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. 2025 Jul 20;56(4):1059–1068. [Article in Chinese] doi: 10.12182/20250760501

α-玉米赤霉醇预防性调控骨髓间充质干细胞分化改善骨质疏松大鼠骨量丢失

Preventive Regulation of Bone Marrow Mesenchymal Stem Cell Differentiation by α-Zearalenol Ameliorates Bone Loss in Osteoporotic Rats

Xueling HE 1, Mingyue BAO 2, Min TANG 2, Xiaolin YAO 2, Liang LI 2,Δ
PMCID: PMC12531007  PMID: 41113407

Abstract

Objective

To evaluate the inhibitory effects of prophylactic administration of α-zearalanol (α-ZAL) on bone microarchitecture and bone resorption activity in ovariectomized osteoporotic rats, and to investigate its regulatory effects on the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

Methods

A total of 60 6-month-old unmated female Sprague-Dawley (SD) rats weighing (300 ± 20) g were randomly divided into the sham surgery group (Sham group), ovariectomy group (OVX group), solvent group (Oil group), estradiol benzoate treatment group (Post-E2 group), α-ZAL prevention group (Pre-ZAL group), and α-ZAL treatment group (Post-ZAL group), with 10 rats in each group. An osteoporosis rat model was established using the ovariectomy method. Rats in the Sham group underwent the same surgical procedures except for ovarian removal. Seventy-two hours after ovarian removal, the Oil group received intramuscular injections of 0.5 mL of oil solvent, and the Pre-ZAL group received intramuscular injections of α-ZAL (1.5 mg·kg－1), administered every 3 days for 120 consecutive days. The Post-E2 group and Post-ZAL group began intramuscular injections of estradiol benzoate (1.5 mg·kg－1) and α-ZAL (1.5 mg·kg－1), respectively, 90 days after ovariectomy, administered every 3 days for 120 consecutive days. After drug administration, bone density and bone tissue microstructure morphology were analyzed using a micro-CT small animal in vivo imaging system and staining methods. Osteoclasts were isolated and their activity was detected. Femoral BMSCs were obtained to assess their osteoblast and adipocyte differentiation capabilities, and uterine tissue morphological changes were observed via histological sections.

Results

Compared with the OVX group, BMD in the Sham group, Post-E2 group, Pre-ZAL group, and Post-ZAL group increased by 133.12%, 75.97%, 69.64%, and 24.69%, respectively (all P < 0.01). BMD in the Pre-ZAL group was 36.09% higher than in the Post-ZAL group (P < 0.01), and there was no significant difference in BMD between the Post-E2 and Pre-ZAL groups (P > 0.05). Tb.N in the Sham group, Post-E2 group, Pre-ZAL group, and Post-ZAL group increased by 160.08%, 118.14%, 94.76%, and 46.76%, respectively, compared with the OVX group (all P < 0.01). Tb.Ar increased by 324.21%, 203.83%, 177.99%, and 82.71%, respectively (all P < 0.01). Tb.N in the Pre-ZAL group increased by 32.71% compared to the Post-ZAL group (P < 0.05), while Tb.Ar increased by 52.15% (P < 0.01). Tb.Sp in the Sham, Post-E2, and Pre-ZAL groups decreased by 58.53%, 42.18%, and 35.61%, respectively, compared with the OVX group (all P < 0.01). The MAR of the upper tibial cancellous bone in the Sham, Post-E2, and Pre-ZAL groups increased by 257.81%, 156.72%, and 142.63%, respectively, compared with the OVX group (all P < 0.01), BFR increased by 192.19%, 137.23%, and 88.13%, respectively (all P < 0.01). MAR and BFR in the Pre-ZAL group increased by 58.10% and 43.63%, respectively, compared with the Post-ZAL group (both P < 0.01). There were no significant differences in MAR and BFR between the Post-E2 group and the Pre-ZAL group (P > 0.05). MMP-9, TRAP, and CK mRNA expression was significantly downregulated in both the Post-E2 group and the Pre-ZAL group (P < 0.01). The osteoblast differentiation capacity of BMSCs in the Post-E2 group and all Post-ZAL groups was enhanced, with a significant increase in the number of mineralized nodules, and the expression levels of OCN, COL1, and OPN mRNA were significantly increased (P < 0.01), while the ability to differentiate into adipocytes was weakened. The number of intracellular lipid droplets in BMSCs was significantly reduced, the lipid droplet volume was smaller, and the expression levels of PPAR-γ2 and aP2 mRNA were decreased (P < 0.05). There were no significant differences between the Post-E2 group and the Pre-ZAL group (P > 0.05). There was no significant increase in body weight in the Post-E2, Pre-ZAL, and Post-ZAL groups, but uterine weight significantly increased in the Post-E2 group (P < 0.05), with marked uterine epithelial hyperplasia. Uterine weight in the Pre-ZAL and Post-ZAL groups showed no significant difference compared to the OVX group (P > 0.05), and no significant changes were observed in uterine epithelium.

