Ling Jia-Yin, Wang Qiu-Lan, Liang Hao-Nan, Liu Qing-Bo, Yin Dong-Hong, Lin Li
First School of Clinical Medicine, Gansu University of Chinese Medicine, Lanzhou, 730000, China.
Gansu Provincial Hospital, Lanzhou, 730000, China.
Chin J Integr Med. 2023 Mar;29(3):213-223. doi: 10.1007/s11655-022-3679-4. Epub 2022 Aug 31.
To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer.
Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay.
Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05).
Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.
评价白花蛇舌草黄酮对人胃癌细胞的凋亡及周期阻滞作用，确定其与控制活性氧（ROS）产生的线粒体依赖性信号转导途径相关的作用机制，并评估小鼠异种移植模型的药效学，为黄酮类化合物在胃癌防治中的应用提供参考。
采用酶解-超声辅助法提取黄酮类化合物，并用D-101树脂纯化。通过高效液相色谱法对生物活性成分进行表征。用不同浓度的黄酮类化合物（64、96、128、160μg/mL）处理人胃癌细胞系MKN-45、AGS和人胃黏膜上皮细胞系GES-1。采用MTT法评价黄酮类化合物对细胞活力的影响，并通过Hoechst染色观察细胞核形态。通过流式细胞术检测凋亡率和细胞周期阶段，用激光共聚焦显微镜检测ROS的产生，用荧光显微镜观察线粒体膜电位（MMP），并通过免疫印迹法检测与线粒体途径激活相关的凋亡蛋白的表达。将MKN-45细胞移植到BALB/c裸鼠体内建立异种移植瘤模型。用苏木精-伊红染色显示皮下肿瘤组织。测量肿瘤体积和肿瘤重量，通过免疫组织化学检测增殖标志物增殖细胞核抗原（PCNA）和Ki-67的表达水平，并用酶联免疫吸附测定法测量CA72-4的表达水平。
白花蛇舌草黄酮抑制MKN-45和AGS人胃癌细胞的增殖，使细胞周期阻滞在G/S期，在凋亡过程中诱导ROS积累，并改变MMP。此外，黄酮类化合物增加了凋亡蛋白酶激活因子-1（Apaf-1）、裂解的半胱天冬酶-3（Cleaved-Caspase-3）和 Bax蛋白的表达，降低了细胞周期蛋白A（Cyclin A）、细胞周期蛋白依赖性激酶2（Cdk2）、Bcl-2、原半胱天冬酶-9（Pro-Caspase-9）和线粒体细胞色素C（Mitochondrial Cytochrome C）的表达（P<0.05）。MKN-45细胞小鼠异种移植模型进一步明确了黄酮类化合物对肿瘤的生长抑制作用。各黄酮剂量组PCNA和Ki-67的表达水平降低，CA72-4的表达水平降低（P<0.05）。
白花蛇舌草黄酮可通过激活线粒体控制的信号转导途径抑制人胃癌细胞的增殖并诱导其凋亡。
