Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats

doi:10.12307/2025.304

Abstract

Abstract:
BACKGROUND: Eucommia ulmoides has a certain osteogenic effect, which can promote the proliferation and differentiation of osteoblasts. However, it is unclear whether Eucommia ulmoides has effects on alveolar bone formation and Wnt/β-Catenin signaling pathway.
OBJECTIVE: To investigate the mechanism by which Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats based on the Wnt/β-Catenin signaling pathway.
METHODS: Sixty female Sprague-Dawley rats were selected and randomly divided into five groups: blank control group, sham-operation group, model group, low-dose group Eucommia ulmoides group, and high-dose Eucommia ulmoides group, with twelve rats in each group. Osteoporosis animal models were constructed by bilateral oophorectomy in the model group and the low-dose and high-dose Eucommia ulmoides groups. The sham-operation group underwent the same method to remove adipose tissue of equal mass around the bilateral ovaries. Three months after surgery, the low- and high-dose Eucommia ulmoides groups were given 2.1 g/kg/d and 4.2 g/kg/d Eucommia ulmoides by gavage, respectively. The sham-operation group and model group were given the same amount of physiological saline by gavage. After 12 weeks of drug intervention, the changes in alveolar bone mass of rats in each group were observed through Micro-CT; hematoxylin-eosin staining was used to observe the pathological structural changes of alveolar bone in rats; enzyme linked immunosorbent assay was used to detect the expression levels of alkaline phosphatase and osteocalcin in the serum of rats; western blot was used to detect the expression levels of β-Catenin and Frizzled9 receptor proteins in the alveolar bone of rats; and real-time fluorescence quantitative PCR was used to detect the expression of osteocalcin, Runt-related transcription factor 2 (Runx2), alkaline phosphatase, β-catenin, and frizzled9 mRNAs in alveolar bone tissues of rats.
RESULTS AND CONCLUSION: Compared with the blank control group, bone volume fraction, trabecular number, trabecular thickness, and bone mineral density were reduced in the model group (P < 0.05), and trabecular separation was elevated (P < 0.05). Pathological observation showed that the arrangement of trabeculae was disordered and irregular, the trabeculae were thinned or broken, and the marrow cavity was enlarged in the model group, with a significant reduction in bone volume; the level of alkaline phosphatase in the serum was increased (P < 0.05), and the level of osteocalcin was decreased (P < 0.05); mRNA expression of alkaline phosphatase, osteocalcin, Runx2, β-catenin, and frizzled9 were decreased (P < 0.05); protein expression of β-Catenin and Frizzled9 was decreased (P < 0.05). Compared with the model group, the low- and high-dose Eucommia ulmoides groups showed an increase in bone volume fraction, trabecular number, trabecular thickness, and bone mineral density (P < 0.05) and a decrease in trabecular separation (P < 0.05). In the low- and high-dose Eucommia ulmoides groups, bone trabeculae were slightly aligned and thickened, with a significant increase in bone mass. Compared with the model group, the serum level of alkaline phosphatase was reduced (P < 0.05) and the serum level of osteocalcin was elevated (P < 0.05) in the low- and high-dose Eucommia ulmoides groups. Compared with the model group, the mRNA expression of alkaline phosphatase, osteocalcin, Runx2, β-catenin, and frizzled9 were increased in the low- and high-dose Eucommia ulmoides groups (P < 0.05). Compared with the model group, the protein expression of Frizzled9 was increased in the low-dose Eucommia ulmoides group (P < 0.05), while the protein expression of β-Catenin and Frizzled9 was increased in the high-dose Eucommia ulmoides group (P < 0.05). Compared with the low-dose Eucommia ulmoides group, the high-dose Eucommia ulmoides group had a more significant improvement in the above indexes. To conclude, Eucommia ulmoides can effectively promote the alveolar bone formation, and its mechanism of action might be related to the activation of the Wnt/β-catenin signaling pathway.

Key words:

Eucommia ulmoides, alveolar bone, bone formation, ovariectomized rat, Wnt/ β-catenin signaling pathway, osteoporosis

CLC Number:

Zheng Lin, Jin Wenjun, Luo Shanshan, Huang Rui, Wang Jie, Cheng Yuting, An Zheqing, Xiong Yue, Gong Zipeng, Liao Jian. Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(6): 1159-1167.

