Huang Jiangfa, Zeng Ningjing, Xu Shuchai, Lv Yang, Li Xing, Yang Peng, Liu Yan
Department of Sports Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 261 Datong Road, Yuexiu District, Guangzhou, 510105 Guangdong China.
The Second Clinical College of Guangzhou, University of Chinese Medicine, Guangzhou, Guangdong China.
Cytotechnology. 2025 Feb;77(1):35. doi: 10.1007/s10616-024-00702-6. Epub 2025 Jan 4.
Cartilage and joint damage can lead to cartilage degeneration. Bone marrow mesenchymal stem cells (BMSCs) have the potential to address cartilage damage. Hence, this study probed the mechanism of BMSC-extracellular matrix (BMSC-ECM) in promoting damaged chondrocyte repair by regulating the Notch1/RBPJ pathway. Human immortalized chondrocytes were cultured in vitro and treated with Notch1 small interfering (si)RNA, pCDNA3.1-Notch1, RBPJ siRNA and their negative controls (NCs). Damaged chondrocytes were constructed. Damaged chondrocyte-BMSC co-culture system was established and treated with lentiviral vector carrying short hairpin-Notch1 and its NC. Cell viability and apoptosis were assessed by CCK-8 and flow cytometry assays. Levels of glycosaminoglycan (GAG), Notch1 and RBPJ mRNA, and Notch1, RBPJ, Col2α1, mmp3, Hes1 and Hey1 were determined by a kit, RT-qPCR and Western blot. NICD nuclear translocation was detected by immunofluorescence. Damaged chondrocytes exhibited down-regulated Notch1 expression, reduced cell viability, and enhanced apoptosis. Further Notch1 knockdown aggravated chondrocyte damage, whereas its overexpression enhanced chondrocyte viability and decreased apoptosis. NICD translocated into the nucleus and bound to RBPJ to activate the Notch1 pathway. RBPJ silencing partly annulled Notch1-regulated damaged chondrocyte apoptosis. BMSC-damaged chondrocyte co-culture up-regulated Notch1, RBPJ, Col2α1, mmp3, Hes1, Hey1 and GAG levels, enhanced cell viability, and reduced apoptosis in chondrocytes, which were partly negated by Notch1 suppression, indicating that BMSC-ECM facilitated damaged chondrocyte repair by activating the Notch1/RBPJ pathway. BMSC-ECM promoted the repair of damaged chondrocytes by promoting NICD translocation into the nucleus and binding to RBPJ to activate the Notch1 pathway.
The online version contains supplementary material available at 10.1007/s10616-024-00702-6.
软骨和关节损伤可导致软骨退变。骨髓间充质干细胞（BMSCs）有修复软骨损伤的潜力。因此，本研究探讨了骨髓间充质干细胞-细胞外基质（BMSC-ECM）通过调节Notch1/RBPJ通路促进受损软骨细胞修复的机制。体外培养人永生化软骨细胞，并用Notch1小干扰（si）RNA、pCDNA3.1-Notch1、RBPJ siRNA及其阴性对照（NCs）处理。构建受损软骨细胞。建立受损软骨细胞-BMSC共培养体系，并用携带短发夹Notch1及其NC的慢病毒载体处理。通过CCK-8和流式细胞术检测细胞活力和凋亡。用试剂盒、RT-qPCR和蛋白质免疫印迹法检测糖胺聚糖（GAG）、Notch1和RBPJ mRNA水平，以及Notch1、RBPJ、Col2α1、mmp3、Hes1和Hey1水平。通过免疫荧光检测NICD核转位。受损软骨细胞Notch1表达下调，细胞活力降低，凋亡增加。进一步敲低Notch1加重软骨细胞损伤，而过表达则增强软骨细胞活力并减少凋亡。NICD转位到细胞核并与RBPJ结合以激活Notch1通路。RBPJ沉默部分消除了Notch1调节的受损软骨细胞凋亡。BMSC-受损软骨细胞共培养上调Notch1、RBPJ、Col2α1、mmp3、Hes1、Hey1和GAG水平，增强细胞活力，并减少软骨细胞凋亡，Notch1抑制部分抵消了这些作用，表明BMSC-ECM通过激活Notch1/RBPJ通路促进受损软骨细胞修复。BMSC-ECM通过促进NICD转位到细胞核并与RBPJ结合以激活Notch1通路，促进受损软骨细胞的修复。
在线版本包含可在10.1007/s10616-024-00702-6获取的补充材料。
