Ni Feifei, Zhang Tao, Xiao Wanan, Dong Hong, Gao Jian, Liu YaFeng, Li Jianjun
Department of Orthopaedics, Shengjing Hospital of China Medical University, Shenyang, China.
Liaoning Qifu Stem Cell Biotechnology Co, Ltd, Shenyang, China.
Front Cell Dev Biol. 2021 Dec 17;9:748831. doi: 10.3389/fcell.2021.748831. eCollection 2021.
To investigate the role of IL-18 in the regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). To assess whether IL-18 affects the osteogenic differentiation of hBMSCs through the c-MYC/SLC7A5 axis, IL-18 dose-response and time-course experiments were performed to evaluate its impact on osteogenic differentiation. To confirm osteogenic differentiation, alizarin red staining calcium measurement were performed. RT-qPCR and western blotting were used to determine the expression levels of bone-specific markers ALP, RUNX2, and BMP2, as well as those of SLC7A5 and c-MYC. Furthermore, SLC7A5 and c-MYC expression was evaluated immunofluorescence. To elucidate the roles of SLC7A5 and c-MYC in osteoblast differentiation, cells were transfected with SLC7A5 or c-MYC siRNAs, or treated with the SLC7A5-specific inhibitor JPH203 and c-MYC-specific inhibitor 10058-F4, and the expression of SLC7A5, c-MYC, and bone-specific markers ALP, RUNX2, and BMP2 was assessed. Our results demonstrated that IL-18 increased calcium deposition in hBMSCs, and upregulated the expression of SLC7A5, c-MYC, ALP, RUNX2, and BMP2. Silencing of SLC7A5 or c-MYC using siRNA reduced the expression of ALP, RUNX2, and BMP2, while IL-18 treatment partially reversed the inhibitory effect of siRNA. Similar results were obtained by treating hBMSCs with SLC7A5 and c-MYC specific inhibitors, leading to significant reduction of the osteogenesis effect of IL-18 on hBMSCs. In conclusion, our results indicate that IL-18 promotes the osteogenic differentiation of hBMSCs the SLC7A5/c-MYC pathway and, therefore, may play an important role in fracture healing. These findings will provide new treatment strategies for delayed fracture healing after splenectomy.
为了研究白细胞介素-18（IL-18）在人骨髓间充质干细胞（hBMSCs）成骨分化调节中的作用。为了评估IL-18是否通过c-MYC/SLC7A5轴影响hBMSCs的成骨分化，进行了IL-18剂量反应和时间进程实验，以评估其对成骨分化的影响。为了确认成骨分化，进行了茜素红染色钙测量。采用逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法来测定骨特异性标志物碱性磷酸酶（ALP）、 runt相关转录因子2（RUNX2）和骨形态发生蛋白2（BMP2）以及SLC7A5和c-MYC的表达水平。此外，通过免疫荧光评估SLC7A5和c-MYC的表达。为了阐明SLC7A5和c-MYC在成骨细胞分化中的作用，用SLC7A5或c-MYC小干扰RNA（siRNAs）转染细胞，或用SLC7A5特异性抑制剂JPH203和c-MYC特异性抑制剂10058-F4处理细胞，并评估SLC7A5、c-MYC和骨特异性标志物ALP、RUNX2和BMP2的表达。我们的结果表明，IL-18增加了hBMSCs中的钙沉积，并上调了SLC7A5、c-MYC、ALP、RUNX2和BMP2的表达。使用siRNA沉默SLC7A5或c-MYC可降低ALP、RUNX2和BMP2的表达，而IL-18处理可部分逆转siRNA的抑制作用。用SLC7A5和c-MYC特异性抑制剂处理hBMSCs也获得了类似的结果，导致IL-18对hBMSCs的成骨作用显著降低。总之，我们的结果表明，IL-18通过SLC7A5/c-MYC途径促进hBMSCs的成骨分化，因此可能在骨折愈合中发挥重要作用。这些发现将为脾切除术后延迟骨折愈合提供新的治疗策略。
