冬凌草甲素通过ROS/JNK信号通路诱导结直肠癌细胞凋亡

ORI induces apoptosis in colorectal cancer cells by ROS/JNK signaling pathway

摘要:

目的 探讨冬凌草甲素通过活性氧/c-Jun氨基末端激酶（ROS/JNK）信号通路诱导结直肠癌细胞凋亡 的机制。方法 MTT法检测冬凌草甲素对人结直肠癌 Caco2和 SW480细胞的作用，Hoechst 33342染色和流式细 胞仪检测细胞的凋亡情况，DCFH-DA（2′，7′-二氯荧光素二乙酸酯）染色检测细胞活性氧（ROS）水平。采用网络药 理学和分子对接技术预测冬凌草甲素通过 ROS诱导结直肠癌细胞凋亡的潜在靶点，并用 Western blot对其潜在作 用靶点进行验证。结果 冬凌草甲素分别作用 Caco2、SW480 结直肠癌细胞后能够抑制细胞的增殖（P < 0.01 或 0.05）。Hoechst 33342 染色显示细胞核固缩，呈明显的亮蓝色，表现凋亡特征。随着冬凌草甲素浓度增加，凋亡的 细胞数增加，裂解的胱天蛋白酶3和多聚腺苷二磷酸核糖聚合酶以及Bcl-2相关蛋白X（Bax）、磷酸化c-Jun氨基末 端激酶（p-JNK）表达亦显著增加，而Bcl-2表达则明显减少，同时细胞内ROS水平增加（P < 0.01或0.05）。ROS抑制 剂 N-乙酰半胱氨酸能够降低细胞内 ROS 水平，下调 p-JNK 蛋白的表达，减少细胞凋亡，增加细胞活力（P < 0.01）。 网络药理学、分子对接及 Western blot结果显示 JNK通路可能是冬凌草甲素通过 ROS诱导结直肠癌细胞凋亡的潜 在靶点。结论 冬凌草甲素能够诱导结直肠癌细胞凋亡，作用机制可能与其调控ROS/JNK信号有关。

Abstract:

Objective To investigate the mechanism of apoptosis in colorectal cancer cells induced by oridonin through the ROS/JNK signaling pathway. Methods The MTT assay was used to detect the effect of oridonin on human colorectal cancer Caco2 and SW480 cells. Morphologic changes in cells were observed using Hoechst 33342 staining. Apoptotic cells were evaluated by flow cytometry, ROS were analyzed using DCFH-DA staining. Network pharmacology and molecular docking were conducted to predict potential ROS-related targets of oridonin (ORI) in colorectal cancer (CRC), which were verified by Western blot analysis. Results After Caco2 and SW480 cells were treated with oridonin, respectively, cell viability significantly decreased (P < 0.01 or 0.05). Chromatin condensation and loss of nuclear struction were observed using Hoechst 33342, which exhibited apoptotic characteristics. With the increase of oridonin concentration, the levels of the apoptotic cells, as well as the protein levels of Cleaved Caspase-3, Cleaved PARP, Bax, and p-JNK in CRC cells, were elevated, while Bcl-2 levels were downregulated (P < 0.01 or 0.05). ORI could dose-dependently increase ROS level in CRC cells (P < 0.01). However, N-Acetyl cysteine (NAC), the ROS scavenger, significantly reduced ROS levels induced by oridonin, downregulated p-JNK protein expression, abolished cell apoptosis, and increased cell viability (P < 0.01). The results of network pharmacology, molecular docking and Western blot indicated that the JNK pathway might be a target of ORI-induced apoptosis in CRC cells through ROS. Conclusion ORI can induce apoptosis in CRC cells via the ROS/JNK signaling pathway.