龟甲提取物干扰NF-κB-NLRP3炎症体正反馈回路改善老年性骨质疏松的研究  

Effective component of Plastrum testudinis extract alleviates senile osteoporosis by disrupting positive feedback circuit of NF-κB-NLRP3 inflammasome

作　　者：陈弘林 余富勇 尚奇[1,2] 张鹏[1,2] 朱广晔 陈桂锋 刘宇[1,2] 沈耿杨[2,3] 任辉[2,3] 江晓兵[2,3] CHEN Hong-lin;YU Fu-yong;SHANG Qi;ZHANG Peng;ZHU Guang-ye;CHEN Gui-feng;LIU Yu;SHEN Geng-yang;REN Hui;JIANG Xiao-bing(Guangzhou University of Chinese Medcine,Guangzhou 510405,China;Lingnan Medical Research Center ofGuangzhou University of Chinese Medicine,Guangzhou 510405,China;The First Affiliated Hospital ofGuangzhou University of Chinese Medicine,Guangzhou 510405,China)

机构地区：[1]广州中医药大学,广州510405 [2]广州中医药大学岭南医学研究中心,广州510405 [3]广州中医药大学第一附属医院,广州510405

出　　处：《中华中医药杂志》2023年第5期2437-2443,共7页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.82205230,No.82274615,No.82205137)。

摘　　要：目的:探讨龟甲提取物对NF-κB-NLRP3炎症体正反馈回路的调节作用及改善老年性骨质疏松的起效机制。方法:超高效液相联用质谱技术完成龟甲水提液中有效成分的鉴定。体外实验中,TRAP染色法检测龟甲提取物对骨髓源性巨噬细胞破骨分化的影响,使用NF-κB信号通路激动剂与抑制剂验证龟甲提取物对NF-κB信号通路的影响,使用NLRP3炎症体激动剂与抑制剂验证龟甲提取物对NLRP3炎症体活性的影响,RT-qPCR检测Nfatc1、Ctsk基因的表达量,Western Blot法检测p50、p-p50、NLRP3、IL-1β、pro-IL-1β的表达量。体内实验中,将6只3月龄C57BL/6小鼠作为对照组,12只22月龄自然衰老小鼠随机分为老年性骨质疏松模型组、龟甲提取物干预组,每组6只,连续干预8周。结果:筛选出龟甲水提液有效成分肉豆蔻酸。体外实验:与对照组比较,肉豆蔻酸显著抑制破骨分化(P<0.01),Nfatc1、Ctsk基因的表达量被显著抑制(P<0.05,P<0.01),p-p50、NLRP3、IL-1β、pro-IL-1β的表达量显著下调(P<0.01)。体内实验:与模型组比较,肉豆蔻酸有效提高骨体积分数(P<0.05),抑制骨髓腔中炎症体的表达量(P<0.01)。结论:龟甲提取物肉豆蔻酸改善老年性骨质疏松可能与干扰NF-κB-NLRP3炎症体正反馈回路,抑制破骨细胞形成相关。Objective:To investigate the effecst of the effective component of Plastrum testudinis extract(PTE)on the positive feedback circuit of NF-κB-NLRP3 inflammasome and the mechanism of ameliorating senile osteoporosis.Methods:UPLC-MS technology was used to identify the effective components in PTE.In vitro experiments,TRAP staining was used to detect the effects of effective component of PTE on osteoclast differentiation of bone marrow derived macrophages,NF-κB signaling pathway agonist and inhibitor were used to verify the effects of effective component of PTE on NF-κB signaling pathway,and NLRP3 inflammasome activity was used to verify the effects of effective component of PTE on NLRP3 inflammasome activity.RT-qPCR was used to detect the expression of Nfatc1 and Ctsk genes;proteins expression levels of p50,p-p50,NLRP3,IL-1βand pro-IL-1βwere detected by Western Blot.In vivo experiments,six 3-month-old C57BL/6 mice were divided into control group,and 12 naturally aging mice aged 22 months were randomly divided into the senile osteoporosis model group and the effective component of PTE intervention group,with 6 mice in each group.The intervention lasted for 8 weeks.Results:Myristic acid(MA)was the effective component of PTE.In vitro experiments,compared with the control group,myristic acid significantly inhibited osteoclast differentiation(P<0.01),the expression of Nfatc1 and Ctsk genes was significantly inhibited(P<0.05,P<0.01),and the expression of p-p50,NLRP3,IL-1βand pro-IL-1βwas significantly down-regulated(P<0.01).In vivo experiments:Compared with the model group,myristic acid effectively increased the bone volume fraction(P<0.05)and inhibited the expression of inflammasomes in the bone marrow cavity(P<0.01).Conclusion:MA,the effective component of PTE,ameliorates senile osteoporosis possibly by disrupting NF-κB-NLRP3 inflammasome positive feedback circuit and inhibiting osteoclast formation.

关 键 词：核因子-ΚB NLRP3炎症体 破骨分化 龟甲提取物 肉豆蔻酸 老年性骨质疏松 

分 类 号：R259[医药卫生—中西医结合]