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

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).

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.

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.

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.