| 卞峰,方红育,李宏亮,等.二甲双胍下调Bhlhe40抑制破骨细胞分化.骨科,2025,16(1): 40-48. |
| 二甲双胍下调Bhlhe40抑制破骨细胞分化 |
| Metformin inhibits osteoclast differentiation by down regulating bhlhe40 |
| 投稿时间：2024-04-24 |
| DOI：10.3969/j.issn.1674-8573.2025.01.007 |
| 中文关键词: 骨质疏松 二甲双胍 Bhlhe40 破骨分化 |
| 英文关键词: Osteoporosis Metformin Bhlhe40 Osteoclastic differentiation |
| 基金项目:武汉市医学科研项目（WX19D14） |
|
| 摘要点击次数: 762 |
| 全文下载次数: 527 |
| 中文摘要: |
| 目的 探究二甲双胍对Bhlhe40表达及破骨细胞分化功能的影响。方法 C57BL/6小鼠（雄性，4周龄）股骨中提取原代骨髓单核细胞，RNA测序分析预测二甲双胍影响破骨细胞分化的相关基因，并通过实时荧光定量PCR验证。诱导原代骨髓单核细胞向破骨细胞分化（OC组），诱导分化的同时加入低浓度二甲双胍药物干预（OC+MET组），免疫荧光激光共聚焦检测对比两组间Bhlhe40表达差异，蛋白免疫印迹试验（Western blot）检测两组间Bhlhe40蛋白表达差异。抗酒石酸酸性磷酸酶染色（TRAP）分析Bhlhe40基因敲除小鼠（Bhlhe40−/−组）与野生型小鼠（WT组）原代骨髓单核细胞经诱导后形成的破骨细胞的面积，Micro-CT对比Bhlhe40−/−组与WT组小鼠股骨的骨量及相关参数，免疫组化染色对比两组股骨远端的TRAP表达量。结果 经RNA测序预测及实时荧光定量PCR验证，诱导原代骨髓单核细胞向破骨细胞分化后Bhlhe40高表达，经二甲双胍干预后，Bhlhe40表达降低。免疫荧光激光共聚焦检测结果可见OC组破骨细胞中Bhlhe40高表达，而OC+MET组中Bhlhe40表达量降低；Western blot结果显示OC+MET组中Bhlhe40蛋白表达明显较OC组下调。TRAP染色结果显示Bhlhe40−/−组破骨细胞面积较WT组减少；Micro-CT结果显示Bhlhe40−/−组小鼠股骨骨小梁增厚，密度升高，骨量增加；免疫组化染色结果显示Bhlhe40−/−组小鼠股骨TRAP阳性细胞数目减少。结论 Bhlhe40调控破骨细胞形成及分化，促进其骨吸收功能，二甲双胍可通过下调Bhlhe40表达抑制破骨细胞分化及其骨吸收能力。 |
| 英文摘要: |
| Objective To explore the effect of metformin on the expression of Bhlhe40 and the differentiation function of osteoclasts. Methods Primary bone marrow mononuclear cells were extracted from the femur of C57BL/6 mice (male, 4 weeks old), and RNA sequencing was used to predict the related genes of metformin affecting osteoclast differentiation, which were validated by real-time fluorescence quantitative PCR. Primary bone marrow monocytes were induced to differentiate into osteoclasts (OC group), while low concentration metformin was added into OC+MET group. The expression differences in Bhlhe40 were detected by immunofluorescence staining, and Western blotting was used to detect the protein expression differences in Bhlhe40 between the two groups. Tartrate resistant acid phosphatase (TRAP) staining was used to analyze the area of osteoclasts in the primary bone marrow monocytes from Bhlhe40 gene knockout mice (Bhlhe40−/− group) and wild-type mice (WT group). Micro CT was used to compare the bone mass of femur between Bhlhe40−/− group and WT group. Immunohistochemical staining was used to compare the number of TRAP labeled osteoclasts between the two groups. Results After RNA sequencing prediction and real-time fluorescence quantitative PCR verification, it was found that primary bone marrow monocytes were induced to differentiate into osteoclasts with high expression of Bhlhe40. However, after intervention with metformin, the expression of Bhlhe40 decreased. The results of immunofluorescence laser confocal detection showed that Bhlhe40 was highly expressed in OC group, and decreased in the OC+MET group. The results of Western blotting showed that the protein expression of Bhlhe40 were significantly down regulated in OC+MET group compared to OC group. The TRAP staining results showed that number and area of osteoclasts decreased in Bhlhe40−/− group compared to WT group. Micro CT results showed thickening of femoral trabeculae, increased density, and increased bone mass in Bhlhe40−/− group. The immunohistochemical staining results showed a decrease in the number of TRAP positive cells in the femur of Bhlhe40−/− group mice. Conclusion Bhlhe40 regulates the formation and differentiation of osteoclasts and promotes bone resorption. Metformin can inhibit the differentiation of osteoclasts and bone resorption by down regulating the expression of Bhlhe40. |
|
查看全文
下载PDF阅读器 |
| 关闭 |
