中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (28): 4491-4497.doi: 10.12307/2024.469

• 脊柱组织构建 spinal tissue construction • 上一篇    下一篇

不同氧浓度下髓核细胞中PI3K/Akt/HIF-1α信号通路表达对延缓椎间盘退变的意义

周明瀚，张  慧，郑先波，徐无忌   

  1. 湖南中医药大学第二附属医院骨伤二科，湖南省长沙市  410208
  • 收稿日期:2023-08-14 接受日期:2023-09-15 出版日期:2024-10-08 发布日期:2023-11-27
  • 通讯作者: 郑先波，硕士，副主任医师，湖南中医药大学第二附属医院骨伤二科，湖南省长沙市 410208 徐无忌，博士，主任医师，湖南中医药大学第二附属医院骨伤二科，湖南省长沙市 410208
  • 作者简介:周明瀚，男，1999年生，江苏省苏州市人，汉族，湖南中医药大学在读硕士，主要从事中西医结合防治脊柱退行性病变的研究。 张慧，女，1996年生，湖南省衡阳市人，汉族，湖南中医药大学在读硕士，主要从事中西医结合防治脊柱退行性病变的研究。
  • 基金资助:
    湖南省自然科学基金项目(2022JJ30449)，项目负责人：徐无忌；湖南省教育厅科学研究重点项目(21A0249)，项目负责人：徐无忌；湖南中医药大学中医学一流学科开放基金项目(2022ZYX25)，项目负责人：徐无忌

Significance of PI3K/Akt/HIF-1α signaling pathway expression in nucleus pulposus cells at different oxygen concentrations in delaying intervertebral disc degeneration

Zhou Minghan, Zhang Hui, Zheng Xianbo, Xu Wuji   

  1. Department of Orthopedics, the Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
  • Received:2023-08-14 Accepted:2023-09-15 Online:2024-10-08 Published:2023-11-27
  • Contact: Zheng Xianbo, Master, Associate chief physician, Department of Orthopedics, the Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China Xu Wuji, MD, Chief physician, Department of Orthopedics, the Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
  • About author:Zhou Minghan, Master candidate, Department of Orthopedics, the Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China Zhang Hui, Master candidate, Department of Orthopedics, the Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
  • Supported by:
    Natural Science Foundation of Hunan Province, No. 2022JJ30449 (to XWJ); Scientific Research Key Project of Hunan Provincial Department of Education, No. 21A0249 (to XWJ); First-class Discipline Open Fund Project for Chinese Medicine of Hunan University of Chinese Medicine, No. 2022ZYX25 (to XWJ)

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摘要：


文题释义：

椎间盘退变：椎间盘由髓核、纤维环、上下软骨终板组成，具有传递和吸收脊柱轴向应力并保持脊柱的多轴灵活性的作用，是脊柱的重要组成部分。健康的椎间盘处于一种低氧、低营养的微环境，当这种微环境为氧化应激、衰老、机械应力压迫、炎性反应等多种因素所破坏时，就容易导致髓核细胞凋亡，引起椎间盘退变，最终导致相应节段病变，引起肩颈、腰腿疼痛。
PI3K/Akt/HIF-1α信号通路：PI3K及其下游分子Akt所组成的信号通路在细胞增殖和凋亡中发挥重要作用，其下游因子HIF-1α是维持椎间盘微环境稳态的重要因子，该通路在氧化应激环境下可被激活，抑制细胞凋亡，促进细胞外基质生成，从而延缓椎间盘退变。


背景：椎间盘退变是一种由于细胞凋亡、氧化应激、炎症反应等多种因素共同作用导致的疾病，目前认为其核心在于髓核细胞退变与凋亡，然而具体病理机制尚不明确。

目的：通过探究不同氧浓度下髓核细胞中磷脂酰激醇-3-激酶(phosphatidylinositol-3kinase，PI3K)/蛋白激酶B(protein kinase B，Akt)/缺氧诱导因子1α(hypoxia-inducible factor 1-alpha，HIF-1α)信号通路的表达及髓核细胞凋亡情况，明确不同氧浓度下髓核细胞的生物学特性，探讨椎间盘退变的机制。
方法：取人正常及退变髓核细胞，取第2代细胞用于影像学鉴定，取第3代细胞分别采用37 ℃、空气、100%湿度环境，100 μmol/L CoCl2浓度的Leibovitz’s培养基、100 μmol/L H2O2浓度的10%胎牛血清培养基构建常氧、低氧及氧化应激环境，将细胞分为正常髓核细胞+低氧环境、正常髓核细胞+常氧环境、正常髓核细胞+氧化应激环境、退变髓核细胞+低氧环境、退变髓核细胞+常氧环境、退变髓核细胞+氧化应激环境6组；采用CCK-8法检测细胞活力及增殖情况，流式细胞术检测细胞凋亡率，RT-PCR、Western-blot检测PI3K、AKT、HIF-1α的蛋白及mRNA表达情况。

