五劳七损方调控Wnt/β-catenin信号通路对AS成纤维细胞增殖及成骨分化的影响  

Effect of Wulao Qisun Prescription on Proliferation and Osteogenic Differentiation of AS Fibroblasts by Regulating Wnt/β-catenin Signaling Pathway

作　　者：杨娟娟 陈平[2] 王海东[2] 王振东 李浩林 张智敏 杨玉萍 程伟刚 苏瑾 宋静静 芦栋生 YANG Juanjuan;CHEN Ping;WANG Haidong;WANG Zhendong;LI Haolin;ZHANG Zhimin;YANG Yuping;CHENG Weigang;SU Jin;SONG Jingjing;LU Dongsheng(Clinical College of Chinese Medicine,Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Provincial Hospital of Traditional Chinese Medicine,Lanzhou 730050,China)

机构地区：[1]甘肃中医药大学中医临床学院,兰州730000 [2]甘肃省中医院,兰州730050

出　　处：《中国实验方剂学杂志》2025年第2期67-73,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：甘肃省科技计划项目(23JRRA1535);国家中医药管理局高水平中医药重点学科建设项目(国中医药人教函[2022]226号)。

摘　　要：目的:探讨五劳七损方对强直性脊柱炎(AS)病理性新骨形成的作用及可能机制。方法:将分离的AS患者髋关节滑膜成纤维细胞用显微镜观察细胞形态;并用免疫荧光染色法鉴定;将分离的AS成纤维细胞分为5组[空白组、低含药血清组(2.5%)、中含药血清组(5%)、高含药血清组(10%)和塞来昔布组(5%)],药物干预后,通过细胞增殖与活性检测(CCK-8)法检测观察AS成纤维细胞增殖情况,筛选最佳干预时间;碱性磷酸酶法测定检测碱性磷酸酶(ALP)活性;蛋白免疫印迹法(Western blot)检测骨钙素(OCN)、骨桥蛋白(OPN)和Runt相关转录因子2(Runx2)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测Wnt5a、β-连环蛋白(β-catenin)和Dickkopf-1(DKK-1)mRNA表达。结果:与空白组比较,五劳七损方各含药血清组及塞来昔布组均可抑制AS成纤维细胞增殖并降低ALP的表达(P<0.01)。与空白组比较,五劳七损方低含药血清组可下调β-catenin mRNA的表达(P<0.05),五劳七损方中、高含药血清组及塞来昔布组可下调Wnt5a和β-catenin mRNA的表达(P<0.05,P<0.01),其中,塞来昔布组下调更明显(P<0.01);五劳七损方高含药血清组和塞来昔布组可显著上调DKK-1 mRNA的表达(P<0.01)。与空白组比较,五劳七损方低含药血清组可抑制OPN、Runx2蛋白的表达(P<0.05,P<0.01),五劳七损方中、高含药血清组和塞来昔布组均可抑制OCN、OPN、Runx2蛋白的表达(P<0.05,P<0.01)。结论:五劳七损方可以抑制AS成纤维细胞增殖及成骨分化,以达到延缓AS病理新骨形成的目的,其机制可能与五劳七损方调控Wnt/β-catenin相关基因的表达,进一步抑制下游靶基因的转录相关。Objective:To investigate the effect and underlying mechanism of the Wulao Qisun prescription on pathological new bone formation in ankylosing spondylitis(AS).Methods:Synovial fibroblasts were isolated from the hip joints of AS patients and observed under a microscope to assess cell morphology.The cells were identified using immunofluorescence staining.The isolated AS fibroblasts were divided into blank group,low drug-containing serum group,medium drug-containing serum group,high drug-containing serum group,and positive drug group.After drug intervention,cell proliferation was measured using the cell counting kit-8(CCK-8)assay to observe fibroblast growth and determine the optimal intervention time.Alkaline phosphatase(ALP)activity was measured using the alkaline phosphatase assay.Protein expression of osteocalcin(OCN),osteopontin(OPN),and runt-related transcription factor 2(Runx2)was detected by Western blot.The mRNA expression levels of Wnt5a,β-catenin,and Dickkopf-1(DKK-1)were measured by real-time quantitative polymerase chain reaction(Real-time PCR).Results:Compared with the blank group,each drug-containing serum group of Wulao Qisun prescription and the positive drug group inhibited the proliferation of AS fibroblasts and reduced ALP expression(P<0.01).Compared with the blank group,the low drug-containing serum group of Wulao Qisun prescription downregulatedβ-catenin mRNA expression(P<0.05).The medium and high drug-containing serum groups and the positive drug group significantly downregulated Wnt5a andβ-catenin mRNA expression(P<0.05,P<0.01),with the positive drug group showing the most pronounced effect(P<0.01).The high drug-containing serum group and the positive drug group significantly upregulated DKK-1 mRNA expression(P<0.01).Compared with the blank group,the low drug-containing serum group of Wulao Qisun prescription inhibited the expression of OPN and Runx2 proteins(P<0.05,P<0.01),while the medium and high drug-containing serum groups and the positive drug group inhibited the expression of OCN,OPN

关 键 词：五劳七损方 强直性脊柱炎 Wnt/β-连环蛋白(β-catenin)信号通路 成纤维细胞 成骨分化 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R274