| 引用本文: |
何甜甜, 吴柯楠, 朱洁, 黄成银, 侯宝龙, 王征, 梁艳妮.熊果酸对非小细胞肺癌H1975细胞的体内外抑制作用研究[J].湖南中医药大学学报,2025,45(5):825-835[点击复制] |
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| 熊果酸对非小细胞肺癌H1975细胞的体内外抑制作用研究 |
| 何甜甜,吴柯楠,朱洁,黄成银,侯宝龙,王征,梁艳妮 |
| (陕西中医药大学陕西中药资源产业化省部共建协同创新中心/陕西省创新药物研究中心, 陕西 咸阳 712083) |
| 摘要: |
| 目的 研究熊果酸（UA）对非小细胞肺癌（NSCLC）H1975细胞的体内外抑制作用，探讨其对移植瘤裸鼠内源性代谢物的影响。方法 以H1975细胞为研究对象，MTT法筛选UA（0~75 μg/mL）作用细胞的最佳浓度。将细胞分为对照组及UA低（9.27 μmol/L）、中（18.53 μmol/L）、高（27.8 μmol/L）剂量组作用细胞48 h，划痕法检测细胞迁移能力，流式细胞仪检测细胞周期和凋亡，Western blot法检测组织金属蛋白酶抑制因子-2（TIMP-2）、促凋亡B淋巴细胞瘤-2相关X蛋白（Bax）、活化的胱天蛋白酶-3（Cleaved-Caspase-3）、基质金属蛋白酶（MMP）-2、MMP-9和抗凋亡蛋白B淋巴细胞瘤-2（Bcl-2）蛋白的表达。在裸鼠背部皮下注射H1975细胞建立移植瘤模型，随机分为对照组、吉非替尼组（50 mg/kg）和UA低（100 mg/kg）、中（200 mg/kg）、高（400 mg/kg）剂量组，每3天给药一次，连续18 d，记录裸鼠体质量和移植瘤生长情况。采用UPLC-MS/MS法检测裸鼠血清内源性代谢物水平。结果 UA抑制H1975细胞增殖IC50（48 h）为27.8 μmol/L。与对照组比较，UA中、高剂量组阻滞细胞周期在G0/G1期、细胞凋亡率增加、细胞迁移能力下降（P＜0.05）；TIMP-2、Bax、Cleaved-Caspase-3蛋白表达上调，MMP-2、MMP-9、Bcl-2蛋白表达下调（P＜0.05）。与UA低剂量组比较，UA高剂量组阻滞细胞周期在G0/G1期、细胞凋亡率增加、细胞迁移能力下降（P＜0.05）；TIMP-2、Bax、Cleaved-Caspase-3蛋白表达上调，MMP-2、MMP-9、Bcl-2蛋白表达下调（P＜0.05）。在体内实验中，与对照组比较，UA各剂量组与吉非替尼组对移植瘤裸鼠的肿瘤生长有抑制作用（P＜0.05）；与UA低、中剂量组比较，UA高剂量组对移植瘤裸鼠肿瘤生长有抑制作用（P＜0.05）。代谢组学分析筛选出5S，15S-二羟基-6E，8Z，10Z，13E-二十碳四烯酸、4-羟基苯甲醇、氧化三甲胺等22个差异代谢物，主要涉及不饱和脂肪酸的生物合成、亚油酸代谢和酪氨酸代谢通路。结论 UA在体内外对NSCLC H1975细胞均有明显抑制作用，其在体内的抑制作用可能与调节不饱和脂肪酸的生物合成、亚油酸代谢和酪氨酸代谢相关。 |
| 关键词: 熊果酸 H1975细胞 细胞凋亡 细胞增殖 细胞迁移 血清代谢组学 |
| DOI：10.3969/j.issn.1674-070X.2025.05.007 |
| 投稿时间：2024-12-20 |
| 基金项目:陕西省重点产业创新链项目（2018ZDCXL-SF-01-02-02）；陕西省教育厅创新团队项目（22JP019，24JP047）；陕西高校青年创新团队项目（陕教〔2019〕90号）。 |
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| In vitro and in vivo inhibitory effects of ursolic acid on non-small cell lung cancer H1975 cells |
| HE Tiantian, WU Kenan, ZHU Jie, HUANG Chengyin, HOU Baolong, WANG Zheng, LIANG Yanni |
| (Shaanxi University of Chinese Medicine, Co-construction Collaborative Innovation Center for Chinese Medicine Resources Industrialization by Shaanxi & Education Ministry/Shaanxi Innovative Drug Research Center, Xianyang, Shaanxi 712083, China) |
| Abstract: |
| Objective To investigate the in vitro and in vivo inhibitory effects of ursolic acid (UA) on non-small cell lung cancer (NSCLC) H1975 cells, and explore its effects on endogenous metabolites in tumor-xenografted nude mice. Methods H1975 cells were used as the research object, and the optimal concentration of UA (0-75 μg/mL) for cell treatment was screened using the MTT assay. Cells were divided into control group, and low-, medium-, and high-dose UA (9.27 μmol/L, 18.53 μmol/L, 27.8 μmol/L) groups and all groups were treated for 48 hours. Cell migration was assessed by the scratch