Conclusion

α-ZAL can effectively protect bone mass, improve bone microstructure, and reduce estrogen-related uterine adverse reactions by regulating the osteogenic/adipogenic differentiation balance of BMSCs, providing a potential new therapeutic strategy for the prevention and treatment of postmenopausal osteoporosis.

Keywords: α-zearalanol, Osteoporosis, Bone marrow mesenchymal stem cells, Differentiation, Preventive intervention

绝经后骨质疏松症（postmenopausal osteoporosis, PMOP）是一种与内分泌功能衰退相关的骨骼代谢性疾病，其主要病理特征表现为骨量减少、骨微结构退化及骨力学强度下降，进而显著提升骨折风险[1]。流行病学研究显示，随着社会老龄化程度加剧，我国50岁以上人群骨质疏松症患病率约为19%，其中女性高达32.1%[2]。该疾病不仅严重影响老年女性的生活质量和心理健康，还给公共卫生系统带来巨大的挑战。

研究表明，骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）分化失衡（即成骨分化抑制与脂肪分化增强）是导致骨形成障碍的重要因素[3-4]。临床观察发现，雌激素缺乏不仅与骨质疏松相关，还与脂肪代谢异常存在关联性[4-5]。PMOP患者常伴随脂肪组织异常堆积和中心性肥胖，而外源性雌激素补充可改善这一现象[6]。这表明雌激素可能通过调控BMSCs的分化平衡来影响骨代谢。

目前临床主要采用雌激素替代疗法（estrogen replacement therapy, ERT）进行干预，其通过激活骨组织中的雌激素受体（estrogen receptor, ER），双向调节成骨细胞增殖和破骨细胞凋亡，从而维持骨代谢平衡[7-8]。然而，该疗法可能诱发子宫内膜增生、乳腺肿瘤等严重不良反应[9]。因此，开发具有靶向性强且安全性高的新型治疗方案，尤其是针对早期骨形成下降的干预策略，具有重要的临床意义。

植物雌激素因其结构与内源性雌激素相似而备受关注。α-玉米赤霉醇（α-zearalanol, α-ZAL）作为镰刀菌毒素ZEA的衍生物，其空间构象与雌二醇高度相似[10]。研究表明，α-ZAL可通过ER信号通路调节骨代谢标志物（如骨钙素、碱性磷酸酶等）的表达水平，改善骨微环境[11]。体外实验证实α-ZAL对乳腺细胞增殖无显著刺激作用[12]。本课题组前期研究也发现，α-ZAL能有效改善去卵巢大鼠模型生物力学性能，其效果与雌二醇无明显差异[13]。但目前尚缺乏关于α-ZAL预防性用药效果的系统评价，尤其是其改善去卵巢诱导骨丢失的机制有待阐明。

本研究拟通过建立卵巢切除（ovariectomized, OVX）骨质疏松大鼠模型，重点探讨预防性给予α-ZAL对骨微结构和骨吸收活性的影响，对BMSCs分化命运的调控作用，以及其对子宫安全性特征。研究成果将为开发新型抗骨质疏松药物提供理论依据。