结果与结论：①在不同的氧浓度下，正常及退变髓核细胞的增殖率随氧浓度升高而下降，凋亡率随氧浓度升高而上升(P < 0.05)，以正常髓核细胞+低氧环境为对照组，细胞正常与否对凋亡率影响极为显著(P < 0.001)，氧浓度对髓核细胞增殖率与凋亡率影响极为显著(P < 0.001)，细胞是否退变与氧浓度二者交互作用对髓核细胞增殖率与凋亡率影响显著(P < 0.05)。②在不同的氧浓度下，正常髓核细胞中低氧环境下PI3K、AKT、HIF-1α的蛋白及mRNA表达最高，氧化应激环境下其次，常氧环境下最低；退变髓核细胞中3项指标的蛋白和mRNA表达随氧浓度升高而下降；正常髓核细胞中PI3K、Akt的蛋白、mRNA表达均显著高于退变髓核细胞(P < 0.05)。以正常髓核细胞+低氧环境为对照组，髓核细胞正常或退变及氧浓度对上述3项指标的蛋白和mRNA表达影响均极为显著，二者交互作用对3项指标的蛋白和mRNA表达影响极为显著(P < 0.001)。③结果表明，髓核细胞的增殖、凋亡与氧浓度和细胞自身功能状态密切相关，PI3K/Akt/HIF-1α信号通路在不同细胞功能状态及不同氧浓度环境下具有拮抗调节作用，其机制可能与PI3K/Akt信号通路被激活，并翻译转录HIF-1α，从而维持活性氧自由基代谢平衡有关。该通路对于抑制氧化应激，拮抗髓核细胞凋亡，进而延缓椎间盘退变具有重要意义。

https://orcid.org/0009-0003-3671-5024(周明瀚)；https://orcid.org/0000-0001-9097-4535(张慧)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 氧浓度, 髓核细胞, 细胞凋亡, 氧化应激, 生物学特性, 信号通路

Abstract: BACKGROUND: Intervertebral disc degeneration is a condition caused by the combined effects of various factors, such as cellular apoptosis, oxidative stress, and inflammatory responses. Currently, it is believed that the core of this condition lies in the degeneration and apoptosis of nucleus pulposus cells (NPCs). However, the specific pathological mechanisms remain unclear.
OBJECTIVE: By investigating the expression of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/hypoxia-inducible factor 1-alpha (HIF-1α) signaling pathway and the apoptosis of NPCs under different oxygen concentrations, to clarify the biological characteristics of NPCs under varying oxygen levels, and to explore the mechanisms of intervertebral disc degeneration.
METHODS: Normal and degenerated NPCs were collected. The second-generation cells were used for imaging identification. The third-generation cells were cultured in the following conditions: 37°C, air, 100% humidity. Leibovitz’s medium containing 100 μmol/L CoCl2 and 10% fetal bovine serum medium containing 100 μmol/L H2O2 were used to create distinct oxygen concentration environments for the third-generation cells, allowing for the investigation of cellular responses and behaviors under normal, low oxygen, and oxidative stress conditions. The third-generation cells were divided into six groups: normal NPCs+hypoxia, normal NPCs+normoxia, normal NPCs+oxidative stress, degenerated NPCs+hypoxia, degenerated NPCs+normoxia, and degenerated NPCs+oxidative stress groups. Cell viability and proliferation were assessed using the cell counting kit-8 method. Cell apoptosis was detected using flow cytometry. RT-PCR and western blot assay were utilized to examine the protein and mRNA expression levels of PI3K, AKT, and HIF-1α.
RESULTS AND CONCLUSION: At different oxygen concentrations, the cell proliferation rate of both normal and degenerated NPCs decreased as the oxygen concentration increased. Conversely, the apoptosis rate increased as the oxygen concentration rose (P < 0.05). With the normal NPCs+hypoxia group as the control group, the effect of degenerated NPCs on the apoptosis rate was highly significant (P < 0.001). Oxygen concentration had a highly significant impact on both NPC proliferation rate and apoptosis rate (P < 0.001). The interaction between cell degeneration and oxygen concentration significantly affected both NPC proliferation and apoptosis rates (P < 0.05). At different oxygen concentrations, the protein and mRNA expression levels of PI3K, AKT, and HIF-1α in normal NPCs were highest under low oxygen concentration, followed by oxidative stress environment, and lowest under normoxia. In degenerated NPCs, the protein and mRNA expression levels of PI3K, AKT, and HIF-1α decreasd as the oxygen concentration increased. The protein and mRNA expression levels of PI3K and Akt in normal NPCs were significantly higher than those in degenerated NPCs (P < 0.05). With the normal NPCs+hypoxia group as the control group, the effects of normal or degenerated NPCs, as well as oxygen concentration, on the protein and mRNA expression of PI3K, AKT, and HIF-1α were highly significant. The interaction between cell degeneration and oxygen concentration also had an extremely significant effect on the protein and mRNA expression of PI3K, AKT, and HIF-1α (P < 0.001). To conclude, there is a close correlation between the proliferation and apoptosis of NPCs and both oxygen concentration and the cellular functional state. The PI3K/Akt/HIF-1α signaling pathway exhibits antagonistic regulatory effects under various cellular functional states and different oxygen concentration environments. The mechanism may be associated with the activation of the PI3K/Akt signaling pathway, which consequently transcribes HIF-1α, thus maintaining the balance of reactive oxygen species metabolism. This pathway plays a significant role in inhibiting oxidative stress, antagonizing NPCs apoptosis, and consequently delaying intervertebral disc degeneration.

Key words: oxygen concentration, nucleus pulposus cell, cell apoptosis, oxidative stress, biological characteristics, signaling pathway

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