摘要
探究珠子参总皂苷对人宫颈癌细胞系HeLa增殖、凋亡和自噬的影响.利用紫外-可见分光光度计检测珠子参总皂苷中皂苷的含量;分别运用CCK-8、DAPI染色法及流式细胞术检测珠子参总皂苷对HeLa细胞活力、细胞核形态变化、细胞周期与凋亡的影响;Western blot技术检测HeLa细胞中凋亡蛋白B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化的半胱氨酸蛋白酶蛋白-9(cleaved caspase-9)、活化的半胱氨酸蛋白酶蛋白-3(cleaved caspase-3),自噬相关蛋白苄氯素1(Beclin-1)和SQSTM1(p62),磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)和丝裂原活化蛋白激酶(MAPK)信号通路相关蛋白表达的影响.研究发现,珠子参总皂苷的得出率、皂苷质量分数分别为6.3％、78.3％;珠子参总皂苷可显著抑制HeLa细胞的增殖(P＜0.001),影响HeLa细胞核形态,阻滞HeLa细胞G0/G1期周期,诱导细胞凋亡,且呈现一定的浓度依赖性.进一步发现珠子参总皂苷可上调促凋亡蛋白Bax、cleaved caspase-9和cleaved caspase-3和自噬相关蛋白p62的表达(P＜0.05),下调抗凋亡蛋白Bcl-2和自噬相关蛋白Beclin-1的表达(P＜0.01),促进p-p38丝裂原活化蛋白激酶(p38 MAPK,简称 p38)/p38、p-c-Jun 氨基末端激酶(JNK)/JNK、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR 水平(P＜0.05),对p-ERK/ERK影响不显著.结果表明,珠子参总皂苷可能通过激活PI3K/Akt/mTOR、JNK和p38信号通路,抑制HeLa细胞自噬,促进其细胞凋亡,表明了珠子参可能具有抗宫颈癌的潜在作用.
Abstract
This paper investigated the effect of total saponins from Rhizoma Panacis Majoris on the proliferation,apoptosis,and au-tophagy of human cervical carcinoma HeLa cells.The saponin content was detected by ultraviolet-visible spectrophotometry.Cell coun-ting kit-8(CCK-8)assay,4,6-diamidino-2-phenylindole(DAPI)staining,and flow cytometry were used to detect the effects of total saponins of Panacis Majoris Rhizoma on cell viability,morphology,cell cycle and apoptosis of HeLa cells.Western blot was used to detect the expression of apoptosis-related proteins B cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved caspase-9,and cleaved caspase-3,autophagy-related proteins Beclin-1 and SQSTMl(p62),and the proteins related to the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)and mitogen-activated protein kinase(MAPK)signaling pathways.It was found that the yield and saponin content of total saponins from Rhizoma Panacis Majoris were 6.3％and 78.3％,re-spectively.Total saponins from Rhizoma Panacis Majoris could significantly inhibit the proliferation(P＜0.001),effect the nuclear morphology,block the G0/G1 cycle,and induce cell apoptosis in HeLa cells with a concentration-dependent manner.In addition,total saponins from Rhizoma Panacis Majoris up-regulated the expression of pro-apoptotic proteins Bax,cleaved caspase-9,and cleaved caspase-3,and autophagy-related protein p62(P＜0.05),while down-regulated the expression of anti-apoptotic protein Bcl-2 and auto-phagy-related protein Beclin-1(P＜0.01).Total saponins from Rhizoma Panacis Majoris could promote the expression of p-p38/p38,p-Jun N-terminal kinase(JNK)/JNK,p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR proteins in PI3K/Akt/mTOR and MAPK signa-ling pathways(P＜0.05).In contrast,the effect on p-ERK/ERK expression was not obvious.Therefore,total saponins from Rhizoma Panacis Majoris may inhibit autophagy and promote apoptosis of HeLa cells through the activation of the PI3K/Akt/mTOR,c-JNK,and p38 MAPK signaling pathways,which indicates that total saponins from Rhizoma Panacis Majoris may have a potential role in cervical cancer treatment.
基金项目
国家自然科学基金青年科学基金项目(81803946)
2023年度陕西中医药大学科技创新团队项目(2023-CXTD-04)
清华大学化学系生命有机磷化学及化学生物学教育部重点实验室开放课题()
NETL
NSTL
万方数据