半夏泻心汤含药肠吸收液对胃癌微环境中PMN-MDSCs迁移侵袭的影响  

Effect of Banxia Xiexintang-containing Intestinal Absorption Solution on Migration and Invasion of PMN-MDSCs in Gastric Cancer Microenvironment

作　　者：魏晶晶 朱中博 刘喜平[1] 李沛清[1] 陈启明[2] 戴丽蓉 施丽娟 段海婧[1] 王庆苗[1] WEI Jingjing;ZHU Zhongbo;LIU Xiping;LI Peiqing;CHEN Qiming;DAI Lirong;SHI Lijuan;DUAN Haijing;WANG Qingmiao(Gansu Engineering Laboratory for New Products of Traditional Chinese Medicine(TCM),Gansu Key Laboratory of TCM Excavation and Innovative Transformation,Gansu University of Chinese Medicine,Lanzhou 730000,China;The First Hospital of Lanzhou University,Lanzhou 730013,China)

机构地区：[1]甘肃中医药大学甘肃省中药新产品创制工程实验室,甘肃省中医方药挖掘与创新转化重点实验室,兰州730000 [2]兰州大学第一医院,兰州730013

出　　处：《中国实验方剂学杂志》2023年第10期48-57,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81860813,81860782);甘肃省高等学校创新基金项目(2020B-007);甘肃省委组织部陇原青年创新创业人才项目(2020RCXM183);甘肃省中医方药挖掘与创新转化重点实验室开放基金项目(ZYFY-2020-003)。

摘　　要：目的:观察半夏泻心汤含药肠吸收液对胃癌微环境中多形核髓系来源抑制细胞(PMN-MDSCs)迁移侵袭的影响。方法:制备含生药量0.63 g·mL^(-1)的半夏泻心汤含药肠吸收液,以Transwell小室将胃癌细胞与PMN-MDSCs非接触共培养建立胃癌微环境模型,采用细胞增殖与活性检测-8(CCK-8)法筛选半夏泻心汤含药肠吸收液对PMN-MDSCs的最佳干预浓度及时间,用于后续实验,分为空白组、模型组、FAK抑制剂组及半夏泻心汤组(26%、18%、10%半夏泻心汤含药肠吸收液),采用细胞划痕实验和Transwell实验检测PMN-MDSCs的迁移和侵袭能力,酶联免疫吸附测定法(ELISA)检测肿瘤微环境中血管内皮细胞生长因子(VEGF)、基质金属蛋白酶-9(MMP-9)细胞因子表达,蛋白免疫印迹法(Western blot)检测PMN-MDSCs通路相关蛋白局部黏着斑激酶(FAK)、磷酸化(p)-FAK、蛋白酪氨酸激酶(Src)、磷酸化(p)-Src蛋白表达水平。结果:与24 h比较,作用48 h 5%、50%、75%、100%半夏泻心汤含药肠吸收液对PMN-MDSCs的抑制率均有升高(P<0.05,P<0.01),与作用48 h比较,培养72 h 50%半夏泻心汤含药肠吸收液对PMN-MDSCs的抑制率低于48 h(P<0.01),5%、100%半夏泻心汤含药肠吸收液略高于48 h(P<0.05,P<0.01),其余浓度抑制率差异无统计学意义,且48 h半数抑制浓度(IC_(50))为18.09%,说明18%半夏泻心汤含药肠吸收液在48 h为最佳干预浓度及时间;与空白组比较,模型组PMN-MDSCs迁移距离显著增大(P<0.01),迁移及侵袭数量显著增多(P<0.01);与模型组比较,FAK抑制剂组、半夏泻心汤含药肠吸收液组PMN-MDSCs的迁移距离均显著减小(P<0.01),迁移及侵袭数量显著减少(P<0.01),以26%含药肠吸收液组最为显著(P<0.01);与空白组比较,模型组PMNMDSCs通路相关蛋白FAK、p-FAK、Src、p-Src表达显著升高(P<0.01),VEGF、MMP-9细胞因子表达均显著上升(P<0.01)。与模型组比较,FAK抑制剂组、半夏泻心汤含药肠吸收液组(26%、18%、10%)PMN-MDSCObjective:To observe the effect of Banxia Xiexintang containing intestinal absorption solution(BXCIAS)on migration and invasion of polymorphonuclear myeloid-derived suppressor cells(PMN-MDSCs)in gastric cancer microenvironment.Method:The complex solution(containing 0.63 g·mL^(-1) crude drug)was prepared.Gastric cancer cells were subjected to non-contact co-culture with PMN-MDSCs in Transwell chamber to create gastric cancer microenvironment.Cell counting kit-8(CCK-8)assay was used to screen the optimal intervention concentration and time of BXCIAS on PMN-MDSCs for subsequent experiment.The blank group,model group,FAK inhibitor group,and BXCIAS groups(26%,18%,and 10%)were designed.Scratch assay and Transwell assay were employed to detect the migration and invasion ability of PMN-MDSCs,and enzyme-linked immunosorbent assay(ELISA)to measure the expression of vascular endothelial cell growth factor(VEGF)and matrix metalloproteinase-9(MMP-9)in tumor microenvironment.The expression levels of PMN-MDSCs pathway-related proteins FAK,phosphorylated(p)-FAK,protein tyrosine kinase(Src),and p-Src were detected by Western blot.Result:The inhibition rates of PMN-MDSCs by 5%,50%,75%,and 100%BXCIAS at 48 h were higher than those at 24 h(P<0.05,P<0.01).The inhibition rate of PMN-MDSCs by 50%BXCIAS at 72 h was lower than that at 48 h(P<0.01),and the inhibition rates by 5%and 100%BXCIAS at 72 h were higher than those at 48 h(P<0.05,P<0.01).There was no significant difference in the inhibition rate by other concentration levels at 48 h.The half-maximal inhibitory concentration(IC_(50))at 48 h was 18.09%,indicating that 18%BXCIAS and 48 h were the optimal concentration and time,respectively.The migration distance of PMN-MDSCs was large(P<0.01),and the number of migrating and invading cells increased(P<0.01)in the mode group compared with those in the blank group.Compared with model group,FAK inhibitor and BXCIAS at different concentration decreased the migration distance of PMN-MDSCs(P<0.01),and the number of migrating and invading c

关 键 词：胃癌微环境 髓源性抑制细胞 半夏泻心汤 黏着斑激酶(FAK)信号通路 

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