| 引用本文: |
陈培, 简功辉, 杨雷, 陈振华, 张天择, 叶政显, 齐新宇, 熊辉, 陆小龙.桃红四物汤通过GLUL/NOTCH通路促进BMMSCs成骨分化及核糖体生成研究[J].湖南中医药大学学报,2025,45(9):1644-1652[点击复制] |
|
| |
|
|
| 本文已被：浏览 1431次 下载 766次 |
| 桃红四物汤通过GLUL/NOTCH通路促进BMMSCs成骨分化及核糖体生成研究 |
| 陈培,简功辉,杨雷,陈振华,张天择,叶政显,齐新宇,熊辉,陆小龙 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南中医药大学, 湖南 长沙 410208;湖南中医药大学第二附属医院, 湖南 长沙 410005) |
| 摘要: |
| 目的 探究桃红四物汤（THSWD）促进骨髓间充质干细胞（BMMSCs）成骨分化影响骨折愈合的具体作用机制。方法 体外培养大鼠BMMSCs，取生长良好的BMMSCs分为对照（CONTROL）组、THSWD干预（THSWD）组、THSWD干预+谷酰胺合成酶（GLUL）敲低或过表达空白对照（THSWD+sh-NC或THSWD+OE-NC）组、THSWD干预+GLUL敲低或过表达（THSWD+sh-GLUL或THSWD+OE-GLUL）组。CONTROL组以BMMSCs完全培养基培养BMMSCs;THSWD组加入100 ng/mL THSWD含药血清干预；THSWD+sh-NC组及THSWD+OE-NC组经100 ng/mL THSWD含药血清干预后，分别转染sh-NC、OE-NC慢病毒；THSWD+shGLUL组和THSWD+OE-GLUL组经100 ng/mL THSWD含药血清干预后，分别转染sh-GLUL、OE-GLUL慢病毒。通过Western blot检测碱性磷酸酶（ALP）、骨骼特异性转录因子（Osx）、Runt相关转录因子2(Runx2)、GLUL、Notch胞内结构域（NICD）、神经源性位点Notch同源蛋白1(NOTCH1)及毛状和增强子分裂1(HES1)蛋白表达水平；采用茜素红染色检测细胞钙化情况；qRT-PCR检测5S rRNA、5.8S rRNA、18S rRNA、28S rRNA表达；利用网络药理学分析并筛选THSWD调控核糖体生成影响BMMSCs的关键靶点GLUL；采用CCK-8法检测细胞活力；采用Transwell检测细胞迁移距离并进行定量分析。结果 与CONTROL组相比，THSWD组BMMSCs中ALP、Osx、Runx2蛋白表达水平增加（P<0.01）；茜素红染色BMMSCs结果为阳性；THSWD组核糖体生成关键因子5S rRNA、5.8S rRNA、18S rRNA、28S rRNA表达增加（P<0.01）。网络药理学分析发现，GLUL是THSWD靶向的关键因子。细胞验证实验发现，与THSWD+sh-NC组相比，THSWD+sh-GLUL组BMMSCs细胞增殖能力，ALP、Osx、Runx2蛋白表达水平，细胞迁移能力及愈合率，5S rRNA、5.8S rRNA、18S rRNA、28S rRNA及NICD、NOTCH1、HES1表达水平均下降（P<0.01）；与THSWD+OE-NC组相比，THSWD+OE-GLUL组BMMSCs细胞增殖能力，ALP、Osx、Runx2蛋白表达水平，细胞迁移能力及愈合率，5S rRNA、5.8S rRNA、18S rRNA、28S rRNA及NICD、NOTCH1、HES1表达水平均增加（P<0.01）。结论 THSWD可能通过上调GLUL的表达，激活NOTCH信号通路的表达，促进核糖体生成，从而促进BMMSCs成骨分化。 |
| 关键词: 骨折愈合 桃红四物汤 骨髓间充质干细胞 核糖体生成 NOTCH通路 |
| DOI：10.3969/j.issn.1674-070X.2025.09.008 |
| 投稿时间：2025-05-09 |
| 基金项目:湖南中医药大学“刘良院士工作站”指导项目(22YS004); 湖南创新型省份建设重点领域研发计划项目(2023SK2047); 中医药防治筋骨疾病基础研究创新平台(kh2201056); 国家自然科学基金面上项目(81874478); 湖南省自然科学基金项目(2024JJ9419); 湖南省教育厅科学研究项目(23A0311); 长沙市自然科学基金项目(kq2208197)。 |
|
| Taohong Siwu Decoction promoting osteogenic differentiation and ribosome biogenesis of BMMSCs through the CLUL/NOTCH pathway |
| CHEN Pei, JIAN Gonghui, YANG Lei, CHEN Zhenhua, ZHANG Tianze, YE Zhengxian, QI Xinyu, XIONG Hui, LU Xiaolong |
| (Chinese Medicine, Changsha, Hunan 410208, China;The Second Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410005, China) |
| Abstract: |
| Objective To explore the specific mechanisms of Taohong Siwu Decoction（THSWD） in promoting osteogenic differentiation of bone marrow mesenchymal stem cells（BMMSCs） and affecting fracture healing. Methods BMMSCs from rats were cultured in vitro. Well-growing BMMSCs were selected and divided into the following groups: the control（CONTROL） group, the THSWD intervention（THSWD） group, the THSWD intervention +glutamine synthetase（GLUL） knockdown or overexpression blank control（THSWD+sh-NC or THSWD+OE-NC） group, and the THSWD intervention+GLUL knockdown or overexpression（THSWD+shGLUL or THSWD+OE-GLUL） group. Cells in the CONTROL group were cultured in complete BMSC medium. The THSWD group was treated with 100 ng/mL THSWD-containing serum. The THSWD+sh-NC and THSWD+OE-NC groups were incubated with 100ng/m L THSWD-containing serum and then transfected with sh-NC and OE-NC lentiviruses, respectively. The THSWD+sh-GLUL and THSWD+OE-GLUL groups were also treated with 100 ng/mL THSWD-containing serum followed by transduction with sh-GLUL and OE-GLUL lentiviruses, respectively. The protein expression levels of alkaline phosphatase（ALP）, Osterix（Osx）, runt-related transcription factor 2（Runx2）, GLUL, NOTCH intracellular domain（NICD）, neurogenic locus notch homolog protein 1（NOTCH1）,and hairy and enhancer of split-1（HES1） were detected by Western blot. Mineralization was assessed using Alizarin Red staining.The expression of 5S rRNA, 5.8S r RNA, 18S r RNA, and 28S rRNA was measured via qRT-PCR. Network pharmacology analysis was employed to predict and identify GLUL as a key target through which THSWD regulates ribosome biogenesis to affect BMMSCs. Cell viability was evaluated using the CCK-8 assay, and cell migration distance was quantified using the Transwell assay. Results Compared with the CONTROL group, the expression levels of ALP, Osx, and Runx2 significantly increased in the THSWD group（P＜0.01）. Alizarin Red staining results were positive in BMMSCs of the THSWD group. The expression of key ribosome biogenesis factors, including 5S rRNA, 5.8S rRNA, 18S rRNA, and 28S rRNA, was also markedly upregulated in the THSWD group（P＜0.01）. Network pharmacology analysis identified GLUL as a key target of THSWD. Cellular validation experiments showed that,compared with the THSWD +sh-NC group, the THSWD +sh-GLUL group exhibited decreased cell proliferation ability, protein expression levels of ALP, Osx, and Runx2, cell migration capacity and wound healing rate, expression of 5S/5.8S/18S/28S rRNAs,and protein expression levels of NICD, NOTCH1, and HES1（all P＜0.01）. Conversely, compared with the THSWD+OE-NC group, the THSWD +OE-GLUL group showed increases in all these parameters（all P＜0.01）. Conclusion THSWD may promote osteogenic differentiation of BMMSCs by upregulating the expression of GLUL, activating the expression of the NOTCH signaling pathway, and thereby enhancing ribosome biogenesis. |
| Key words: fracture healing Taohong Siwu Decoction bone marrow mesenchymal stem cells ribosome biogenesis NOTCH signaling pathway |
|
二维码（扫一下试试看！） |
|
|
|
|
