尿石素A介导p38/MAPK通路抑制破骨细胞活性  

Urolithin A mediates p38/MAPK pathway to inhibit osteoclast activity

作　　者：黄浩然 凡一诺 卫杨文祥 江梦钰 方汉军 王海彬[1] 陈镇秋[1] 刘予豪 周驰[1] Huang Haoran;Fan Yinuo;Wei-Yang Wenxiang;Jiang Mengyu;Fang Hanjun;Wang Haibin;Chen Zhenqiu;Liu Yuhao;Zhou Chi(First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378,Guangdong Province,China)

机构地区：[1]广州中医药大学第一附属医院,广东省广州市510405 [2]广州中医药大学第三附属医院,广东省广州市510378

出　　处：《中国组织工程研究》2024年第8期1149-1154,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省教育厅项目(2021KTSCX021),项目负责人:周驰;广东省自然科学基金项目(2021A1515011484),项目负责人:刘予豪;广东省中医药局项目(20221136),项目负责人:刘予豪;广东省中医药局项目(20231162),项目负责人:陈镇秋;广东省中医药局项目(20231122),项目负责人:方汉军;广州中医药大学“双一流”与高水平大学学科协同创新团队项目(2021xk46),项目负责人:陈镇秋。

摘　　要：背景:过度活跃的破骨细胞可破坏骨稳态,并在骨质疏松、脆性骨折、骨关节炎等相关性骨骼疾病的病理机制中起重要作用。研究证实,鞣花酸和鞣花单宁有抑制破骨细胞分化的潜能,尿石素A作为其天然代谢产物,具有抗氧化、抗炎、抗增殖及抗癌作用,但其对破骨细胞分化的影响及其潜在的分子机制尚不清楚。目的:探讨尿石素A对核因子κB受体活化因子配体诱导破骨细胞分化的影响及作用机制。方法:体外培养稳定生长的小鼠单核巨噬细胞白血病细胞(RAW264.7),使用细胞毒性MTS实验检测不同浓度尿石素A(0,0.1,0.5,1.5,2.5μmol/L)对RAW264.7细胞的毒性,筛选出安全作用浓度。再使用不同浓度的尿石素A干预核因子κB受体活化因子配体诱导的RAW264.7细胞体外分化过程,进行抗酒石酸酸性磷酸酶染色和肌动蛋白环及细胞核染色,观察尿石素A对破骨细胞形成和功能的影响。最后通过Western Blot及RT-qPCR实验,观察尿石素A干预后丝裂原活化蛋白激酶信号通路中上下游基因和蛋白的表达情况。结果与结论:①尿石素A呈浓度依赖性地抑制体外破骨细胞分化及肌动蛋白环形成,2.5μmol/L的抑制作用最强;②尿石素A可抑制与破骨形成及骨吸收相关的Nfatc1、Ctsk、Mmp9、Atp6v0d2基因的表达及Nfatc1、Ctsk蛋白的合成;③尿石素A可通过抑制p38蛋白的磷酸化,抑制丝裂原活化蛋白激酶信号通路传导,从而抑制破骨细胞的活性。BACKGROUND:Overactive osteoclasts disrupt bone homeostasis and play a bad role in the pathological mechanisms of related skeletal diseases,such as osteoporosis,fragility fractures,and osteoarthritis.Studies have confirmed that ellagic acid and ellagtannin have the potential to inhibit osteoclast differentiation.As their natural metabolites,urolithin A has antioxidant,anti-inflammatory,anti-proliferative and anti-cancer effects,but its effect on osteoclast differentiation and its underlying molecular mechanisms remain unclear.OBJECTIVE:To explore the effect of urolithin A on osteoclast differentiation induced by receptor activator for nuclear factor-κB ligand and its mechanism.METHODS:Mouse mononuclear macrophage leukemia cells(RAW264.7)that grew stably were cultured in vitro.Toxicity of urolithin A(0,0.1,0.5,1.5,2.5μmol/L)to RAW264.7 cells were detected by cytotoxic MTS assay to screen out the safe concentration.Different concentrations of urolithin A were used again to intervene with receptor activator for nuclear factor-κB ligand-induced differentiation of RAW264.7 cells in vitro.Then,tartrate-resistant acid phosphatase staining and F-actin ring and nucleus staining were performed to observe its effect on the formation and function of osteoclasts.Finally,the expressions of urolithin A on upstream and downstream genes and proteins in the MAPK signaling pathway were observed by western blot and RT-qPCR assays.RESULTS AND CONCLUSION:Urolithin A inhibited osteoclast differentiation and F-actin ring formation in a concentration-dependent manner and 2.5μmol/L had the strongest inhibitory effect.Urolithin A inhibited the mRNA expression of Nfatc1,Ctsk,Mmp9 and Atp6v0d2 and the protein synthesis of Nfatc1 and Ctsk,related to osteoclast formation and bone resorption.Urolithin A inhibited the activity of osteoclasts by downregulating the phosphorylation of p38 protein to inhibit the mitogen-activated protein kinase signaling pathway.

关 键 词：尿石素A 破骨细胞 MAPK P38 RANKL 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R34