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| 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 |
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| DOI：10.3969/j.issn.1006-7108.2025.02.007 |
| 中文关键词: TP-064 破骨细胞 RANKL NF-κB 绝经后骨质疏松症 |
| 英文关键词:TP-064 osteoclasts RANKL NF-κB postmenopausal osteoporosis |
| 基金项目:广东省基础与应用基础研究基金项目（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（2021KSYS009） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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-α protein. 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 and attenuate bone loss caused by ovariectomy through suppressing the production of ROS and inhibiting the RANKL-driven NF-κB and PI3K/AKT signaling pathways. TP-064 may potentially prevent and treat osteolytic diseases such as postmenopausal osteoporosis. |
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