大网膜脂肪干细胞旁分泌HGF与加味补阳还五汤抑制腹膜间皮细胞EMT相关性的体外研究  

In Vitro Study on Correlation between HGF Paracrine Secretion by ADSCs in Greater Omentum and Inhibition of EMT in Peritoneal Mesothelial Cells by Supplemented Buyang Huanwu Decoction(加味补阳还五汤)

作　　者：郑敏麟[1] 王亚楠 范文江 詹倩倩 陈锋斌[3] ZHENG Minlin;WANG Yanan;FAN Wenjiang;ZHAN Qianqian;CHEN Fengbin(College of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China;Research Institute of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China;The First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,Fujian,China)

机构地区：[1]福建中医药大学中西医结合学院,福建福州350122 [2]福建中医药大学中西医结合研究院,福建福州350122 [3]福建医科大学附属第一医院,福建福州350005

出　　处：《中华中医药学刊》2025年第1期11-18,I0006,I0007,共10页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(82274516);福建省自然科学基金面上项目(2022J01842);福建中医药大学校管科研课题-重点项目(自然科学)(X2021003-重点)。

摘　　要：目的通过体外研究,探讨加味补阳还五汤抑制的腹膜间皮细胞(PMC)上皮-间充质转化(EMT)防治术后腹腔粘连(PAA)的作用,是否主要通过促进大网膜脂肪干细胞(ADSCs)旁分泌肝细胞生长因子(HGF)。方法实验1明确加味补阳还五汤抑制PMC的EMT作用,是该方对PMC的直接作用,还是通过调控大网膜中的ADSCs旁分泌的间接作用:使用加味补阳还五汤与加味补阳还五汤培养ADSCs后制取其条件培养基(以下称“中药培养基”)分别干预EMT模型的PMC,Western Blot法检测EMT相关的蛋白E-钙黏蛋白(E-Cad)、α-平滑肌肌动蛋白(α-SMA)的表达水平。实验2明确加味补阳还五汤抑制PMC的EMT作用,是否与大网膜中的ADSCs旁分泌HGF相关:用低表达HGF的ADSCs条件培养基(以下称“低HGF培养基”)、正常ADSCs条件培养基(以下称“普通培养基”)、中药培养基等3种培养基干预PMC,并检测以下指标:(1)用ELISA法检测在干预PMC之前和之后,3种培养基中HGF的含量。(2)Western Blot法、qPCR法、免疫荧光(IF)法检测干预后各组PMC的EMT相关蛋白表达水平,划痕实验检测各组PMC的迁移能力。结果实验1:加味补阳还五汤与中药培养基对EMT相关指标的影响。与模型组相比,加味补阳还五汤组上调E-Cad的蛋白表达(P<0.05),下调α-SMA的蛋白表达(P<0.05);中药培养基组显著上调了E-Cad的蛋白表达(P<0.01),显著下调α-SMA的蛋白表达(P<0.01)。与加味补阳还五汤组相比,中药培养基组上调E-Cad的蛋白表达(P<0.05),显著下调α-SMA的蛋白表达(P<0.01)。实验2:(1)作用于PMC前后,3种不同的培养基中HGF含量的比较:作用于PMC前,相比于普通培养基,低HGF培养基中含量显著降低(P<0.01),中药培养基中含量显著增加(P<0.01);作用于PMC后,各培养基组含量均显著性下降。(2)EMT相关蛋白表达情况:综合Western Blot、qPCR、IF结果,与模型组相比,各培养基组均可升高E-Cad表达(P<0.05或P<0.01),降低α-SMA、VIObjective Through in vitro studies,it explored whether Supplemented Buyang Huanwu Decoction(加味补阳还五汤,SBHD)could promote adipose stem cells(ADSCs)in greater omentum to paracrine hepatocyte growth factor(HGF)and whether this effect could inhibit the epithelial-mesenchymal transition(EMT)of peritoneal mesothelial cells(PMC)in order to initially clarify the pharmacological mechanism and target of this prescription in preventing and treating postoperative abdominal adhesion(PAA).Methods Experiment 1 clarified whether the inhibitory effect of SBHD on the EMT of PMC was a direct effect of the formula on PMC or an indirect effect by regulating the paracrine secretion of ADSCs in the greater omentum.After culturing ADSCs with SBHD,conditional culture media(hereinafter referred to as“Chinese medicine culture media”)were prepared to intervene in the PMC of EMT models,and the expression levels of EMT related proteins E-cadherin(E-Cad)andα-smooth muscle actin(α-SMA)were detected by Western Blot.Experiment 2 clarified whether the inhibitory effect of SBHD on EMT of PMC was related to the secretion of HGF by ADSCs in the greater omentum.The intervention of PMC used three types of media:low HGF expressing ADSCs conditioned medium(hereinafter referred to as“low HGF medium”),normal ADSCs conditioned medium(hereinafter referred to as“normal medium”)and traditional Chinese medicine medium and the following indicators were detected.(1)ELISA was used to detect the content of HGF in the three media before and after intervention in PMC.(2)Western Blot,qPCR and IF methods were used to detect the expression levels of EMT related proteins in each group of PMCs after intervention,and scratch assay was used to detect the migration ability of PMCs in each group.Results Experiment 1:Effect of SBHD and traditional Chinese medicine culture medium on EMT related indicators:Compared with the model group,the SBHD group up-regulated the protein expression of E-Cad(P<0.05)and down-regulated the protein expression ofα-SMA(P

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：R289.5[医药卫生—方剂学]