木犀草素通过增加ROS的产生和下调AKT/mTOR通路及HO-1蛋白表达抑制肺癌A549细胞增殖

doi:10.12122/j.issn.1673-4254.2024.12.12

摘要/Abstract

摘要：

目的探究木犀草素（Lut）对肺癌A549细胞增殖的抑制作用及其内在机制。方法用不同浓度的Lut处理A549细胞48 h，通过MTT法检测细胞活性，通过平板克隆和EdU染色检测细胞增殖，通过DCFH-DA法检测细胞活性氧（ROS）水平，通过Hoechst33258 染色法检测细胞凋亡水平，通过MDC染色法检测细胞自噬水平，通过Western blotting实验检测细胞凋亡相关蛋白Bax、Bcl-2、Cleaved caspase-9，自噬相关蛋白LC3B、Beclin1、P62，AKT/mTOR通路蛋白以及HO-1蛋白的表达。结果 Lut剂量依赖性的抑制A549细胞的活力和增殖能力（P<0.05），引发细胞内ROS水平增加（P<0.05），上调凋亡相关蛋白Bax、Cleaved caspase-9和自噬相关蛋白Beclin1的表达，增加LC3B-II/LC3B-I的比值，下调抗凋亡蛋白Bcl-2和自噬相关蛋白P62的表达，诱导细胞凋亡和自噬（P<0.001）。此外，Lut可显著抑制AKT和mTOR的磷酸化，下调HO-1蛋白的表达（P<0.05）。结论 Lut通过增加细胞内ROS的产生，抑制AKT/mTOR通路以及下调HO-1蛋白水平诱导A549细胞的凋亡和自噬。

关键词: 木犀草素, 活性氧, 凋亡, 自噬, AKT/mTOR通路, HO-1

Abstract:

Objective To investigate the mechanism of luteolin for inhibiting proliferation of lung cancer A549 cells. Methods A549 cells treated with different concentrations of luteolin for 48 h were evaluated for changes in cell viability, proliferation, reactive oxygen species (ROS) production and apoptosis using MTT assay, plate cloning assay, EdU staining, DCFH-DA assay and Hoechst33258 staining. The changes in cell autophagy were examined with MDC staining, and the expressions of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-9), autophagy-related proteins (LC3B, Beclin 1, and P62), AKT/mTOR pathway proteins, and HO-1 protein were detected using Western blotting. Results Treatment with luteolin dose-dependently inhibited the viability and proliferation of A549 cells, increased intracellular ROS levels, up-regulated the expressions of Bax, cleaved caspase-9, and Beclin 1, increased the LC3B-II/LC3B-I ratio, down-regulated the expressions of Bcl-2 and P62, and induced cell apoptosis and autophagy. Luteolin also significantly inhibited the phosphorylation of AKT and mTOR and down-regulated the expression of HO-1 protein in the cells. Conclusion Luteolin induces apoptosis and autophagy to inhibit proliferation of A549 cells by increasing ROS production, inhibiting the AKT/mTOR pathway and down-regulating HO-1 protein expression.

Key words: luteolin, reactive oxygen species, apoptosis, autophagy, AKT/mTOR signaling pathway, HO-1

李欢, 邱紫欣, 徐文洁, 陈雪, 魏典典, 王允. 木犀草素通过增加ROS的产生和下调AKT/mTOR通路及HO-1蛋白表达抑制肺癌A549细胞增殖[J]. 南方医科大学学报, 2024, 44(12): 2367-2374.

Huan LI, Zixin QIU, Wenjie XU, Xue CHEN, Diandian WEI, Yun WANG. Luteolin inhibits proliferation of lung cancer A549 cells by increasing ROS production and inhibiting the AKT/mTOR signaling pathway and HO-1 expression[J]. Journal of Southern Medical University, 2024, 44(12): 2367-2374.

图/表 5

图1 Lut抑制A549细胞的活力、增殖和克隆形成能力

Fig.1 Luteolin (Lut) inhibits viability, proliferation, and clone-forming ability of A549 cells. A: MTT assay of A549 cells treated with different concentrations of luteolin for 48 h. B: MTT assay of A549 and Beas-2B cells treated with 20 and 40 μmol/L luteolin for 48 h. C, D: EdU assay of A549 cells treated with 20 and 40 μmol/L luteolin for 48 h (Original magnification: ×100). E, F: Clone formation assay of A549 cells treated with 20 and 40 μmol/L luteolin for 48 h. *P<0.05, **P<0.01, ***P<0.001 vs control.

图2 Lut诱导A549细胞氧化应激

Fig.2 Luteolin induces oxidative stress in A549 cells. A, B: Detection of intracellular ROS levels in luteolin-treated A549 cells by flow cytometry. C: MTT assay of A549 cells treated with luteolin in the presence or absence of NAC for 48 h. *P<0.05, ***P<0.001vs control; ##P<0.01,###P<0.001 vs NAC- group.

图3 Lut诱导A549细胞凋亡

Fig.3 Luteolin induces apoptosis of A549 cells. A: Hoechst 33258 staining of luteolin-treated A549 cells (×100). B-D: Western blotting for detecting expression levels of Bax, Bcl-2, cleaved-caspase-9 in luteolin-treated A549 cells. ***P<0.001 vs control.

图4 Lut诱导A549细胞自噬

Fig.4 Luteolin induces autophagy in A549 cells. A, B: MDC staining for detecting autophagy in luteolin-treated A549 cells (×100). C-F: Western blotting for detecting expression levels of autophagy-related proteins Beclin 1, P62, and LC3B in luteolin-treated A549 cells. *P<0.05, ***P<0.001 vs control.

图5 Lut对A549细胞AKT/mTOR通路、HO-1蛋白水平的改变

Fig.5 Luteolin-induced changes in AKT/mTOR signaling and HO-1 protein levels in A549 cells. A-D: Western blotting for detecting expression levels of AKT, P-AKT, mTOR, P-mTOR, and HO-1 proteins in luteolin-treated A549 cells. E, F: Western blotting for detecting HO-1 protein expression levels in A549 cells treated with luteolin in the presence or absence of LY294002 for 48 h. G-I: Western blotting of P-AKT and P-mTOR protein expression levels in A549 cells treated with luteolin in the presence or absence of NAC for 48 h. ***P<0.001 vs control; #P<0.05, ###P<0.001 vs Lut group.

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