木犀草素对脉络膜恶性黑色素瘤细胞的影响

doi:10.13438/j.cnki.jdzk.2023.01.010

摘要/Abstract

摘要：为探讨木犀草素调节Wnt/β-catenin信号通路对人脉络膜恶性黑色素瘤细胞（MuM-2C）凋亡、迁移和侵袭的影响，采用CCK-8法测定0，5，10，15，20，25 μmol/L浓度木犀草素处理24 h后的MuM-2C活力，以筛选合适的药物作用浓度.将体外培养的MuM-2C随机分为3组：对照组、木犀草素组及木犀草素+氯化锂组，木犀草素组以15 μmol/L木犀草素处理，木犀草素+氯化锂组以15 μmol/L木犀草素和10 μmol/L的氯化锂联合处理.采用流式细胞技术和Hoechst 33258染色检测MuM-2C的凋亡情况，采用划痕实验和Transwell实验检测各组MuM-2C的迁移、侵袭情况，采用免疫荧光检测各组MuM-2C中凋亡蛋白BAX与BCL-2表达，采用免疫印记检测各组MuM-2C中EMT标志蛋白（E-cadherin，N-cadherin，Vimentin）和Wnt/β-catenin信号相关蛋白（Wnt1，β-catenin）表达，结果表明：不同剂量木犀草素均可抑制MuM-2C生长，并在一定范围内随剂量升高而作用增强；与对照组比较，木犀草素组细胞核形态固缩而大小不一，着色不均匀，部分呈现明亮的蓝色荧光，表现为明显的凋亡病理现象，细胞凋亡率、细胞BAX/BCL-2比值与E-cadherin蛋白表达显著升高（P＜0.05），细胞迁移率与侵袭数、细胞蛋白（N-cadherin，Vimentin，Wnt1，β-catenin）表达显著降低（P＜0.05）；与木犀草素组比较，木犀草素+氯化锂组细胞核的凋亡病理现象明显减轻，细胞凋亡率、细胞BAX/BCL-2比值与E-cadherin蛋白表达显著降低（P＜0.05），细胞迁移率与侵袭数、细胞蛋白（N-cadherin，Vimentin，Wnt1，β-catenin）表达显著升高（P＜0.05）.说明木犀草素可通过抑制Wnt/β-catenin信号途径传导而降低MuM-2C活力，促进其凋亡，并抑制其迁移和侵袭.

关键词： 木犀草素, Wnt/β-catenin, 人脉络膜恶性黑色素瘤细胞, 凋亡, 迁移, 侵袭

Abstract: The influences of luteolin on the apoptosis,migration and invasion of human choroidal malignant melanoma cells by regulating Wnt/β-catenin signaling pathway have been investigated.The cell viability of human choroidal malignant melanoma cells MuM-2C treated with luteolin at 0,5,10,15,20,25 μmol/L concentrations for 24 h was determined by CCK-8 method to screen the appropriate concentration of drug action.The MuM-2C cells cultured in vitro were randomly grouped into three groups:control group,luteolin group and luteolin + lithium chloride group.After treatment with 15 μmol/L luteolin and 10 μmol/L lithium chloride,flow cytometry and Hoechst 33258 staining were performed to detect the apoptosis of MuM-2C cells;scratch test and Transwell test were performed to detect the migration and invasion of MuM-2C cells in each group;immunofluorescence was applied to detect the expression of apoptotic proteins BAX and BCL-2 in MuM-2C cells in each group;Western blotting was performed to detect the expression of EMT marker proteins (E-cadherin,N-cadherin,Vimentin) and Wnt/β-catenin signaling-related proteins (Wnt1,β-catenin) in MuM-2C cells in each group.The results showed that different doses of luteolin could inhibit the growth of MuM-2C cells,and the effect was enhanced with increasing doses within a certain range.Compared with the control group,the nuclei in the luteolin group were pyknotic,different in size,uneven in coloration,and showed bright blue fluorescence and obvious pathological phenomena of apoptosis,with the apoptosis rate,the ratio of BAX/BCL-2 and the expression of E-cadherin protein obviously increasing (P＜0.05) and the cell migration rate and invasion number,and the expression of N-cadherin,Vimentin,Wnt1 and β-catenin proteins in cells obviously decreasing (P＜0.05).Compared with the luteolin group,the pathological phenomenon of nuclear apoptosis in the luteolin + lithium chloride group was obviously reduced,with the apoptosis rate,the ratio of BAX/BCL-2 and the expression of E-cadherin protein obviously decreasing (P＜0.05) and the cell migration rate and invasion number,and the expression of N-cadherin,Vimentin,Wnt1 and β-catenin proteins in cells obviously increasing (P＜0.05).It can be concluded that luteolin can reduce the viability of MuM-2C cells,promote their apoptosis,and inhibit their migration and invasion by inhibiting the Wnt/β-catenin signaling pathway transduction.

Key words: luteolin, Wnt/β-catenin, human choroidal malignant melanoma cells, apoptosis, migration, invasion

唐静, 袁小波, 阳帆. 木犀草素对脉络膜恶性黑色素瘤细胞的影响[J]. 吉首大学学报（自然科学版）, 2023, 44(1): 68-76.

TANG Jing, YUAN Xiaobo, YANG Fan. Influences of Luteolin on Choroidal Malignant Melanoma Cells[J]. Journal of Jishou University(Natural Sciences Edition), 2023, 44(1): 68-76.

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