assay, cell cycle distribution and apoptosis were analyzed by flow cytometry, and protein expression levels of tissue inhibitor of metalloproteinases-2 (TIMP-2), pro-apoptotic Bcl-2-associated X protein (Bax), cleaved-caspase-3, matrix metalloproteinases (MMP)-2, MMP-9, and anti-apoptotic Bcl-2 were determined by Western blot. A xenograft tumor model was established by subcutaneous injection of H1975 cells into the back of nude mice, which were randomly divided into control, gefitinib (50 mg/kg), and low-, medium-, and high-dose UA (100 mg/kg, 200 mg/kg, 400 mg/kg) groups. Drugs were administered every three days for 18 consecutive days, and nude mouse body weight and tumor growth were recorded. UPLC-MS/MS was used to determine endogenous metabolite levels in nude mouse serum. Results TheIC50 value of UA for inhibiting H1975 cell proliferation at 48 h was 27.8 μmol/L. Compared with the control group, medium- and high-dose UA groups arrested the cell cycle at the G0/G1 phase, and showed increased apoptosis rate and reduced cell migration ability (P<0.05). Additionally, the protein expression levels of TIMP-2, Bax, and Cleaved Caspase-3 were upregulated, while those of MMP-2, MMP-9, and Bcl-2 were downregulated (P<0.05). Compared with the low-dose UA group, the high-dose UA group arrested the cell cycle at the G0/G1 phase, and exhibited elevated apoptosis rate, reduced cell migration ability (P<0.05), and upregulated TIMP-2, Bax, and Cleaved-Caspase-3 protein expression levels, as well as downregulated MMP-2, MMP-9, and Bcl-2 protein expression levels (P<0.05). In vivo, compared with the control group, all dose groups of UA and the gefitinib group significantly inhibited tumor growth in tumor-xenografted nude mice (P<0.05). Furthermore, the high-dose UA group demonstrated enhanced tumor growth inhibition compared with the low- and medium-dose UA groups (P<0.05). Metabolomics analysis identified 22 differential metabolites, including 5S, 15S-dihydroxy-6E, 8Z, 10Z, 13E-eicosatetraenoic acid, 4-hydroxybenzyl alcohol, and trimethylamine N-oxide, primarily involving pathways related to unsaturated fatty acid biosynthesis, linoleic acid metabolism, and tyrosine metabolism. Conclusion UA exhibits significant inhibitory effects on NSCLC H1975 cells both in vitro and in vivo. Its in vivo inhibitory effects may be associated with the regulation of unsaturated fatty acid biosynthesis, linoleic acid metabolism, and tyrosine metabolism pathways. |
| Key words: ursolic acid H1975 cell apoptosis cell proliferation cell migration serum metabolomics |
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