1. 材料与方法

1.1. 实验材料

1.1.1. 实验动物

60只6月龄未交配雌性Sprague-Dawley（SD）大鼠，体质量（300±20） g，购自四川大学实验动物中心〔生产许可证编号SCXK（川）2018-026〕。所有大鼠饲养于标准实验室内。本研究严格遵守四川大学华西医院动物中心伦理委员会的要求，批准号20240221049。

1.1.2. 主要试剂和设备

α-ZAL由戴顺龄教授（中国医学科学院基础医学研究所）惠赠。苯甲酸雌二醇（estradiol benzoate, E2）购自上海通用药业；DMEM-LG培养基、胎牛血清（fetal bovine serum, FBS）均购自Invitrogen公司；成骨诱导培养基、成脂诱导培养基均购自Cyagen公司；茜素红S染色液（alizarin red S）、油红O染色液、钙黄绿素、Masson-Goldner trichrome 染色液均购自Sigma-Aldrich公司；抗酒石酸酸性磷酸酶检测试剂盒购自碧云天生物公司；qRT-PCR试剂盒购自Thermo Fisher。vivaCT80 micro-CT小动物活体影像系统（Scanco Medical公司）、RM2155型硬组织切片机（Leica公司）、ProFlex PCR 系统（Thermo Fisher公司）、BX53荧光显微镜（Olympus公司）。

1.2. 方法

1.2.1. 实验分组及骨质疏松模型构建

所有大鼠在实验室适应性饲养1周后开始实验。60只SD大鼠随机分为假手术组（Sham组）、模型组（OVX组）、溶剂组（Oil组）、苯甲酸雌二醇组（Post-E2组）、α-ZAL预防组（Pre-ZAL组）、α-ZAL治疗组（Post-ZAL组），每组10只。大鼠经1%异氟烷吸入麻醉后，除Sham组外，其余大鼠均摘除双侧卵巢，构建PMOP大鼠模型。Sham组大鼠除不摘除卵巢外，其余接受相同的手术操作。卵巢摘除72 h后，Oil组肌内注射0.5 mL油溶剂、Pre-ZAL组肌内注射α-ZAL（1.5 mg/kg），每3 d注射 1次，连续120 d；Post-E2组、Post-ZAL组于摘除卵巢90 d后分别肌内注射E2（1.5 mg/kg）、α-ZAL （1.5 mg/kg），每3 d注射1次，连续120 d。给药结束后，安乐死大鼠，micro-CT小动物活体影像系统分析骨密度、骨组织微结构形态，获取股骨BMSCs检测其成骨细胞和成脂肪细胞分化能力，分离破骨细胞检测活性，并通过病理切片观察子宫组织形态学变化。

1.2.2. 骨组织微结构形态检测

使用micro-CT80扫描仪评估胫骨的骨小梁结构。计算骨密度（bone mineral density, BMD）、骨小梁面积百分数（percent trabecular area, Tb.Ar）、骨小梁数量（trabecular number, Tb.N）、骨小梁分离度（trabecular separation/spacing, Tb.Sp）等形态学参数。胫骨用硬组织切片机制成5 μm厚切片，采用Masson-Goldner trichrome 染色观察骨小梁微结构形态变化。

1.2.3. 骨形成能力检测

所有大鼠分别在实验结束前第11天和第4天腹腔注射钙黄绿素（10 mg/kg）进行体内双荧光标记。实验结束后，取出股骨使用硬组织切片机制成30 μm厚切片，在荧光显微镜下观察钙黄绿素双荧光带，分析骨矿化沉积率（mineralization deposition rate, MAR）和骨形成速率（bone formation rate, BFR）。

