茯苓酸对人骨肉瘤细胞系MG-63的增殖凋亡、迁移侵袭影响及PERK/CCAAT信号通路调控作用  

Effects of pachymic acid on proliferation,apoptosis and invasion of osteosarcoma cells and its regulatory effect on PERK/CHOP signaling pathway

作　　者：施擎宇 黄帆[1] 王冬明 SHI Qingyu;HUANG Fan;WANG Dongming(Department of Oncology,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai China)

机构地区：[1]上海中医药大学附属龙华医院肿瘤科,上海200032

出　　处：《山东医药》2023年第30期1-5,共5页Shandong Medical Journal

摘　　要：目的观察茯苓酸(PA)对人骨肉瘤细胞系MG-63增殖、迁移、凋亡和侵袭的影响,并探讨PA对蛋白激酶R样内质网激酶(PERK)/CCAAT增强子结合蛋白同源蛋白(CHOP)信号通路的调控作用。方法体外培养MG-63细胞,分为对照组、低浓度PA组(PA-L组)、中浓度PA组(PA-M组)、高浓度PA组(PA-H组)、高浓度PA+蛋白激酶R样内质网激酶(PERK)抑制剂GSK2606414组(PA-H+GSK2606414组),PA-L组、PA-M组及PA-H组分别用含20、40及80μmol/L PA的培养基培养,PA-H+GSK2606414组用含80μmol/L的PA和5μmol/L的GSK2606414培养基培养,对照组用空白培养基培养。分别于培养24、48、72 h时采用MTT法观察各组细胞增殖情况;培养24 h时采用细胞划痕实验观察各组细胞迁移情况;培养48 h时采用Transwell实验观察各组细胞侵袭情况;培养48 h时采用流式细胞术测算各组细胞凋亡率;培养48 h时,采用RT-qPCR法检测PERK及CHOP的mRNA表达,并采用WESTERN Blotting法检测PERK/CHOP信号通路相关蛋白及凋亡相关蛋白真核生物起始因子2α(eIF2α)、活化转录因子4(ATF4)、CHOP、BCL2相关X蛋白(Bax)及B淋巴细胞瘤-2(Bcl-2)。结果与对照组比较,PA-M组及PA-H组培养48与72 h时细胞OD_(570nm)值低、细胞迁移率低、细胞侵袭数少、细胞凋亡率高(P均<0.05);与PA-M组相比,PA-H组培养48及72 h时的细胞OD_(570nm)值低、细胞迁移率低、细胞侵袭数少、细胞凋亡率高(P均<0.05);与PA-H组相比,PA-H+GSK2606414组细胞培养48及72 h时的OD_(570nm)值高、细胞迁移率高、细胞侵袭数多、细胞凋亡率低(P均<0.05)。与对照组比较,PA-M组及PA-H组细胞PERK及CHOP mRNA相对表达量高,PERK及eIF2α磷酸化水平高,ATF4、CHOP、Bax蛋白相对表达量高,Bcl-2相对表达量低(P均<0.05);与PA-M组相比,PA-H组细胞PERK及CHOP mRNA相对表达量高,PERK及eIF2α磷酸化水平高,ATF4、CHOP、Bax蛋白相对表达量高,Bcl-2相对表达量低(P均<0.05);与PA-H组相比,PA-H+GSK2606414组细胞PERK及CHOP mObjective To investigate the effects of pachymic acid(PA)on the proliferation,apoptosis and invasion of osteosarcoma cells and to explore its regulatory effect on the protein kinase R-like endoplasmic reticulum kinase(PERK)/CCAAT enhancer binding protein homologous protein(CHOP)signaling pathway.Methods MG-63 cells were cultured in vitro and were divided into the control group,low-concentration PA group(PA-L group),medium-concentration PA group(PA-M group),high-concentration PA group(PA-H group),and high-concentration PA+PERK inhibitor GSK2606414 group(PA-H+GSK2606414 group),respectively.Cells in the PA-L group,PA-M group and PA-H group were cultured in medium containing 20,40 and 80μmol/L PA;cells in the PA-H+GSK2606414 group were cultured in medium containing 80μmol/L PA and 5μmol/L GSK2606414,and cells in the control group were cultured in the blank medium.The cell proliferation of each group was observed by MTT assay at 24,48 and 72 h,respectively;cell migration in each group was observed by cell Scratch test after 24 h culture;the cell invasion of each group was observed by Transwell assay after 48 h culture;the apoptosis rate of each group was measured by flow cytometry after 48 h;after 48 h of culture,the mRNA expression levels of PERK and CHOP were detected by RT-qPCR,PERK/CHOP signaling pathway related-proteins and apoptosis-related proteins eukaryotic initiation factor 2α(eIF2α),activating transcription factor 4(ATF4),CHOP,BCL2-associated X protein(Bax),and B-cell lymphoma-2(Bcl-2)were detected by Western blotting.Results Compared with the control group,the OD_(570nm) values(48 h,72 h),cell mobility rates,invasion cells were lower,and the apoptosis rates were higher in the PA-M and PA-H groups(all P<0.05).Compared with the PA-M group,the OD_(570nm) values(48 h,72 h),cell mobility rates,invasion cells were lower,and the apoptosis rates were higher in the PA-H group(all P<0.05).Compared with the PA-H group,PA-H+GSK2606414 group had higher OD_(570nm) values(48 h,72 h),higher cell mobility rate,more in

关 键 词：茯苓酸 骨肉瘤 细胞增殖 细胞凋亡 细胞侵袭 蛋白激酶R样内质网激酶 CCAAT增强子结合蛋白同源蛋白 

分 类 号：R285[医药卫生—中药学]