加味补阳还五汤调控大网膜脂肪干细胞旁分泌防治术后腹腔粘连的体外研究  

Modified Buyang Huanwu Decoction prevents and treats postoperative abdominal adhesion by regulating paracrine signaling by ADSCs in the omentum majus:An in vitro study

作　　者：郑敏麟[1] 王亚楠 范文江 詹倩倩 ZHENG Minlin;WANG Yanan;FAN Wenjiang;ZHAN Qianqian(College of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;Research Institute of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中西医结合学院,福州350122 [2]福建中医药大学中西医结合研究院

出　　处：《北京中医药大学学报》2024年第2期223-237,共15页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.82274516);福建省自然科学基金面上项目(No.2022J01842);福建中医药大学校管科研课题-重点项目(No.X2021003)。

摘　　要：目的明确加味补阳还五汤调控大网膜脂肪干细胞(ADSCs)旁分泌肝细胞生长因子(HGF)和转化生长因子⁃β1(TGF⁃β1)的作用机制,探讨该作用对腹膜间皮细胞TGF⁃β1/Sma和Mad相关蛋白(Smad)3信号通路及其下游上皮-间充质转化(EMT)、细胞外基质的影响,为临床防治术后腹腔粘连(PAA)提供依据。方法用0、5、10、20 g/L加味补阳还五汤培养ADSCs取得其条件培养基,酶联免疫吸附测定(ELISA)检测以上各浓度的培养基中HGF、TGF⁃β1含量变化。制作中药培养基(以上述最佳质量浓度的加味补阳还五汤培养的ADSCs培养基)、普通培养基(正常ADSCs培养基)、低HGF培养基(低表达HGF的ADSCs培养基),ELISA法检测不同培养基干预腹膜间皮细胞前后HGF的含量。3种培养基分别干预TGF⁃β1诱导的腹膜间皮细胞EMT模型,运用蛋白质印迹法(Western blotting)、免疫荧光(IF)、qPCR方法,检测腹膜间皮细胞的TGF⁃β1/Smad信号通路相关蛋白、EMT相关蛋白和纤维连接蛋白(FN)的表达水平。结果与加味补阳还五汤0 g/L相比,加味补阳还五汤5、10、20 g/L中HGF含量增加(P<0.01),TGF⁃β1含量降低(P<0.01)。与普通培养基相比,中药培养基中HGF含量增加(P<0.01),低HGF培养基中HGF含量降低(P<0.01)。综合Western blotting、qPCR、IF结果,与模型组相比,各组培养基均可降低Smad3磷酸化水平(P<0.05,P<0.01),下调锌指转录因子1表达(P<0.05,P<0.01),效果依次为中药培养基组>普通培养基组>低HGF培养基组;各组培养基均可升高E⁃cad表达(P<0.05,P<0.01),降低波形蛋白、FN表达(P<0.05,P<0.01);各组培养基均可抑制腹膜间皮细胞EMT,减少FN沉积,效果依次为中药培养基组>普通培养基组>低HGF培养基组。结论大网膜中ADSCs旁分泌HGF水平低下,可能是容易发生PAA的重要因素之一;加味补阳还五汤可能主要通过促进ADSCs旁分泌HGF,减少其旁分泌TGF⁃β1,从上游阻断TGF⁃β1/Smad3信号通路,从而抑�Objective We aimed to elucidate the effect of Modified Buyang Huanwu Decoction on Journal of Beijing University of Traditional Chinese Medicine adipose⁃derived stem cells(ADSCs)in the omentum majus by analyzing the hepatocyte growth factor(HGF)and transforming growth factor⁃β1(TGF⁃β1)levels,and explore its influence on the TGF⁃β1/Smad signaling pathway and epithelial⁃mesenchymal transition(EMT)of peritoneal mesothelial cells and the extracellular matrix,in order to provide a theoretical basis for the clinical prevention and treatment of postoperative abdominal adhesion(PAA).Methods ADSCs were cultured with different concentrations(0、5、10、20 g/L)of Modified Buyang Huanwu Decoction and their conditioned medium(CM)was obtained,HGF and TGF⁃β1 levels in the CM were determined by ELISA.The CM of ADSCs cultured with Modified Buyang Huanwu Decoction(TCM⁃ADSC⁃CM),CM of control ADSCs(ADSC⁃CM),and conditioned medium of ADSCs with low expression of HGF(Low⁃HGF⁃ADSC⁃CM)were prepared,and their HGF content was detected by ELISA before and after treatment of peritoneal mesothelial cells.TCM⁃ADSC⁃CM,ADSC⁃CM,and Low⁃HGF⁃ADSC⁃CM were used to treat an EMT model of peritoneal mesothelial cells.Western blotting,immunofluorescence(IF),qPCR,and other methods were used to determine the expression levels of proteins related to the TGF⁃β1/Smad signaling pathway and EMT and FN in peritoneal mesothelial cells.Results Compared with the normal group(with 0 g/L Modified Buyang Huanwu Decoction),after treatment with different concentrations of TCM(5,10,and 20 g/L),the content of HGF in TCM⁃ADSC⁃CM was significantly increased(P<0.01)and the content of TGF⁃β1 was significantly decreased(P<0.01).Compared with ADSC⁃CM,before treatment of peritoneal mesenchymal cells,the HGF content in TCM⁃ADSC⁃CM was significantly increased(P<0.01),and the HGF content in Low⁃HGF⁃ADSC⁃CM was significantly decreased(P<0.01).Based on the Western blotting,qPCR,and IF results,compared with the model grou

关 键 词：加味补阳还五汤 术后腹腔粘连 腹膜间皮细胞 脂肪间充质干细胞 肝细胞生长因子 上皮-间充质转化 

分 类 号：R285.5[医药卫生—中药学]