1.2.4. BMSCs的分离与分化

获取大鼠股骨中骨髓，用含青霉素（100 IU/mL）/链霉素（100 μg/mL）、15%胎牛血清的DMEM-LG培养基培养12 h，除去未贴壁细胞，添加新鲜培养基继续培养，每周更换2次培养基，当细胞融合度达约80%时进行传代。将第3代细胞接种于6孔板中的盖玻片上，并培养至约80%融合状态，将培养基更换为成骨诱导培养基或成脂诱导培养基，继续培养21 d，每周更换2次培养基。经成骨诱导培养的盖玻片，用新鲜配制的0.5%茜素红S溶液染色；经成脂诱导培养的盖玻片，用0.5%油红O染色。收集细胞总RNA，检测如骨钙素（osteocalcin, OCN）、骨桥蛋白（osteopontin, OPN）和Ⅰ型胶原蛋白（collagen Ⅰ, COL1）等成骨细胞分化相关基因以及如过氧化物酶体增殖物激活受体γ2（peroxisome proliferator-activated receptorγ2, PPAR-γ2）和脂肪细胞脂肪酸结合蛋白（adipocyte lipid binding protein, aP2）等成脂肪细胞分化相关基因mRNA表达水平。

1.2.5. 破骨细胞培养及活性检测

无菌条件下从大鼠长骨中获取混合骨细胞群，培养于牛皮质骨切片（厚度约600 µm）4 h后，除去未贴附细胞，将富含破骨细胞的贴壁细胞以4×10⁶ mL－1密度在α-MEM培养基中培养24 h后，分别采用抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase, TRAP）染色和鬼笔环肽（Phalloidin）进行纤维状肌动蛋白（F-actin）荧光标记。同时，破骨细胞连续培养9 d，每天检测破骨细胞活性。吸弃细胞培养基，加入10%甲醛，室温固定10 min；弃甲醛，乙醇固定2 min后移除，加入TRAP活性检测试剂，37 ℃孵育40 min后，加入终止反应液，在酶标仪415 nm波长处记录吸光度值。培养至第9天收集细胞总RNA，检测基质金属蛋白酶-9（matrix metalloproteinase-9, MMP-9）、TRAP和组织蛋白酶 K（cathepsin K, CK）mRNA表达水平。

1.2.6. RNA提取与实时荧光定量PCR（qRT-PCR）

使用Trizol法提取总RNA，定量并使用逆转录试剂盒获得cDNA。检测成骨细胞相关基因OCN、COL1、OPN，脂肪细胞相关基因PPAR-γ2、aP2，破骨细胞相关基因MMP-9、TRAP、CK mRNA表达水平。引物由Primer premier 5.0软件设计，由上海生工生物工程有限公司合成，OCN上游引物：5'-gaccctctctctgctcactc-3'，下游引物：5'-gctagctcgtcacaattggg-3'；aP2上游引物：5'-gcttacaaaatgtgcgacgc-3'，下游引物：5'-cacgcccagtttgaaggaaa-3'； COL1a上游引物：5'-tcaagatggtggccgttact-3'，下游引物：5'-catcttgaggtcacggcatg-3'；OPN上游引物：5'-aacagtatcccgatgccaca-3'，下游引物：5'-agctgacttgactcatggct-3'；PPAR-γ2上游引物：5'-tcaaactccctcatggccat-3'，下游引物：5'-gcattgtgagacatccccac-3'；MMP-9上游引物：5'-tgacaagtacttcacccgct-3'，下游引物：5'-atgagtggatagctcggtgg-3'；TRAP上游引物：5'-caacggctacctacgctttc-3'，下游引物：5'-tccctccctcagacccatta-3'；CK上游引物：5'-acacctctgcttcccttctc-3'，下游引物：5'-atagaagacaggggcttggg-3'；GAPDH上游引物：5'-gagacagccgcatcttcttg-3'，下游引物：5'-tgactgtgccgttgaacttg-3'。以GAPDH为内参基因，采用2－ΔΔCt法计算基因相对表达量。

1.2.7. 大鼠体质量及子宫形态观察

实验结束后，记录大鼠体质量，处死大鼠并分离整个子宫、记录质量，于体积分数为10%中性甲醛中固定72 h，经梯度乙醇溶液脱水后石蜡包埋，切片厚度为5 µm，进行苏木精-伊红染色法（hematoxylin and eosin staining, HE）染色，显微镜下观察。