Figures/Tables 6

2.1 实验动物数量分析 60只大鼠全部进入结果分析。 2.2 验证骨质疏松动物模型构建成功 2.2.1 SD大鼠造模前后体质量变化造模前，从空白对照组及模型组各随机抽取5只大鼠称取体质量并记录，摘除双侧卵巢后第1，2，3个月时分别记录体质量。最初各组大鼠体质量、体型相似，体质量在(261.90±7.07) g范围内。卵巢摘除3个月后，相较于空白对照组的(294.10±11.43) g，模型组大鼠体质量明显增加(P < 0.05)，为(340.40±9.92) g(图2A)。 2.2.2 Micro-CT验证骨质疏松动物模型构建成功双侧卵巢摘除术3个月后，Micro-CT检测结果显示空白对照组下颌牙槽骨骨小梁致密，排列整齐；模型组卵巢摘除后骨量丢失，骨小梁变细变窄，小梁骨间离散程度增加(图2B)。 2.3 杜仲对骨质疏松大鼠下颌牙槽骨骨组织结构的影响 Micro-CT检测结果显示各组大鼠下颌第一磨牙根尖隔牙槽骨区域，空白对照组及假手术组下颌牙槽骨骨小梁致密，排列整齐；模型组卵巢摘除后骨量丢失，骨小梁变细窄，小梁骨间离散程度增加；给予低剂量杜仲治疗后部分骨量得以恢复，可见骨小梁数目增加，排列稍致密，离散程度减小；给予高剂量杜仲治疗后骨量恢复明显，骨小梁排列致密，离散程度明显减小，见图3A。通过 Micro-CT 扫描分析研究区域得出牙槽骨骨微结构参数，与空白对照组及假手术组比较，模型组大鼠牙槽骨骨体积分数、骨小梁数目、骨小梁厚度、骨密度均降低(P < 0.01)，骨小梁分离度增加(P < 0.01)；与模型组比较，杜仲低、高剂量组大鼠牙槽骨骨体积分数、骨小梁数目、骨小梁厚度、骨密度升高(P < 0.05)，骨小梁分离度降低(P < 0.05)；与杜仲低剂量组比较，杜仲高剂量组大鼠牙槽骨骨体积分数、骨小梁数目、骨小梁厚度表达升高(P < 0.05)，骨小梁分离度降低(P < 0.05)，见图3B。 2.4 杜仲对骨质疏松大鼠下颌牙槽骨骨组织形态的影响苏木精-伊红染色结果显示，空白对照组及假手术组大鼠牙槽骨组织结构完整，骨小梁排列均匀；模型组大鼠牙槽骨骨小梁排列紊乱、不规则，骨小梁断裂，骨小梁变细，骨髓腔变大，破骨细胞数量增多，骨量明显减少；与模型组相比，杜仲低剂量组大鼠牙槽骨骨小梁排列稍整齐，骨小梁断裂减少，骨髓腔间隙稍改善；杜仲高剂量组大鼠牙槽骨骨小梁较粗大，排列均匀且致密，骨髓腔间隙减少，骨细胞增多，骨量增多(图4)。 2.5 杜仲对骨质疏松大鼠血清中碱性磷酸酶、骨钙素的影响与空白对照组及假手术组比较，模型组大鼠血清中碱性磷酸酶表达水平上升(P < 0.01)，骨钙素表达水平下降(P < 0.01)；与模型组相比，杜仲低、高剂量组大鼠血清中碱性磷酸酶表达水平下降(P < 0.05)，骨钙素表达水平上升(P < 0.05)，并且存在剂量依赖性，见图5。表明杜仲可以影响血清中成骨相关因子碱性磷酸酶及骨钙素的表达来改善骨代谢，促进骨生成。 2.6 杜仲对骨质疏松大鼠牙槽骨骨组织中碱性磷酸酶、骨钙素、Runx2及β-连环蛋白、卷曲蛋白9 mRNA表达的影响 RT-qPCR检测结果显示，与空白对照组及假手术组比较，模型组大鼠牙槽骨组织中碱性磷酸酶、骨钙素、Runx2、β-连环蛋白、卷曲蛋白9 的mRNA表达均降低(P < 0.05或P < 0.01)；与模型组比较，杜仲低剂量组大鼠牙槽骨组织中碱性磷酸酶、骨钙素、β-连环蛋白、卷"

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