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

何学令; 包明月; 唐敏; 姚晓琳; 李良

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

摘要

摘要:
目的评价预防性应用α-玉米赤霉醇（α-zearalanol, α-ZAL）对卵巢摘除骨质疏松大鼠骨微结构、骨吸收活性的抑制作用，探究其对骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）成骨分化和脂肪分化的调控作用。
方法选用6月龄未交配雌性Sprague-Dawley（SD）大鼠60只，体质量（300±20） g，随机分为假手术组（Sham组）、卵巢摘除组（OVX组）、溶剂组（Oil组）、苯甲酸雌二醇治疗组（Post-E2组）、α-ZAL预防组（Pre-ZAL组）、α-ZAL治疗组（Post-ZAL组），每组10只，采用卵巢摘除法建立骨质疏松大鼠模型。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组分别于摘除卵巢后90 d开始分别肌内注射苯甲酸雌二醇（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，采用micro-CT小动物活体影像系统和染色方法分析骨密度、骨组织微结构形态，分离破骨细胞并检测其活性，获取股骨BMSCs检测其成骨细胞和成脂肪细胞分化能力，并通过病理切片观察子宫组织形态学变化。
结果与OVX组相比较，Sham组、Post-E2组、Pre-ZAL组和Post-ZAL 组BMD分别增加了133.12%、75.97%、69.64%和24.69%（均P<0.01）； Pre-ZAL组骨密度（BMD）较Post-ZAL组增加了36.09%（P<0.01），Post-E2组和Pre-ZAL组之间BMD未见明显差异（P>0.05）；Sham组、Post-E2组、Pre-ZAL组和Post-ZAL组骨小梁数量（Tb.N）较OVX组分别增加160.08%、118.14%、94.76%和46.76%（均P<0.01）；骨小梁面积百分数（Tb.Ar）分别增加324.21%、203.83%、177.99%和82.71%（均P<0.01）；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）；Sham组、Post-E2组和Pre-ZAL组骨小梁分离度（Tb.Sp）较OVX组分别降低58.53%、42.18%和35.61%（均P<0.01）；Sham组、Post-E2组和Pre-ZAL组大鼠胫骨上段松质骨骨矿化沉积率（MAR）分别较OVX组增加257.81%、156.72%和142.63%（均P<0.01），骨形成速率（BFR）分别提高192.19%、137.23%和88.13%（均P<0.01）；Pre-ZAL组MAR、BFR分别较Post-ZAL组增加58.10%和43.63%（均P<0.01）；Post-E2组和Pre-ZAL组之间MAR、BFR均未见明显差异（P>0.05）；Post-E2组与Pre-ZAL组的MMP-9、TRAP及CK mRNA表达下调（P<0.01）；Post-E2组和各Post-ZAL组BMSCs向成骨细胞分化能力增强，矿化结节形成数量明显增加，OCN、COL1与OPN mRNA表达水平增高（P<0.01），而向脂肪细胞分化能力减弱，BMSCs胞内脂滴数量明显减少，脂滴体积较小，PPAR-γ2与aP2 mRNA表达水平降低（P<0.05），Post-E2组与Pre-ZAL组间无明显差异（P>0.05）；Post-E2组、Pre-ZAL组及Post-ZAL组大鼠体质量增加不明显，但Post-E2组大鼠子宫质量增加（P<0.05），子宫上皮增生明显；Pre-ZAL组和Post-ZAL组大鼠子宫质量与OVX组无明显差异（P>0.05），子宫上皮未见明显改变。
结论 α-ZAL可通过调控BMSCs的成骨/成脂分化平衡有效保护骨量、改善骨微结构，并减少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。

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.

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