吴茱萸碱抑制破骨细胞分化延缓骨丢失的研究

目的探讨吴茱萸碱对破骨细胞分化与骨吸收功能的调控及对骨质疏松症的治疗作用。 方法取小鼠原代骨髓来源巨噬细胞分别给予0、10、20、50、100、200 μmol/L吴茱萸碱处理，CCK8检测细胞活力；然后利用原代骨髓来源巨噬细胞给予小鼠重组可溶性核因子κB受体活化因子配体与集落刺激因子行破骨细胞分化诱导，分别给予20与50 μmol/L吴茱萸碱干预。抗酒石酸酸性磷酸酶（TRAP）染色检测破骨细胞形成能力，荧光定量PCR分析破骨细胞分化相关基因表达，免疫荧光检测F肌动蛋白（F-actin）形成，扫描电镜观察破骨细胞骨吸收能力。7月龄C57BL/6小鼠灌胃给予100与200 mg/kg吴茱萸碱，给药3个月后Micro-CT检测小鼠骨密度与骨质量。采用单因素方差分析和t检验进行统计学分析。 结果CCK8结果显示，与对照组相比，给予10、20、50、100 μmol/L吴茱萸碱处理后细胞活力无明显变化，差异无统计学意义（P > 0.05）；而给予200 μmol/L吴茱萸碱的细胞活力下降（100.64±0.18比47.54±5.58），差异具有统计学意义（P < 0.01）。与对照组相比，20 μmol/L吴茱萸碱的TRAP染色阳性细胞数（200.57±28.35）比（142.29±19.21）个]、Trap （1.00±0.13比0.55±0.16）、组织蛋白酶K（Ctsk） （1.01±0.17比0.59±0.11）mRNA水平、骨吸收面积比（1.00±0.15比0.79±0.19）均减少，差异有统计学意义（P < 0.05）。与对照组相比，50 μmol/L吴茱萸碱的TRAP阳性细胞数（200.57±28.35）比（112.71±12.18）个]、Trap （1.00±0.13比0.46±0.17）、Ctsk（1.01±0.17比0.49±0.12）、树突状细胞-特异性跨膜蛋白（DC- Stamp） （1.00±0.10比0.55±0.14）、c-Fos （1.01±0.10比0.58±0.14）、活化T细胞核因子c1 （Nfatc1） （1.00±0.10比0.59±0.14）、H+转运ATP酶v0亚基d2 （Atp6v0d2）的mRNA表达（1.00±0.10比0.59±0.18）、F-actin数量（165.00± 18.50）比（98.33±21.15）个]和骨吸收面积比（1.00±0.15比0.62±0.10）均降低，差异有统计学意义（P < 0.05）。Micro-CT结果显示，与生理盐水组相比，100 mg/kg吴茱萸碱组小鼠骨密度有一定升高（0.19±0.03）比（0.21±0.01）g/cm³]，但差异无统计学意义（P > 0.05）；与生理盐水组相比，200 mg/kg吴茱萸碱组小鼠胫骨的骨密度（0.19±0.03）比（0.23±0.01）g/cm³]、骨体积比（9.79±1.39）﹪比（11.62±1.18）﹪]、骨小梁数量（2.43±0.29）比（3.08±0.43）/mm]上升，骨小梁分离度（0.44±0.06）比（0.27±0.05）mm]下降，差异具有统计学意义（P < 0.05）。 结论吴茱萸碱通过抑制破骨细胞分化与骨吸收功能延缓小鼠骨量丢失。

Evodiamine regulates osteoclast differentiation and inhibits bone loss

ObjectiveTo investigate the regulation of evodiamine on osteoclast (OC) differentiation and bone resorption, and its therapeutic effect on osteoporosis. MethodsThe mouse primary bone marrow-derived macrophages were treated with 0, 10, 20, 50, 100, 200 μmol/L evodiamine respectively, and the cell viability was detected by CCK8. Primary bone marrow-derived macrophages were used to induce osteoclast differentiation in mice by administering recombinant soluble nuclear factor-κB receptor activator factor and colony-stimulating factor, and were intervened by 20 and 50 μmol/L evodiamine, respectively. The formation ability of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining, the expression of osteoclast differentiation-related genes was analyzed by fluorescence quantitative PCR, the formation of F-actin was detected by immunofluorescence, and the bone resorption ability of osteoclasts was observed by scanning electron microscope. 7-month-old C57BL/6 mice were orally administrated with evodiamine daily at 100 or 200 mg/kg for 3 months. Bone density and bone quality of the mice were measured by Micro-CT. One-way ANOVA and t-test was used for statistical analysis. ResultsCompared with the control group, CCK8 results showed that there was no significant change in cell viability in the 10, 20, 50, and 100 μmol/L evodiamine-treated groups, and the difference was not statistically significant (P > 0.05) . 200 μmol/L evodiamine inhibited the cell viability (100.64±0.18 vs 47.54±5.58) , and the difference was statistically significant (P < 0.01) . 20 μmol/L evodiamine decreased the number of TRAP staining positive cells (200.57±28.35 vs 142.29±19.21) , the mRNA levels of Trap (1.00±0.13 vs 0.55±0.16) and cathepsin K (Ctsk) (1.01±0.17 vs 0.59±0.11) , the area of bone resorption (1.00±0.15 vs 0.79±0.19) , compared with the control group, and the difference was statistically significant (P < 0.05) . 50 μmol/L evodiamine reduced the number of TRAP staining positive cells (200.57±28.35 vs 112.71±12.18) , the mRNA levels of Trap (1.00±0.13 vs 0.46±0.17) , Ctsk (1.01±0.17 vs 0.49±0.12) , DC-Stamp (1.00±0.10 vs 0.55±0.14) , c-Fos (1.01±0.10 vs 0.58±0.14) , Nfatc1 (1.00±0.10 vs 0.59±0.14) and Atp6v0d2 (1.00±0.10 vs 0.59±0.18) , the number of F-actin (165.00±18.50 vs 98.33±21.15) and bone resorption area was also decreased (1.00±0.15 vs 0.62±0.10) , and the difference was statistically significant (P < 0.05) . Micro-CT results showed the bone density of the mice in the 100 mg/kg evodiamine group were somewhat elevated compared with control group (0.19±0.03) vs (0.21±0.01) g/cm³], but the difference was not statistically significant (P > 0.05) .The bone density (0.19±0.03) vs (0.23±0.01) g/cm³], bone volume ratio (9.79±1.39) ﹪ vs (11.62±1.18) ﹪], and the number of trabecular bones (2.43±0.29) vs (3.08±0.43) mm] in the evodiamine 200 mg/kg group were increased, and the separation of trabecular bones (0.44±0.06) vs (0.27±0.05) mm] was decreased, compared with the control group, and the difference was statistically significant (P < 0.05) . ConclusionEvodiamine inhibited bone loss by inhibiting osteoclast differentiation and bone resorption.