to BAC
inducing apoptosis by promoting excessive autophagy of cells.
KEYWORDS Benzoyl aconitine；Non-small cell lung cancer；A549 cells；Proliferation；Autophagy；Apoptosis

肺癌是全球发病率和致死率最高的恶性肿瘤，其中 根据凋亡相关因子B淋巴细胞瘤因子2（Bcl-2）、Bcl-2凋
非小细胞肺癌（Non-small cell lung cancer，NSCLC）患者 亡相关 X 蛋白（Bax）、胱天蛋白酶 3（Caspase-3）和自噬
约占肺癌病例总数的80％，且其发病隐匿、预后不良，约 相关因子Beclin1、LC3、P62的基因及蛋白表达水平的变
75％的NSCLC患者被确诊时已到晚期，总体5年生存率 化，从自噬途径探索BAC可能的抗肿瘤分子机制，为中
仅为20％ [1-2] 。自噬是细胞内蛋白降解的主要途径之一， 药活性单体用于NSCLC的治疗提供新思路和新靶点。
是一种“自食”过程，其通过自噬-溶酶体途径将细胞质
蛋白、复合物或细胞器吞入自噬体，然后使自噬体进入
晚期内体中，或者将自噬体转运至溶酶体融合以产生自
溶酶体，然后通过酸性水解酶释放使自噬体降解 [3-4] 。有
研究报道，诱导自噬可抑制人肺癌A549细胞的增殖、促
进其凋亡 [5-6] 。
近年来的研究显示，从植物药中提取的许多天然单
体成分已表现出抗肿瘤活性，而多种中药配方及其提取 图1 BAC的化学结构式
物更已被证实对NSCLC具有可靠的治疗效果 [7-9] 。乌头 Fig 1 Chemical structure of BAC
类中药在临床应用广泛，具有抗炎、降血压和抗肿瘤等 1 材料
活性，临床上常用来治疗风湿性关节炎、高血压、肿瘤等 1.1 仪器
[10]
多种疾病 。苯甲酰乌头原碱（Benzoyl aconitine，BAC） Heal Force型超净工作台、HF90型CO2培养箱（上海
是主要存在于制川乌、草乌和附子等中药中的活性单 力新仪器有限公司）；MLS-3020 型高压蒸汽灭菌器（日
体，其分子式为 C32H45NO10，分子量为 603.705（化学结构 本 Sanyo 公司）；DMi8 型倒置显微镜（日本 Olympus 公
式见图 1）。已有研究证实，BAC 能有效抑制人胎盘绒 司）；BDFACS AriaⅢ型流式细胞仪（美国Becton Dickin-
膜癌BeWo细胞生长 ，但其对NSCLC细胞增殖和凋亡 son公司）；Pultton P200+型超微量紫外分光光度计（北京
[11]
的影响少见报道。因此，本课题组以人肺癌 A549 细胞 五洲东方科技发展有限公司）；BL-150型电子天平（德国
为研究对象，考察BAC对该细胞增殖与凋亡的影响，并 Sartorius 公 司）；7900 Real-Time 型 聚 合 酶 链 式 反 应

中国药房 2019年第30卷第20期 China Pharmacy 2019 Vol. 30 No. 20 ·2783 ·