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骨质疏松症是一种全身性骨代谢疾病,其典型特征是骨密度下降、骨脆性增加和骨微环境被破坏[1]。骨稳态失衡是其发生的主要病理学基础。骨稳态是指成骨细胞行使的骨形成功能和破骨细胞行使的骨吸收功能处在一个相对平衡的过程[2]。破骨细胞分化及其功能的过度活化是导致骨稳态失衡的重要因素[3]。中国骨质疏松症流行病学调查显示,我国50岁以上人群骨质疏松发病率为19.2%,65岁以上人群发病率为32%[4]。目前临床上治疗骨质疏松症的药物主要是骨吸收抑制剂,其在抑制骨吸收的同时,也干扰骨形成进程。因而发掘更好的治疗骨质疏松的药物是迫切需要的。
骨髓来源的巨噬细胞(BMMs)向破骨细胞分化需要重组小鼠巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)的持续刺激[5]。M-CSF增加了早期BMMs的增殖,RANKL与受体RANK结合激活肿瘤坏死因子受体相关因子6(TRAF6),进而激活细胞凋亡信号调节激酶1(ASK1)和NF-κB抑制物激酶(IKKs),活化的ASK1和IKKs磷酸化JNK、ERK和P38以及NF-κB特异性抑制因子IκB特定部位的丝氨酸,激活MAPK和NF-κB信号。活化的MAPK和NF-κB使c-Fos、NFATc1表达增加,促进DC-STAMP、ATP6V0d2、TRAP、CTSK等破骨细胞特异性基因的转录与表达,导致破骨细胞分化[6]。研究表明,减弱破骨细胞分化及功能,能够有效地治疗骨质疏松症[7]。
冬虫夏草是一味传统中药,有增强免疫、抗炎、抗氧化和延缓衰老等作用[8]。先前的研究表明,富含锶的冬虫夏草菌丝发酵液对去卵巢骨质疏松大鼠有良好的治疗效果,其机制是提高了血清中的雌二醇水平,但是该研究仅基于整体水平解释了冬虫夏草作用于骨质疏松症的机制,对冬虫夏草的菌种也未作鉴定,并且野生的冬虫夏草提取液在骨质疏松症中的作用也未见报道[9-11]。本研究旨在探讨冬虫夏草提取液(CSE)对去卵巢小鼠的治疗作用以及对破骨细胞分化和功能的影响,为CSE防治骨质疏松症提供实验依据。
Cordyceps sinensis extract protects against the ovariectomy-induced bone loss via the action on osteoclasts
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摘要:
目的 探讨冬虫夏草提取液(Cordyceps sinensis extract,CSE)对去卵巢小鼠骨量流失的保护作用以及对核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)诱导的破骨细胞分化及其功能的影响。 方法 从C57BL/6小鼠骨髓中提取巨噬细胞(bone marrow-derived macrophages,BMMs);在破骨细胞分化过程中,加入CSE干预处理,通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色分析破骨细胞数量;将接近成熟的破骨细胞种于羟基磷灰石板,观测骨陷窝面积;使用DAPI和鬼笔环肽对破骨细胞肌动蛋白环(F-actin)进行染色,观测环内细胞核数量和环形态;使用q-PCR检测DC-STAMP、ATP6V0d2、TRAP、CTSK、NFATc1的表达;使用Western blot检测MAPK通路蛋白的表达;构建去卵巢小鼠,每天灌胃给予CSE,治疗6周取小鼠股骨进行形态学分析以及ELISA检测外周血中骨碱性磷酸酶(bone alkaline phosphatase,ALP)、骨钙素(BGP)、TRAP含量。 结果 CSE显著性地抑制了破骨细胞的分化,且呈剂量依赖性;并且主要作用于破骨细胞分化的早期阶段;也抑制了F-actin环的形成,减少了骨陷窝面积;同时抑制DC-STAMP、ATP6V0d2、TRAP、CTSK、NFATc1的表达和MAPK通路JNK、ERK和P38的激活;CSE治疗也可减缓小鼠的骨量流失,提高血清中ALP、BGP含量,降低TRAP含量。 结论 CSE通过抑制MAPK通路激活,从而抑制破骨细胞分化及其功能,并对去卵巢小鼠的骨量流失具有良好的保护作用。 Abstract:Objective To explore the effects of Cordyceps sinensis extract (CSE) on osteoporosis and RANKL-mediated osteoclastogenesis. Methods Bone marrow-derived macrophages (BMMs) was isolated from the bone marrow of C57BL/6 mice. CSE was added in osteoclast differentiation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP). The nearly mature osteoclasts were planted on hydroxyapatite plates and the area of bone lacunae was observed by microscope. The F-actin belt was stained by DAPI and phylloeptide and the number of nuclei was observed by confocal microscopy. The expressions of DC-STAMP, ATP6V0D2, TRAP, CTSK, and NFATC1 were detected by q-PCR. The protein expression of the MAPK pathway was detected by Western Blot. The in vivo experiments were carried out by administering CSE to the ovariectomized mice daily through gavage. After 6 weeks of intervention, mouse femurs were taken for morphological analysis. Peripheral blood was taken for ELISA. Results CSE represses osteoclastogenesis, bone resorption, F-actin belts formation, osteoclast specific gene expressions and MAPK signaling pathways in vitro. In vivo study indicated that CSE prevents OVX-induced osteoporosis and preserves bone volume by repressing osteoclast activity and function. It also increases the serum ALP, BGP content, and reduces TRAP content. Conclusion CSE can attenuate osteoclast formation and OVX-induced osteoporosis, suggesting potential clinical therapeutic effects for osteoporosis. -
Key words:
- Cordyceps sinensis extract /
- osteoclast /
- differentiation /
- function /
- mechanism /
- ovariectomized mice
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