芒柄花素抑制脂多糖诱导髓核间充质干细胞的炎症反应  

Formononetin inhibits lipopolysaccharide-induced inflammation in nucleus pulposus mesenchymal stem cells

作　　者：于庆贺 蔡子鸣 田禾 李翩 阮烨 梁金柱 林淑惠 林文平 Yu Qinghe;Cai Ziming;Tian He;Li Pian;Ruan Ye;Liang Jinzhu;Lin Shuhui;Lin Wenping(Department of Spine Surgery,Shenzhen Pingle Orthopedic Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518118,Guangdong Province,China;Shenzhen Pingle Orthopedic Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518118,Guangdong Province,China;Department of Gynecology,Shenzhen Pingle Orthopedic Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518118,Guangdong Province,China;Third School of Clinical Medicine,Guangzhou University of Chinese Medicine.Guangzhou 510006,Guangdong Province,China)

机构地区：[1]广州中医药大学附属深圳平乐骨伤科医院脊柱外科,广东省深圳市518118 [2]广州中医药大学附属深圳平乐骨伤科医院,广东省深圳市518118 [3]广州中医药大学附属深圳平乐骨伤科医院妇科,广东省深圳市518118 [4]广州中医药大学第三临床医学院,广东省广州市510006

出　　处：《中国组织工程研究》2025年第25期5328-5334,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81771323),项目负责人:林文平;广东省自然科学基金面上项目(2021A1515010722),项目负责人:林文平;深圳市自然科学基金面上项目(JCYJ20190813112401660),项目负责人:林文平。

摘　　要：背景:芒柄花素具有抗炎、抗氧化能力,但其对髓核间充质干细胞是否有保护作用尚不清楚。目的:探讨芒柄花素对炎症微环境下髓核间充质干细胞的作用及机制。方法:①从SD大鼠尾椎椎间盘中提取髓核间充质干细胞,流式细胞术鉴定间充质干细胞表面标志;②CCK-8法检测脂多糖、芒柄花素对髓核间充质干细胞增殖活力的影响,以确定后续处理细胞应用的芒柄花素浓度;③在DMEM/F-12完全培养基中添加5μg/mL脂多糖模拟炎症微环境,然后用不同浓度芒柄花素处理髓核间充质干细胞24 h,通过Western blot、实时荧光定量PCR、免疫荧光检测炎症标志物表达,Western blot检测核因子κB信号通路蛋白水平。结果与结论:①贴壁培养的髓核间充质干细胞呈梭形,生长状态良好,流式细胞术结果显示间充质干细胞表面标志物CD29、CD44、CD90呈阳性,造血干细胞表面标志物CD34、CD45呈阴性。②12.5,25,50,100,200μmol/L芒柄花素处理24 h对髓核间充质干细胞无明显增殖抑制效果。与脂多糖组相比,12.5,25,50μmol/L芒柄花素处理24 h后髓核间充质干细胞活力显著提高,因此后续选用12.5,25,50μmol/L为芒柄花素低、中、高浓度处理髓核间充质干细胞。③与脂多糖组相比,芒柄花素低、中、高浓度组髓核间充质干细胞中基质金属蛋白酶3、基质金属蛋白酶13、肿瘤坏死因子α蛋白和mRNA表达均明显降低(P<0.05),且具有剂量依赖性。④与脂多糖组相比,芒柄花素低、中、高浓度组髓核间充质干细胞中磷酸化核因子κB和磷酸化核因子κB抑制蛋白的表达显著降低(P<0.05),且具有剂量依赖性。上述结果说明芒柄花素可能通过抑制核因子κB信号通路减轻脂多糖诱导的大鼠髓核间充质干细胞炎症。BACKGROUND:Formononetin demonstrates potent anti-inflammatory and antioxidant abilities.However,its protective effect on nucleus pulposus mesenchymal stem cells is not yet clear.OBJECTIVE:To investigate the effect and mechanism of formononetin on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment.METHODS:(1)Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral discs of SD rats,and flow cytometry was performed to identify the surface markers of mesenchymal stem cells.(2)The CCK-8 assay was employed to evaluate the impact of lipopolysaccharide and formononetin on the proliferation viability of nucleus pulposus mesenchymal stem cells,aiming to determine the appropriate concentration of formononetin for subsequent cell treatments.(3)An inflammatory microenvironment was simulated by adding 5μg/mL lipopolysaccharide to the DMEM/F-12 culture medium,and nucleus pulposus mesenchymal stem cells were treated with different concentrations of formononetin for 24 hours.Levels of inflammation markers were detected using western blot assay,real-time quantitative PCR,and immunofluorescence.Western blot assay was conducted to measure the protein levels of the nuclear factor kappa B signaling pathway.RESULTS AND CONCLUSION:(1)The nucleus pulposus mesenchymal stem cells cultured in adherent wall were shuttle-shaped with good growth status.The results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD29,CD44,and CD90,and the surface markers of hematopoietic stem cells were negative for CD34 and CD45.(2)The treatment with formononetin at 12.5,25,50,100,and 200μmol/L concentrations for 24 hours had no significant proliferation inhibitory effect on nucleus pulposus mesenchymal stem cells.Compared with the lipopolysaccharide group,the cell viability of nucleus pulposus mesenchymal stem cells treated with 12.5,25,and 50μmol/L formononetin for 24 hours was significantly increased,so formononetin at 12.5,25,and 50μmol/L concentratio

关 键 词：芒柄花素 髓核间充质干细胞 炎症 椎间盘退变 核因子ΚB 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R681.5