1.2.8. 统计学方法

所有数据分析均使用SPSS 25.0进行统计学分析。定量数据以Inline graphic表示。多组间比较采用ANOVA方差分析，两组间比较采用Bonferroni法，P<0.05为差异有统计学意义。

2. 结果

2.1. α-ZAL改善大鼠胫骨松质骨骨小梁微结构

各组大鼠胫骨松质骨骨微结构见图1A、1B。骨微结构定量分析，与OVX组相比较，Sham组、Post-E2组、Pre-ZAL组和Post-ZAL组均显著提升大鼠BMD与骨微结构参数。Sham组、Post-E2组、Pre-ZAL组和Post-ZAL组BMD分别较OVX组增加了133.12%、75.97%、69.64%和24.69%（均P<0.01）； Pre-ZAL组BMD较Post-ZAL组增加了36.09%（P<0.01）；OVX组与Oil组之间、Post-E2组与Pre-ZAL组之间BMD均未见明显差异（P>0.05，图1C）。

图 1.

图 1

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α-ZAL improves the microstructure of trabecular bone in rat tibial cancellous bone

α-ZAL改善大鼠胫骨松质骨骨小梁微结构

A, Three-dimensional reconstruction of rat tibia from micro-CT scans; B, orphology of rat trabecular microarchitecture stained with Masson-Goldner qtrichrome ( × 20); C, uantitative analysis of rat tibial bone mineral density; D-F, quantitative analysis of bone microarchitecture parameters. Sham: pseudosurgical group; OVX: ovariectomized rat model group; Oil: solvent control group; Post-E2: estradiol benzoate treatment group; Pre-ZAL: preventive intervention group; Post-ZAL: α-zearalanol treatment group; BMD: bone mineral density; Tb.N: trabecular number; Tb.Ar: trabecular area; Tb.Sp: trabecular separation/spacing. n = 6. ## P < 0.01, ** P < 0.01.

与OVX组相比较，Sham组、Post-E2组、Pre-ZAL组和Post-ZAL 组Tb.N分别增加160.08%、118.14%、94.76%和46.76%（均P<0.01，图1D）；Tb.Ar分别增加324.21%、203.83%、177.99%和82.71%（均P<0.01，图1E）；Pre-ZAL组较Post-ZAL组Tb.N增加32.71%（P<0.05），而Tb.Ar增加52.15%（P<0.01）；与OVX组相比较，Sham组、Post-E2组和Pre-ZAL组Tb.Sp分别降低58.53%、42.18%和35.61%（均P<0.01，图1F），Pre-ZAL组较Post-ZAL组Tb.Sp降低了28.63%（P<0.05）。Masson-Goldner trichrome染色也显示，Post-E2组、Pre-ZAL组和Post-ZAL组大鼠胫骨上段松质骨BMD、Tb.N、Tb.Ar均增加，Tb.Sp均降低（图1B）。

2.2. α-ZAL提高大鼠股骨松质骨矿化沉积和新骨形成

与OVX组相比，Sham组、Post-E2组和Pre-ZAL组大鼠胫骨上段松质骨MAR分别增加257.81%、156.72%和142.63%（均P<0.01，图2A、图2B）；BFR分别提高192.19%、137.23%和88.13%（均P<0.01，图2A、图2C）；Pre-ZAL组大鼠胫骨上段松质骨MAR、BFR分别较Post-ZAL组分别增加58.10%和43.63%（均P<0.01）；Post-E2组和Pre-ZAL组之间大鼠胫骨上段松质骨MAR、BFR均未见明显差异（P>0.05）。

图 2.

图 2

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α-ZAL increases MAR and BFR in rat femurs

α-ZAL提高大鼠股骨松质骨矿化沉积和新骨形成

A, Calcein xanthophyll fluorescence double-labeled images of rat femur tissue ( × 200); B, mineral apposition rate (MAR); C, bone formation rate (BFR). n = 6. ## P < 0.01, ** P < 0.01.

