精氨酸甲基转移酶抑制剂TP-064调控破骨细胞研究  

Study of arginine methyltransferase inhibitor TP-064 modulates osteoclasts

作　　者：苏珮茹 罗香雅 李舸 钟宣丽 曾春平 杨明理 宋仁生 周琳 SU Peiru;LUO Xiangya;LI Ge;ZHONG Xuanli;ZENG Chunping;YANG Mingli;SONG Rensheng;ZHOU Lin(Department of Endocrinology,the Fifth Affiliated Hospital of Guangzhou Medical University,Key Laboratory of Biological Targeting Diagnosis,Therapy and Rehabilitation of Guangdong Higher Education Institutes,Guangzhou 510700,China;Department of Endocrinology,the Affiliated Shunde Hospital of Jinan University,Foshan 528305,China;Department of Medical Genetics,Zunyi Medical University,Zunyi 563000,China;Department of Cardiovascular,the Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou 510700,China)

机构地区：[1]广州医科大学附属第五医院内分泌科,广东高校生物靶向诊治与康复重点实验室,广东广州510700 [2]暨南大学附属顺德医院内分泌科,广东佛山528305 [3]遵义医科大学医学遗传学教研室,贵州遵义563000 [4]广州医科大学附属第五医院心血管内科,广东广州510700

出　　处：《中国骨质疏松杂志》2025年第2期195-203,共9页Chinese Journal of Osteoporosis

基　　金：广东省医学科学技术研究基金(A2024227);广东省基础与应用基础研究基金(2019A1515110723);暨南大学医学联合基金自由申请项目(MF220206);遵义市科技计划项目(遵市科合HZ字2020-36号);遵义医科大学学术新苗培养及创新探索专项项目(黔科合平台人才[2018]5772-043);广东高校生物靶向诊治与康复重点实验室项目(2021KSYS009)。

摘　　要：目的探讨精氨酸甲基转移酶抑制剂TP-064对绝经后骨质疏松症等溶骨性疾病的调控作用及机制。方法(1)细胞实验:CCK8法检测TP-064对RAW 264.7细胞的增殖活性影响,TRAP染色和鬼笔环肽染色法分别检测TP-064对破骨细胞分化及F-肌动蛋白环形成的影响,利用DCFH-DA法检测细胞内活性氧的水平,荧光素酶报告基因实验检测NF-κB的荧光素酶活性,Western blot检测TP-064对NF-κB和PI3K/AKT信号通路相关蛋白的影响。(2)动物实验:10周龄雌性的C57BL/6小鼠分成假手术组、去卵巢组和去卵巢+TP-064组,构建去卵巢骨质疏松症小鼠模型,按实验分组腹腔注射10 mg/kg TP-064或生理盐水。8周后取材,Micro-CT扫描并分析各组骨微结构参数变化,H&E及TRAP染色检测骨组织的形态及破骨细胞数量的变化。结果(1)TP-064能抑制破骨细胞分化及F-肌动蛋白环形成,且对RAW 264.7细胞增殖活性无抑制作用。TP-064能降低NF-κB的荧光素酶活性,抑制细胞内活性氧的产生,并且抑制IκB-α蛋白降解及AKT、PI3K蛋白的磷酸化。(2)与去卵巢组相比,TP-064治疗后可改善骨密度及骨微结构参数BV/TV及Tb.N的变化,并且减少股骨组织内破骨细胞的数量。结论TP-064通过抑制ROS产生和抑制PI3K/AKT、NF-κB通路来抑制破骨细胞分化,减少卵巢切除后引起的骨量丢失,从而防治绝经后骨质疏松症等溶骨性疾病。Objective To investigate the regulatory effect and mechanism of arginase methyltransferase inhibitor TP-064 on osteolytic diseases,particularly postmenopausal osteoporosis.Methods(1)Cell experiments:CCK8 assay was used to assess the impact of TP-064 on the cell viability of RAW 264.7 cells.TRAP staining and phalloidin staining were employed to assess the effect of TP-064 on osteoclast differentiation and the formation of the F-actin ring,respectively.The level of intracellular reactive oxygen species in osteoclasts was valued by using the fluorescent probe DCFH-DA.The effect of TP-064 on NF-κB transcriptional activity was measured using the luciferase reporter gene.The action of TP-064 on proteins related to the NF-κB and PI3K/AKT pathways was evaluated with Western blotting.(2)Animal experiments:Ten-week-old female C57BL/6 mice were randomly allocated into sham-operated group,ovariectomy(OVX)group,and ovariectomy(OVX)+TP-064 group.An ovariectomized mouse model of osteoporosis was established.Mice were intraperitoneally injected with 10 mg/kg TP-064 or saline according to the experimental grouping.After 8 weeks,micro-CT scanning was used to analyze bone microstructural parameter changes in mice of each group.Changes in bone tissue morphology and osteoclast numbers were observed using H&E and TRAP staining.Results(1)TP-064 suppressed the differentiation of osteoclasts and the generation of the F-actin ring without suppressing the cell activity of RAW 264.7 cells.TP-064 significantly reduced the luciferase activity of NF-κB and inhibited the degradation of IκB-αprotein.Also,TP-064 reduced the phosphorylation of AKT and PI3K proteins in the PI3K/AKT signaling pathway.Moreover,TP-064 inhibited the production of reactive oxygen species in osteoclasts.(2)Compared to the OVX group,the treatment of TP-064 improved bone mineral density and microstructural parameters,such as BV/TV and Tb.N.Besides,TP-064 decreased the number of osteoclasts in the femoral tissue.Conclusion TP-064 may inhibit osteoclast differentiation

关 键 词：TP-064 破骨细胞 RANKL NF-κB 绝经后骨质疏松症 

分 类 号：R589.5[医药卫生—内分泌]