摘要
【目的】运用超高效液相色谱-四极杆-静电场轨道阱串联质谱(UHPLC-QE-MS)技术分析橘荔散结丸醇提物的化学成分,并探讨其对子宫肌瘤细胞增殖的抑制作用及机制。【方法】(1)以UHPLC-QE-MS技术分析橘荔散结丸醇提物化学成分。(2)培养原代人子宫肌瘤细胞。采用细胞计数试剂盒8(CCK-8)测定不同浓度橘荔散结丸醇提物处理不同时间后的细胞增殖抑制率,并筛选后续实验中其2个高、低浓度。实验分为空白对照组,二甲基亚砜(DMSO)组,米非司酮组及橘荔散结丸醇提物高、低浓度组,分别给予相应处理后,采用流式细胞术检测细胞周期,Western Blot或定量聚合酶链反应(qPCR)法分别检测细胞Wnt/糖原合成酶激酶3β(GSK-3β)/β-连环蛋白(β-catenin)信号通路关键蛋白Wnt 5b、GSK-3β、p-GSK-3β、β-catenin、表皮生长因子受体(EGFR)和基质金属蛋白酶2(MMP2)的蛋白、mRNA表达水平。【结果】鉴别橘荔散结丸醇提物主要化学成分91个,橘荔散结丸醇提物冻干粉与中成药橘荔散结片主要化学成分共有22个。橘荔散结丸醇提物各浓度组细胞增殖抑制率均大于空白对照组(P<0.05);橘荔散结丸醇提物高、低浓度组及米非司酮组G0/G1期细胞数均大于空白对照组,而G2/M期细胞数小于空白对照组(P<0.05);橘荔散结丸醇提物高、低浓度组及米非司酮组Wnt 5b、β-catenin、EGFR、MMP2蛋白、mRNA表达水平均低于与空白对照组(均P<0.05),而GSK-3β蛋白及mRNA表达水平与空白对照组无显著性差异(P>0.05)。【结论】UHPLC-QE-MS技术分离橘荔散结丸醇提物化学成分众多,主要成分具有氧化应激调节作用;橘荔散结丸醇提物可有效抑制子宫肌瘤细胞增殖,其分子机制与抑制Wnt/GSK-3β/β-catenin信号通路关键蛋白表达,进而干预子宫肌瘤细胞微环境氧化应激反应有关。
Objective To analyze the chemical composition of the alcoholic extract of Juli Sanjie Pills by using ultra-high performance liquid chromatography-Q exactive-mass spectrometry(UHPLC-QE-MS)and to investigate the inhibitory effect and the mechanism on the proliferation of uterine fibroid cells.Methods(1)The chemical composition of the alcoholic extract of Juli Sanjie Pills was analyzed by UHPLC-QE-MS.(2)Cultured primary human uterine fibroid cells.Cell counting kit 8(CCK-8)was used to determine the inhibition rate of cell proliferation after different concentrations of Juli Sanjie Pills alcohol extracts were treated for different times,and blank control group,dimethyl sulfoxide(DMSO)group,Mifepristone group and Juli Sanjie Pills alcoholic extracts high-and low-concentration groups,respectively,and the corresponding treatments were given,and then the cell cycle was detected by flow cytometry;the protein and mRNA expression levels of the cellular Wnt/glycogen synthase kinase 3β(GSK-3β)/β-catenin signalling pathway key proteins Wnt 5b,GSK-3β,p-GSK-3β,β-catenin,epidermal growth factor receptor(EGFR)and matrix metalloproteinase 2(MMP2)were detected by Western Blot or quantitative polymerase chain reaction(qPCR).Results The inhibition rate of cell proliferation in each concentration group of Juli Sanjie Pills was greater than that of the blank control group(P<0.05);the number of cells in G0/G1 phase in the high-and low-concentration groups of Juli Sanjie Pills was greater than that of the blank control group,while the number of cells in G2/M phase was less than that of the blank control group(P<0.05);the protein and mRNA expression levels of Wnt 5b,β-catenin,EGFR,MMP2 in the high-and low-concentration groups of Juli Sanjie Pills alcoholic extracts and the Mifepristone group were lower than those with the blank control group(all P<0.05),whereas there was no significant difference between the protein and mRNA expression levels of GSK-3βwith the blank control group(P>0.05).Conclusion UHPLC-QE-MS technique was used to
作者
谢卓庭
王乃平
程晓榆
李坤寅
XIE Zhuo-Ting;WANG Nai-Ping;CHENG Xiao-Yu;LI Kun-Yin(Dongguan Hospital of Guangzhou University of Chinese Medicine,Dongguan 523000 Guangdong,China;The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510375 Guangdong,China)
出处
《广州中医药大学学报》
CAS
2024年第2期463-470,共8页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
广东省自然科学基金资助项目(编号:2020A1515010600)
广东省中医药管理局项目(编号:20203003)
广东省普通高校重点领域专项(编号:2021ZDZX2015)
东莞市高水平专项(编号:20231800903501)。