2.3. α-ZAL降低大鼠破骨细胞活性和破骨细胞相关基因表达

TRAP染色显示，破骨细胞形态典型，胞质呈现明显的紫红色（图3A），OVX组和Oil组破骨细胞数量较其他组多，鬼笔环肽染色同样显示破骨细胞形态典型，细胞体积较大，形态不规则（图3B）；OVX组和Oil组破骨细胞数量显著高于其他组，Post-E2组和Pre-ZAL组之间破骨细胞数量无明显差异。各组破骨细胞活性均呈现先降后升再降的趋势，细胞活性在第7天达峰值（图3C）；在观察期间，Post-E2组和Pre-ZAL组破骨细胞活性均低于OVX组、Oil组和Post-ZAL组，Post-E2组和Pre-ZAL组破骨细胞活性无明显差异。与OVX组相比，Sham组、Post-E2组和Pre-ZAL组破骨细胞MMP-9、TRAP和CK mRNA表达水平降低（P<0.01），Post-E2 组和PPre-ZAL组之间无明显差异（P>0.05）。

图 3.

图 3

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α-ZAL decreases osteoclast activity and osteoclast-related gene expression in rats

α-ZAL降低大鼠破骨细胞活性和破骨细胞相关基因表达

A, Tartrate-resistant acid phosphatase (TRAP) stain for osteoclast (the arrow indicates an osteoclast, × 200); B, fluorescent staining of osteoclasts ( × 200); C, osteoclast activity; D, osteoclast-related gene expression. n = 3. ## P < 0.01, vs. OVX group; * P < 0.05, vs. Pre-ZAL group.

2.4. α-ZAL增强大鼠BMSCs成骨细胞分化和抑制成脂肪细胞分化

与OVX组相比较，Sham组、Post-E2组和Pre-ZAL组BMSCs矿化结节形成数量增加（图4A），OCN、COL1和OPN mRNA表达水平均增加（P<0.01，图4B）；Pre-ZAL组OCN、COL1和OPN mRNA表达水平高于Post-ZAL组，但差异不明显（P>0.05），Post-E2组与Pre-ZAL组间无明显差异（P>0.05）。与OVX组相比较，Sham组、Post-E2组、Pre-ZAL组和Post-ZAL组BMSCs胞内脂滴数量较少，且脂滴体积小，PPAR-γ2与aP2 mRNA表达水平均降低（P<0.05），Pre-ZAL组PPAR-γ2与aP2 mRNA表达水平均低于Post-ZAL组（P<0.05），Post-E2 组和Pre-ZAL组之间无明显差异（P>0.05，图4C、4D）。

图 4.

图 4

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α-ZAL enhances osteoblast differentiation and inhibits adipocyte differentiation in rat BMSCs

α-ZAL增强大鼠BMSCs成骨细胞分化和抑制成脂肪细胞分化

A, Osteoblast differentiation (the arrow indicates a calcified nodule, × 100); B, osteoblast differentiation-related gene expression; C, adipocyte differentiation (the arrow indicates a lipid droplet, × 200); D, adipocyte differentiation-related gene expression. n = 3. # P < 0.05, ## P < 0.01, vs. OVX group; * P < 0.05, ** P < 0.01, vs. Pre-ZAL group.

2.5. α-ZAL对大鼠子宫和体质量的影响

与各组初始体质量相比较，实验结束时大鼠体质量均有明显增加，其中OVX组和Oil组增加幅度最大，分别增加29.6%和36.1%，组间差异无统计学意义（P>0.05）,其次是Sham组和Post-ZAL，分别增加17.7%和19.1%，Post-E2组和Pre-ZAL组分别增加12.3%和15.2%（P<0.05，图5A）。与Post-E2组相比较，其他各组大鼠子宫/体质量比较低（P<0.01），其中，Post-E2组大鼠子宫/体质量比是Pre-ZAL组的11.4倍，Pre-ZAL组、Post-ZAL组、OVX组和Oil组无明显差异（P>0.05，图5B）；Post-E2组大鼠子宫上皮增生明显，与Sham组相比较，Pre-ZAL组、Post-ZAL组、OVX组和Oil组大鼠子宫上皮未见明显改变（图5C）。

图 5.

