塞来昔布通过抑制 mTOR 信号通路激活自噬，防止髓核细胞凋亡。
Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells.
机构信息
Department of Orthopaedics, Zhongshan Hospital of Fudan University, 180 Fenglin Road/1609 Xietu Road, 200032, Shanghai, China.
Department of Orthopedic Surgery, Zhongshan Hospital Xiamen Branch, Fudan University, 668 Jinhu Rd, District of Huli, Fujian, 361015, Xiamen, China.
出版信息
BMC Pharmacol Toxicol. 2022 Dec 1;23(1):90. doi: 10.1186/s40360-022-00633-y.
BACKGROUND
Intervertebral disc degeneration results from a variety of etiologies, including inflammation and aging. Degenerated intervertebral discs feature down-regulated extracellular matrix synthesis, resulting in losing their ability to retain water and absorb compression. Celecoxib is a well-known selective cyclooxygenase-2 inhibitor for treating arthritis and relieving pain. Nevertheless, the mechanism of Celecoxib for treating inflammation-related intervertebral disc degeneration has not yet been clarified.
METHOD
Protein synthesis was analyzed by western blot. Fluorescent probes DCFH-DA and MitoSox Red detected reactive oxygen species and were measured by flow cytometry. The activity of the kinase pathway was evaluated by protein phosphorylation. Autophagy was monitored by mRFP-GFP-LC3 transfection and LC3 analysis. Mitochondrial apoptotic proteins were analyzed by western blot and cell membrane integrity was measured by flow cytometry. The autophagic gene was silenced by siRNA.
RESULTS
In this study, interleukin-1β stimulation reduced the synthesis of aggrecan, type I and II collagen and caused excessive production of reactive oxygen species. We looked for a therapeutic window of Celecoxib for nucleus pulposus cells to regain extracellular matrix synthesis and reduce oxidative stress. To look into nucleus pulposus cells in response to stimuli, enhancement of autophagy was achieved by Celecoxib, confirmed by mRFP-GFP-LC3 transfection and LC3 analysis. The mammalian target of rapamycin and a panel of downstream proteins responded to Celecoxib and propelled autophagy machinery to stabilize homeostasis. Ultimately, inhibition of autophagy by silencing autophagy protein 5 disrupted the protective effects of Celecoxib, culminating in apoptosis.
CONCLUSION
In summary, we have demonstrated a new use for the old drug Celecoxib that treats intervertebral disc degeneration by enhancing autophagy in nucleus pulposus cells and opening a door for treating other degenerative diseases.
背景
椎间盘退变是由多种病因引起的，包括炎症和衰老。退变的椎间盘表现为细胞外基质合成减少，从而丧失保持水分和吸收压缩的能力。塞来昔布是一种著名的选择性环氧化酶-2 抑制剂，用于治疗关节炎和缓解疼痛。然而，塞来昔布治疗炎症相关椎间盘退变的机制尚未阐明。
方法
通过 Western blot 分析蛋白质合成。荧光探针 DCFH-DA 和 MitoSox Red 检测活性氧，并通过流式细胞术进行测量。通过蛋白磷酸化评估激酶途径的活性。通过 mRFP-GFP-LC3 转染和 LC3 分析监测自噬。通过 Western blot 分析线粒体凋亡蛋白，通过流式细胞术测量细胞膜完整性。通过 siRNA 沉默自噬基因。
结果
在这项研究中，白细胞介素-1β刺激降低了聚集蛋白、I 型和 II 型胶原的合成，并导致活性氧的过度产生。我们寻找塞来昔布治疗椎间盘细胞恢复细胞外基质合成和减少氧化应激的治疗窗口。为了研究椎间盘细胞对刺激的反应，塞来昔布通过增强自噬来实现，通过 mRFP-GFP-LC3 转染和 LC3 分析得到证实。雷帕霉素的哺乳动物靶标和一组下游蛋白对塞来昔布作出反应，并推动自噬机制来稳定体内平衡。最终，通过沉默自噬蛋白 5 抑制自噬破坏了塞来昔布的保护作用，导致细胞凋亡。
结论
总之，我们已经证明了旧药塞来昔布的新用途，通过增强椎间盘细胞的自噬来治疗椎间盘退变，并为治疗其他退行性疾病开辟了道路。
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