茯苓酸通过Smads调控MMP/TIMP平衡抑制肾癌细胞侵袭转移的机制  

Mechanism of Pachymic Acid in Inhibiting Invasion and Metastasis of Renal Carcinoma Cells via Regulating MMP/TIMP Balance by Smads

作　　者：罗园园 冯心怡 褚泽文 朱虹[1] 刘延庆[1] 王海波[1] LUO Yuanyuan;FENG Xinyi;CHU Zewen;ZHU Hong;LIU Yanqing;WANG Haibo(Medical College,Key Laboratory of Syndrome Differentiation and Treatment of Gastric Cancer of State Administration of Traditional Chinese Medicine,Yangzhou University-Yangzhou Cancer Research Institute,Yangzhou University,Yangzhou 225001,China)

机构地区：[1]扬州大学医学院,国家中医药管理局胃癌毒邪论治重点研究室,扬州大学-扬州市肿瘤研究所,江苏扬州225001

出　　处：《中国实验方剂学杂志》2023年第7期76-83,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82104946、81773944);江苏省自然科学基金项目(BK20210817);江苏省中医药科技发展计划项目(QN202008)。

摘　　要：目的:探讨茯苓中的三萜类化合物茯苓酸(PA),对肾癌侵袭转移的作用及机制。方法:采用细胞增殖与活性检测-8(CCK-8)试剂盒检测PA(0、20、40、80、160μmol·L^(-1))对细胞存活率的影响,并筛选PA浓度用于后续实验。采用克隆形成实验评估PA(0、20、40、80μmol·L^(-1))对细胞增殖的作用。细胞黏附实验用来观察PA(0、20、40、80μmol·L^(-1))对细胞黏附能力的效果。划痕实验和Transwell侵袭实验用来检测PA(0、20、40、80μmol·L^(-1))对细胞侵袭转移的影响。高内涵成像技术进一步动态观察和验证PA(0、20、40、80μmol·L^(-1))对细胞运动的抑制作用。PA(0、20、40、80μmol·L^(-1))对细胞中侵袭转移相关蛋白基质金属蛋白酶/基质金属蛋白酶组织抑制因子(MMP/TIMP)和相关通路关键蛋白Smads表达的影响通过蛋白免疫印迹法(Western blot)来检测。结果:CCK-8结果显示,与空白组比较,PA组细胞存活率显著降低(P<0.01),24 h时PA对ACHN细胞的半数抑制浓度(IC50)为70.42μmol·L^(-1)。克隆形成实验显示,与空白组比较,PA组细胞克隆群数目显著减少(P<0.01)。细胞黏附实验显示,与空白组比较,PA组细胞的黏附数量显著减少(P<0.01)。划痕实验表明,与空白组比较,PA组细胞的划痕愈合率降低(P<0.05,P<0.01)。Transwell侵袭实验显示,与空白组比较,PA组细胞的穿膜数量减少(P<0.01)。高内涵成像技术表明,与空白组比较,PA组细胞累计迁移距离更短(P<0.01)。Western blot结果表明,与空白组比较,PA组MMP-2、MMP-9蛋白的表达降低(P<0.01),TIMP-1蛋白的表达升高(P<0.01)。此外,与空白组比较,PA组Smad2、Smad3蛋白的表达降低(P<0.01)。结论:PA可抑制肾癌细胞的增殖、侵袭转移,其机制可能是通过Smad2/3调控MMP/TIMP的稳态平衡。Objective:To investigate the effect and mechanism of pachymic acid(PA)in Poria on the invasion and metastasis of renal carcinoma cells.Method:The effect of PA(0,20,40,80,160μmol·L^(-1))on cell viability was detected by cell counting kit-8(CCK-8),and the dose of PA was selected for subsequent experiments.The effect of PA(0,20,40,80μmol·L^(-1))on cell proliferation was evaluated by colony formation assay.The effect of PA(0,20,40,80μmol·L^(-1))on cell adhesion ability was observed by cell adhesion assay.The effect of PA(0,20,40,and 80μmol·L^(-1))on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay.The inhibitory effect of PA(0,20,40,80μmol·L^(-1))on cell motility was further observed and verified by high-content imaging technology.The effects of PA(0,20,40,80μmol·L^(-1))on the expression of matrix metalloproteinase(MMP)/tissue inhibitor of metalloproteinasas(TIMP)related to invasion and metastasis and Smads were detected by Western blot.Result:CCK-8 results showed that compared with the blank group,the PA groups showed decreased cell viability(P<0.01),with the half-maximal inhibitory concentration(IC50)of ACHN cells of 70.42μmol·L^(-1)at 24 h.Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group(P<0.01).Cell adhesion assay showed that compared with the blank group,the PA groups displayed reduced cell adhesion(P<0.01).Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group(P<0.05,P<0.01).Transwell invasion assay showed that compared with the blank group,the number of transmembrane cells in PA groups was reduced(P<0.01).High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group(P<0.01).The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased(P<0.01),and TIMP-1 protein expression increased(P

关 键 词：茯苓酸 肾癌 基质金属蛋白酶/基质金属蛋白酶组织抑制因子(MMP/TIMP) SMAD2/3 侵袭和迁移 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5