槲皮素通过抗骨相关细胞衰老作用治疗雌激素缺乏骨质疏松症的初步研究

Quercetin alleviates estrogen deficiency-induced osteoporosis through anti-senescence effect on bone cells

目的通过体内外实验探究槲皮素对骨相关细胞衰老的影响,验证槲皮素通过抗骨相关细胞衰老作用对绝经后骨质疏松症的治疗作用。方法选取小鼠骨细胞样细胞MLO-Y4及成骨细胞样细胞MC3T3-E1,构建体外压力诱导的成熟前衰老(stress induced premature senescence, SIPS)模型,通过qRT-PCR法和细胞衰老相关β-半乳糖苷酶染色确定细胞衰老样变化,利用CCK-8法确定槲皮素体外工作浓度,验证槲皮素在骨相关细胞SIPS中的作用。建立去势小鼠骨质疏松模型,槲皮素灌胃给药,与经典雌激素疗法相比较,利用micro-CT扫描分析骨参数及骨微结构的变化,并通过qPCR检测小鼠皮质骨内衰老相关基因p21、p53,衰老相关分泌表型(senescence associated secretory phenotype, SASP)TNF-α、IL-6以及破骨相关基因TRAP、CTSK和成骨相关基因OCN、RUNX2的表达情况。结果体外实验证明槲皮素能有效抑制骨相关细胞SIPS样改变( P <0.05)。体内实验发现,给药12周后,相较对照组小鼠,槲皮素组小鼠股骨骨表面积与骨体积比值(BS/BV)和骨小梁分离度(Tb.Sp)显著降低( P <0.05),骨小梁数量(Tb.N)显著升高( P < 0.05),骨质疏松程度减轻,较雌激素疗法差异无统计学意义,并伴骨内SIPS相关基因p21、p53及SASP水平下调( P <0.05),成骨水平不受明显抑制。结论槲皮素可通过抑制细胞衰老挽救由雌激素缺乏导致的骨丢失;这可能成为绝经后骨质疏松症治疗的新手段。

Objective To investigate the effect of quercetin on postmenopausal osteoporosis and its relation to anti-senescence of bone cells. Methods Stress induced premature senescence (SIPS) model in vitro was induced by exposure to stress in mouse osteocyte-like MLO-Y4 cells and osteoblast-like MC3T3-E1 cells. Senescence-like changes were determined by qRT-PCR and SA-β-Gal staining;the optimal concentration of quercetin was determined with CCK-8 tests;and the effects of quercetin on SIPS of bone cells was verified. The estrogen deficiency-induced osteoporosis model in vivo was established by bilateral ovariectomy in mice. Quercetin was administered to ovariectomized (OVX) mice by oral gavage, and estrogen therapy was taken as the positive control. Micro-CT was performed to detect the degree of osteoporosis. Senescence-related genes, osteogenic and osteoclast-related genes and senescence associated secretory phenotype (SASP) in cortical bone were detected by qRT-PCR. Results Quercetin suppressed SIPS-like changes in bone cells in vitro , as indicated by attenuation of senescence-related genes expression and decreased numbers of senescent cells. After 12-week administration, quercetin prevented OVX mice from osteoporosis, as indicated by bone surface/bone volume ratio(BS/BV), trabecular spacing (Tb.Sp) and trabecular number (Tb.N)( P <0.05), as well as reduced SIPS level and suppressed SASP genes transcription in cortical bone of OVX mice ( P <0.05). Conclusion Quercetin can reverse estrogen deficiency-induced bone loss by suppressing cellular senescence in mice, suggesting that it might be a potential treatment for postmenopausal osteoporosis.