龟甲通过miR-214调控的Wnt/β-连环蛋白信号通路刺激骨形成。

Plastrum Testudinis Stimulates Bone Formation through Wnt/β-catenin Signaling Pathway Regulated by miR-214.

作者信息

Lin Qing, Zhao Bi-Yi, Li Xiao-Yun, Sun Wei-Peng, Huang Hong-Hao, Yang Yu-Mei, Wang Hao-Yu, Zhu Xiao-Feng, Yang Li, Zhang Rong-Hua

机构信息

College of Traditional Chinese Medicine, Jinan University, Guangzhou, 510632, China.

College of Pharmacy, Jinan University, Guangzhou, 510632, China.

出版信息

Chin J Integr Med. 2025 May 13. doi: 10.1007/s11655-025-4012-9.

DOI:10.1007/s11655-025-4012-9

PMID:40358879

Abstract

OBJECTIVE

To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of Plastrum Testudinis (PT) in the treatment of osteoporosis (OP).

METHODS

Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method, including sham group, ovariectomized group (OVX), ovariectomized groups treated with high-, medium-, and low-dose PT (160, 80, 40 mg/kg per day, respectively), with 6 rats in each group. Except for the sham group, the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment, bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry, Western blot, and quantitative polymerase chain reaction. In addition, Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently, PT extract was used to rescue the effects of Dkk-1 and miR-214, and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated.

RESULTS

PT-M and PT-L significantly reduced the weight gain in OVX rats (P<0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats, and increased the expressions of bone formation-related factors including alkaline phosphatase, bone morphogenetic protein type 2, collagen type I alpha 1, and runt-related transcription factor 2 when compared with the OVX group (P<0.05 or P<0.01). Meanwhile, different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats, and increased the mRNA or protein expressions of Wnt3a, β-catenin, glycogen synthase kinase-3β, and low-density lipoprotein receptor-related protein 5 (P<0.05 or P<0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition, the expression of miR-214 was decreased in OVX rats (P<0.01), and it was negatively correlated with the osteogenic differentiation of BMSCs (P<0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (P<0.05 or P<0.01). Conversely, PT effectively counteracted the effect of miR-214 mimic, thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (P<0.05 or P<0.01).

CONCLUSION

PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway, which may be related to inhibiting miR-214 in BMSCs.

摘要

目的

探讨龟甲提取物（PT）治疗骨质疏松症（OP）的Wnt信号通路及微小RNA（miRNA）机制。

方法

将30只雌性Sprague Dawley大鼠按随机数字表法随机分为5组，包括假手术组、去卵巢组（OVX）、高、中、低剂量PT治疗的去卵巢组（分别为每天160、80、40 mg/kg），每组6只。除假手术组外，其余大鼠行双侧卵巢切除术以模拟OP，并连续10周经口灌胃给予PT。治疗后，采用双能X线吸收法测量骨密度；通过微计算机断层扫描及苏木精-伊红染色分析骨微结构；采用免疫化学、蛋白质印迹法及定量聚合酶链反应检测成骨分化相关因子的表达。此外，使用Dickkopf-1（Dkk-1）抑制骨髓间充质干细胞（BMSC）中的Wnt信号通路，并通过miRNA过表达评估miR-214对BMSC成骨分化的影响。随后，用PT提取物挽救Dkk-1和miR-214的作用，并评估其对BMSC成骨分化相关因子的影响。

结果

PT中、低剂量组显著降低了OVX大鼠的体重增加（P<0.05）。PT还调节了OVX大鼠股骨的骨量和骨微结构，与OVX组相比，增加了包括碱性磷酸酶、骨形态发生蛋白2型、I型胶原α1链和 runt相关转录因子2等骨形成相关因子的表达（P<0.05或P<0.01）。同时，不同剂量的PT显著挽救了OVX大鼠中Wnt信号通路相关因子的抑制，并增加了Wnt3a、β-连环蛋白、糖原合酶激酶-3β和低密度脂蛋白受体相关蛋白5的mRNA或蛋白表达（P<0.05或P<0.01）。PT刺激了被Dkk-1抑制的BMSC的成骨分化并激活了Wnt信号通路。此外，OVX大鼠中miR-214的表达降低（P<0.01），且其与BMSC的成骨分化呈负相关（P<0.01）。miR-214模拟物抑制了BMSC中的Wnt信号通路（P<0.05或P<0.01）。相反，PT有效抵消了miR-214模拟物的作用，从而激活了Wnt信号通路并刺激了BMSC的成骨分化（P<0.05或P<0.01）。