目的 探讨香草酸（vanillic acid,VA）抑制破骨细胞分化的作用机制及其对去卵巢（ovariectomy,OVX）小鼠骨质疏松症的影响。方法 体外实验：通过核因子κB受体活化因子配体（receptor activator for nuclear factor-κB ligand,RANKL）诱导的RAW264.7细胞建立体外破骨细胞分化模型,用VA进行干预,分为阴性对照（negative control,NC）,RANKL分化（differentiation,Diff）组、RANKL+VA50 μmol/L组、RANKL+VA100 μmol/L组、RANKL+VA200μmol/L组。细胞增殖检测试剂盒（cell counting kit-8,CCK8）检测细胞活力;利用抗酒石酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色和免疫印迹分析检测破骨细胞分化及其相关蛋白表达;采用2',7'-二氯荧光素二乙酸酯（2',7'-dichlorodihydrofluorescein diacetate,DCFH-DA）探针检测细胞活性氧（reactive oxygen species, ROS）水平;免疫印迹检测核因子E2相关因子-2（nuclear factor erythroid2-related factor 2,Nrf2）、Kelch 类环氧氯丙烷相关蛋白1（kelch like epichlorohydrin related protein 1,Keap1）、谷氨酸-半胱氨酸连接酶催化亚基（glutamate-cysteine ligase catalytic subunit,GCLC）、超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）蛋白的表达;采用ML385抑制Nrf2表达后检测破骨细胞分化相关蛋白及氧化应激相关蛋白表达。体内实验：通过OVX小鼠建立骨质疏松模型,分为假手术组（Sham）、OVX组、OVX+雌二醇（estradiol,E2）组、OVX+VA50 mg/kg组、OVX+100 mg/kg组,每组6只。小动物成像仪显微CT（micro computed tomography,Micro-CT）检测小鼠股骨骨质结构;酶联免疫吸附试验（enzyme linked immunosorbent assay,ELISA）检测血清中RANKL和骨保护素（osteoprotegerin,OPG）的表达水平;试剂盒检测血清中SOD、CAT、丙二醛（malondialdehyde,MDA）水平;免疫荧光检测股骨切片Nrf2表达水平。结果 体外实验：与NC组相比,1~500 μmol/L VA均对RAW264.7细胞活力无明显影响（P>0.05）,Diff组破骨细胞分化明显增多（P<0.05）,破骨细胞分化蛋白表达和ROS水平明显升高（P<0.05）;与Diff组相比,RANKL+VA200 μmol/L组破骨细胞分化数量明显减少（P<0.05）,破骨细胞分化蛋白表达和ROS水平明显降低（P<0.05）,并且显著促进了Nrf2的入核（P<0.05）,显著增加了抗氧化酶GCLC、SOD、CAT的表达（P<0.05）;与RANKL+VA200 μmol/L组相比,RANKL+VA200μmol/L+ML385组破骨细胞分化蛋白表达明显升高,而抗氧化酶表达明显降低（P<0.05）。体内实验：与Sham组相比,OVX组股骨结构质量明显下降（P<0.05）,血清RANKL、MDA水平明显上升（P<0.05）,血清OPG、SOD、CAT水平明显降低（P<0.05）,Nrf2表达明显降低（P<0.05）;与OVX组相比,OVX+VA100 mg/kg组骨结构质量明显上升（P<0.05）,血清RANKL、MDA水平明显下降（P<0.05）,血清OPG、SOD、CAT水平明显上升（P<0.05）,Nrf2表达明显升高（P<0.05）。结论 VA通过激活Nrf2信号通路抑制破骨细胞分化,从而改善OVX小鼠的骨质疏松症。
Abstract
Objective To investigate the mechanism of action of vanillic acid (VA) in regulating osteoclast differentiation and its effect on osteoporosis in ovariectomized (OVX) mice. Methods In vitro experiment: receptor activator for nuclear factor-κB ligand (RANKL)-treated RAW264.7 cells were used to establish an osteoclast differentiation model. The cells were divided into negative control (NC), RANKL- differentiated (Diff), and RANKL plus VA at 50, 100, and 200 μmol/L groups. Cell viability was assessed using the cell counting kit-8(CCK8) assay. The osteoclast differentiation was detected by tartrate resistant acid phosphatase (TRAP) staining and immunoblot analysis of osteoclast differentiation-related proteins. Cellular reactive oxygen species (ROS) levels were measured using a 2',7'-Dichlorodihydrofluorescein diacetate（DCFH-DA）probe, and Western blotting was performed to detect nuclear factor erythroid2-related factor 2(Nrf2), Kelch like epichlorohydrin related protein 1(Keap1), glutamate-cysteine ligase catalytic subunit (GCLC), superoxide dismutase (SOD), and catalase (CAT) protein expressions. Additionally, ML385 was used to inhibit Nrf2 expression to evaluate its role in osteoclast differentiation and oxidative stress. In vivo experiment: an osteoporosis model was established in OVX mice. The mice were divided into sham, OVX, OVX+estradiol (E2), OVX+VA50 mg/kg, and OVX+VA100 mg/kg (n= 6 per group) groups. Femoral bone structure was analyzed using micro computed tomography（Micro-CT）. Serum RANKL and osteoprotegerin (OPG) levels were assessed by enzyme linked immunosorbent assay (ELISA), and serum oxidative stress markers (SOD, CAT, and malondialdehyde (MDA)) were also quantified. Nrf2 expression in femur sections was evaluated immunofluorescently. Results In vitro: VA at concentrations of 1-500 μmol/L was non-toxic to RAW264.7 cells. Osteoclast differentiation, protein expression, and ROS levels were significantly elevated in the Diff group compared to the NC group (P<0.05). VA (200 μmol/L) significantly inhibited osteoclast differentiation, decreased protein expression and ROS levels, promoted Nrf2 nuclear translocation, and increased antioxidant enzyme (GCLC, SOD, CAT) expression (P<0.05). These changes were reversed after the inhibition of Nrf2 by ML385 (P<0.05). In vivo: compared to the sham group, OVX mice exhibited significantly reduced femoral structural mass, increased serum RANKL and MDA levels, and decreased serum OPG, SOD, and CAT levels, along with reduced Nrf2 expression (P<0.05). VA (100 mg/kg) treatment significantly improved bone structure, reduced serum RANKL and MDA levels, increased serum OPG, SOD, and CAT levels, and elevated Nrf2 expression compared to the OVX group (P<0.05). Conclusion VA mitigates osteoporosis in OVX mice by activating the Nrf2 signaling pathway, and thereby inhibiting osteoclast differentiation and oxidative stress.
关键词
香草酸 /
Nrf2 /
氧化应激 /
破骨细胞 /
骨质疏松
Key words
vanillic acid /
Nrf2 /
oxidative stress /
osteoclast /
osteoporosis
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