沙苑子总黄酮基于RANK/RANKL/OPG信号轴抑制MG-63骨肉瘤细胞生长并促使其凋亡的机制研究  

Mechanism of Flavonoids from Astragalus Complanatus Inhibiting the Growth of MG-63 Osteosarcoma Cells and Promoting Apoptosis Based on the RANK/RANKL/OPG Signaling Axis

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作  者:李婷 张旗 汪国友 李东波 何跃 吕政 石厚银 沈骅睿 Li Ting;Zhang Qi;Wang Guoyou;Li Dongbo;He Yue;LüZheng;Shi Houyin;Shen Huarui(Southwest Medical University,Sichuan,Luzhou 646000,China)

机构地区:[1]西南医科大学,四川泸州646000 [2]四川省合江县中医医院,四川合江646000 [3]西南医科大学附属中医医院,四川泸州646000 [4]四川省康复医院,四川成都610000 [5]成都中医药大学,四川成都610000

出  处:《中国中医急症》2023年第8期1336-1341,共6页Journal of Emergency in Traditional Chinese Medicine

基  金:四川省科技计划联合创新专项(泸州)(2022YFS0609);泸州市人民政府-西南医科大学科技战略合作项目(2021LZXNYD-J31);四川省中医药管理局中医药科研专项课题(2021MS463);西南医科大学——西南医科大学附属中医医院联合专项(2020XYLH-013)。

摘  要:目的观察沙苑子总黄酮(FAC)基于RANK/RANKL/OPG信号轴对MG-63骨肉瘤细胞凋亡的影响。方法不同浓度FAC、顺铂(阳性对照)作用于MG-63骨肉瘤细胞后,采用CCK-8法检测不同时间点的增殖抑制作用,划痕实验检测细胞迁移修复能力,Hoechest染色、流式细胞术检测细胞凋亡,PCR和Western blotting检测细胞RANK、RANKL、OPG和凋亡相关因子Caspase-3 m RNA和蛋白表达。结果CCK-8法显示,除50 mg/LFAC对24、48 h细胞存活率影响相近(P>0.05)外,其余浓度FAC(100、200、300、400 mg/L)均可显著抑制MG-63骨肉瘤细胞的增殖(P<0.05),并呈一定的时间和浓度依赖性。划痕实验显示FAC及顺铂对MG-63骨肉瘤细胞的迁移修复能力有抑制作用,并表现出浓度依赖性,低、中、高浓度组(100、200、300 mg/L)迁移修复率分别为(31.62±0.85)%、(25.38±1.18)%、(6.05±2.96)%,阳性对照组(10 mg/L顺铂)为(14.74±2.58)%。Hoechest荧光染色和流式细胞术显示FAC、顺铂作用48 h可促进MG-63骨肉瘤细胞凋亡。对照组、阳性对照组细胞凋亡率为7.84%、93.61%,低、中、高浓度组凋亡率分别为4.46%、8.51%、98.15%。PCR显示FAC和顺铂降低MG-63骨肉瘤细胞OPG的m RNA表达,增强RANK、RANKL、Caspase-3的m RNA表达。Western blotting显示FAC和顺铂降低MG-63骨肉瘤细胞OPG蛋白表达水平,上调RANK、RANKL、Caspase-3蛋白表达水平。结论FAC在体外具有促进MG-63骨肉瘤细胞凋亡作用,300 mg/L FAC促凋亡作用与10 mg/L顺铂相当,其促凋亡机制可能与RANK/RANKL/OPG信号轴调控有关。Objective:To investigate the apoptotic effect of flavonoids from Astragalus complanatus(FAC)on MG-63 osteosarcoma cells based on RANK/RANKL/OPG signaling axis and its mechanism.Methods:After dif⁃ferent concentrations of FAC and cisplatin(positive control)were applied to MG-63 osteosarcoma cells,the prolif⁃eration inhibition was detected by CCK-8 assay at different time points,cell migration repair ability was detected by scratch test,apoptosis was detected by Hoechest staining and flow cytometry,mRNA and protein expressions of RANK,RANKL,OPG and Caspase-3 in cells were detected by PCR and Western blotting.Results:CCK-8 meth⁃od showed that,except for 50 mg/L FAC which showed no statistically significant difference in cell survival at 24 h and 48 h,residual concentration of FAC(100,200,300,400 mg/L)could significantly inhibit the prolifera⁃tion of MG-63 osteosarcoma cells(P<0.05),and showed a certain time and concentration dependence.The scratch test showed that FAC and cisplatin inhibited the migration and repair of MG-63 osteosarcoma cells in a concentration-dependent manner,the migration repair rates of low,middle and high concentration groups(100,200,300 mg/L)were(31.62±0.85)%,(25.38±1.18)%,(6.05±2.96)%respectively,and the positive control group(10 mg/L cisplatin)was(14.74±2.58)%.Hoechest fluorescence staining and flow cytometry showed that FAC and cisplatin treated for 48 h could promote the apoptosis of MG-63 osteosarcoma cells.The apoptosis rates of control group and positive control group were 7.84%and 93.61%,and the apoptosis rates of low,medium and high concen⁃tration groups were 4.46%,8.51%and 98.15%.PCR showed that FAC and cisplatin decreased the mRNA expres⁃sion of OPG and enhanced the mRNA expression of RANK,RANKL and Caspase-3 in MG-63 osteosarcoma cells.Western blotting showed that FAC and cisplatin decreased the expression of OPG protein in MG-63 osteosar⁃coma cells,and up-regulated the expression of RANK,RANKL and Caspase-3.Conclusion:FAC could promote the apoptosis of MG-63 oste

关 键 词:沙苑子 黄酮 MG-63骨肉瘤细胞 凋亡 RANK/RANKL/OPG 

分 类 号:R730.59[医药卫生—肿瘤]

 

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