Ding Zhili, Du Wei, Huang Jie, Han Jiaheng, Bai Jie, Yang Guangnan, Zhang Yan, Ding Yu
Orthopedics of TCM Senior Department, The Sixth Medical Center of PLA General Hospital, Beijing 100048, China; Navy Clinical College, Fifth School of Clinical Medicine, Anhui Medical University, Hefei, China.
Orthopedics of TCM Senior Department, The Sixth Medical Center of PLA General Hospital, Beijing 100048, China.
Int Immunopharmacol. 2025 Jan 10;144:113700. doi: 10.1016/j.intimp.2024.113700. Epub 2024 Dec 3.
Intervertebral disc degeneration (IVDD) is a common cause of low back pain and spinal issues. Allogeneic platelet lysate (APL) is a blood product for several growth agents. However, only a few studies have revealed that APL can increase autophagy in defective mitochondria by activating the SIRT1-PINK1/parkin pathway while enhancing mitochondrial function to decrease reactive oxygen species (ROS) levels.
To elucidate the mechanism by which APL mediates mitochondrial autophagy via the SIRT1-PINK1/Parkin pathway in the treatment of IVDD in vitro.
Pure platelet-rich plasma (P-PRP) was prepared by two-step centrifugation, and APL was prepared via freeze-thaw cycles. The nucleus pulposus cells of New Zealand white rabbits were harvested and grown. After the third generation, four groups of cells were cultured: (1) control group: standard culture conditions; (2) IL-1β group: intervention; (3) APL group: 24-hour IL-1β intervention followed by 24-hour APL treatment; and (4) APL + EX527 group: SIRT1 inhibitor EX527 24-hour treatment after 24-hour IL-1β and APL treatment. After interventions, cell activity was measured by Trypan blue staining. Apoptosis was measured by flow cytometry in each group. Immunofluorescence labeling measured mitochondrial permeability, ROS, and ROS. RT-PCR evaluated autophagy and inflammation-related gene mRNA expression. Western blot analysis revealed the protein levels of these genes. Electron microscopy reveals mitochondrial autophagy.
APL from P-PRP decreased ROS levels in an IVDD in vitro model, mediated autophagy in dysfunctional mitochondria, and alleviated inflammation via the SIRT1-PINK1/Parkin pathway.
椎间盘退变（IVDD）是腰痛和脊柱问题的常见原因。同种异体血小板裂解物（APL）是一种含有多种生长因子的血液制品。然而，仅有少数研究表明，APL可通过激活SIRT1-PINK1/parkin途径增加缺陷线粒体中的自噬，同时增强线粒体功能以降低活性氧（ROS）水平。
阐明APL在体外治疗IVDD中通过SIRT1-PINK1/Parkin途径介导线粒体自噬的机制。
通过两步离心制备纯富血小板血浆（P-PRP），并经冻融循环制备APL。采集新西兰白兔的髓核细胞并进行培养。第三代后，将细胞分为四组进行培养：（1）对照组：标准培养条件；（2）IL-1β组：进行干预；（3）APL组：先进行24小时IL-1β干预，随后进行24小时APL处理；（4）APL + EX527组：在24小时IL-1β和APL处理后，进行24小时SIRT1抑制剂EX527处理。干预后，通过台盼蓝染色检测细胞活性。采用流式细胞术检测每组细胞的凋亡情况。通过免疫荧光标记检测线粒体通透性、ROS以及线粒体自噬情况。RT-PCR评估自噬和炎症相关基因的mRNA表达。蛋白质印迹分析检测这些基因的蛋白水平。电子显微镜观察线粒体自噬情况。
P-PRP来源的APL在体外IVDD模型中降低了ROS水平，介导功能失调线粒体中的自噬，并通过SIRT1-PINK1/Parkin途径减轻炎症。