图 5

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Effect of alpha-ZAL on the morphology of the rat uterus and changes in rat body mass

α-ZAL对大鼠子宫形态学和大鼠体质量变化的影响

A, Histological morphology of rat uterus ( × 400, × 100，the red arrow indicates rat uterine wall); B, percentage increase in rat body mass; C, ratio of uterine mass to body mass in rats. n = 10. ## P < 0.01, vs. OVX group; && P < 0.05, vs. Sham group; ▲▲ P < 0.01, vs. Post-E2 group.

3. 讨论

骨质疏松症是一种以骨量降低和骨微结构损伤为特征的代谢性骨病，其病理改变导致骨骼机械强度下降和骨折风险增加。在女性群体中，PMOP作为与年龄相关的原发性骨质疏松症，表现出显著的流行病学特征[14]。

BMSCs作为具有多向分化潜能的祖细胞，在骨髓微环境中可分化为成骨细胞和脂肪细胞。这两种分化途径之间存在动态平衡关系，其中BMSCs向成骨细胞的分化过程对维持骨形成与骨吸收的生理平衡具有关键作用。当成脂分化增强时，往往伴随成骨分化的减弱，这种分化失衡被认为是导致骨量减少的重要病理机制[15-16]。在PMOP的发病过程中，特定的分子机制促使BMSCs分化倾向发生改变，表现为成骨分化能力降低而脂肪生成增加。增生的脂肪细胞可通过旁分泌作用促进造血干细胞向破骨细胞分化，进而破坏成骨细胞与破骨细胞之间的生理性偶联平衡。这种级联反应最终导致进行性骨量丢失[17–18]。骨髓微环境中骨形成与脂肪形成之间的复杂相互作用网络，为理解骨质疏松症的发病机制提供了新的视角。

研究表明，体内雌激素水平下降可引起BMSCs成骨分化减少、成脂分化增加，而脂肪细胞刺激更多造血干细胞分化为破骨细胞，继而引起成骨细胞-破骨细胞偶联失衡，最终导致骨量丢失[19-20]。其中， PPAR-γ2作为调控BMSCs成脂分化的关键转录因子，其表达水平直接影响BMSCs的分化方向。当PPAR-γ2表达缺失时，BMSCs的成脂分化受到抑制，同时成骨分化能力增强。研究证实雌二醇可通过调控BMSCs的分化命运促进成骨形成[21]。在雌激素缺乏状态下，BMSCs中PPAR-γ2表达显著上调，而补充雌激素后其表达水平降低[22]。

本研究发现，α-ZAL（尤其是Pre-ZAL组）对OVX骨质疏松大鼠骨骼形态的影响与Post-E2 组相似，能够显著提高BMD、BV/TV、Tb.N及Tb.Th，同时降低SMI和Tb.Sp。同时，结果还显示α-ZAL（尤其是Pre-ZAL组）可显著减少OVX骨质疏松大鼠长骨组织中破骨细胞数量，并抑制其活性，MMP-9、TRAP和CK mRNA表达水平下降。

在BMSCs分化方面，α-ZAL（尤其是Pre-ZAL组）可增强其成骨分化能力，钙结节形成增加，OCN、COL1和OPN mRNA表达水平上调。同时，α-ZAL抑制BMSCs的成脂分化，细胞内脂滴数量减少、体积减小，并下调PPAR-γ2和aP2 mRNA表达。上述结果表明，α-ZAL可通过促进BMSCs成骨分化、抑制成脂分化，减少破骨细胞生成，从而改善骨微结构并减少骨量丢失。

α-ZAL的分子构型与E2具有高度相似性，能够与雌激素受体结合并诱导其二聚化，从而发挥类雌激素活性，提升动物体内雌激素水平[23]。然而，关于其改善卵巢切除诱导的骨质疏松的具体分子机制，包括是否通过经典ERα/ERβ途径，或通过调控Wnt/β-catenin、BMP/Smad、PPARγ等关键信号通路发挥作用，仍需深入阐明，这将成为后续研究的重点方向。

研究发现，绝经初期雌激素水平的急剧下降与多部位骨量快速丢失密切相关。在绝经后5～10年发生骨质疏松，其中以富含松质骨的区域表现尤为显著[24-25]。这种骨量减少伴随着明显的骨小梁微结构恶化，包括小梁数量显著减少、间距增大等形态学改变。而早期干预可有效改善这些病理变化，从而显著降低脆性骨折风险[26]。因此，绝经早期被认为是进行骨质疏松预防性干预的关键时期。在本研究中，不同α-ZAL干预组（尤其是Pre-ZAL组）表现出良好的生物学效应，能有效抑制BMSCs向脂肪细胞分化，维持其成骨分化潜能，进而显著改善大鼠胫骨上段松质骨骨量丢失和骨微结构破坏。

雌激素替代疗法在绝经后骨质疏松防治领域备受关注。虽然该疗法具有独特的治疗优势，但其潜在的子宫内膜癌等不良反应限制了临床应用。近年发现植物雌激素因其与内源性雌激素相似的结构特征而展现出良好的应用前景，在维持骨量的同时表现出更佳的安全性特征。本研究结果进一步证实，Post-E2组OVX骨质疏松大鼠出现显著的子宫质量增加和子宫内膜增生，而各α-ZAL剂量组则未观察到明显的子宫刺激作用，显示了α-ZAL在安全性方面的优势。

综合以上研究结果表明，α-ZAL（特别是预防性应用）可通过调控BMSCs的分化方向，有效预防OVX诱导的骨量丢失，显著改善骨微结构，且不引起雌激素相关的子宫不良反应，为绝经后骨质疏松的防治提供了潜在的新型治疗策略。后续研究将重点关注其分子作用机制、长期用药的安全性和有效性，以及临床转化的可行性等问题。

*　　　　*　　　　*

作者贡献声明　何学令负责数据审编、调查研究、研究方法、研究项目管理、监督指导、可视化和初稿写作，包明月负责数据审编、研究方法、监督指导和初稿写作，唐敏负责正式分析、研究方法和可视化，姚晓琳负责调查研究、研究方法和初稿写作，李良负责论文构思、经费获取、研究方法、研究项目管理、提供资源和审读与编辑写作。所有作者已经同意将文章提交给本刊，且对将要发表的版本进行最终定稿，并同意对工作的所有方面负责。

Author Contribution　HE Xueling is responsible for data curation, investigation, methodology, project administration, supervision, visualization, and writing--original draft. BAO Mingyue is responsible for data curation, methodology, supervision, and writing--original draft. TANG Min is responsible for formal analysis, methodology, and visualization. YAO Xiaolin is responsible for investigation, methodology, and writing--original draft. LI Liang is responsible for conceptualization, funding acquisition, methodology, project administration, resources, and writing--review and editing. All authors consented to the submission of the article to the Journal. All authors approved the final version to be published and agreed to take responsibility for all aspects of the work.

利益冲突　本文作者何学令为编辑部工作人员。本文在编辑评审过程中所有流程严格按照期刊政策进行，且未经作者本人处理。除此之外，所有作者均声明不存在利益冲突。

Declaration of Conflicting Interests　HE Xueling is a staff member of the Editorial Office of the journal. All processes involved in the editing and reviewing of this article were carried out in strict compliance with the journal's policies and there was no inappropriate personal involvement by the authors. Other than this, all authors declare no competing interests.

Funding Statement

国家自然科学基金（No. 11872260）资助

Contributor Information

学令 何 (Xueling HE), Email: hxlscu@126.com.

良 李 (Liang LI), Email: lilianghx@163.com.

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