Rapamycin mitigates inflammation-mediated disc matrix homeostatic imbalance by inhibiting mTORC1 and inducing autophagy through Akt activation

IF 3.4 3区 医学 Q1 ORTHOPEDICS
JOR Spine Pub Date : 2024-01-02 DOI:10.1002/jsp2.1303
Takashi Yurube, William J. Buchser, Zhongying Zhang, Prashanta Silwal, Michael T. Lotze, James D. Kang, Gwendolyn A. Sowa, Nam V. Vo
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Abstract

Background

Low back pain is a global health problem that originated mainly from intervertebral disc degeneration (IDD). Autophagy, negatively regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, prevents metabolic and degenerative diseases by removing and recycling damaged cellular components. Despite growing evidence that autophagy occurs in the intervertebral disc, the regulation of disc cellular autophagy is still poorly understood.

Methods

Annulus fibrosus (rAF) cell cultures derived from healthy female rabbit discs were used to test the effect of autophagy inhibition or activation on disc cell fate and matrix homeostasis. Specifically, different chemical inhibitors including rapamycin, 3-methyladenine, MK-2206, and PP242 were used to modulate activities of different proteins in the PI3K/Akt/mTOR signaling pathway to assess IL-1β-induced cellular senescence, apoptosis, and matrix homeostasis in rAF cells grown under nutrient-poor culture condition.

Results

Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), reduced the phosphorylation of mTOR and its effector p70/S6K in rAF cell cultures. Rapamycin also induced autophagic flux as measured by increased expression of key autophagy markers, including LC3 puncta number, LC3-II expression, and cytoplasmic HMGB1 intensity and decreased p62/SQSTM1 expression. As expected, IL-1β stimulation promoted rAF cellular senescence, apoptosis, and matrix homeostatic imbalance with enhanced aggrecanolysis and MMP-3 and MMP-13 expression. Rapamycin treatment effectively mitigated IL-1β-mediated inflammatory stress changes, but these alleviating effects of rapamycin were abrogated by chemical inhibition of Akt and mTOR complex 2 (mTORC2).

Conclusions

These findings suggest that rapamycin blunts adverse effects of inflammation on disc cells by inhibiting mTORC1 to induce autophagy through the PI3K/Akt/mTOR pathway that is dependent on Akt and mTORC2 activities. Hence, our findings identify autophagy, rapamycin, and PI3K/Akt/mTOR signaling as potential therapeutic targets for IDD treatment.

Abstract Image

雷帕霉素通过抑制 mTORC1 和激活 Akt 诱导自噬,缓解炎症介导的椎间盘基质平衡失调
腰痛是一个全球性的健康问题,主要源于椎间盘退变(IDD)。自噬受磷脂酰肌醇 3- 激酶(PI3K)/Akt/哺乳动物雷帕霉素靶标(mTOR)信号通路的负调控,通过清除和回收受损细胞成分来预防代谢性和退行性疾病。尽管越来越多的证据表明自噬发生在椎间盘中,但人们对椎间盘细胞自噬的调控仍然知之甚少。我们用来自健康雌兔椎间盘的纤维环(rAF)细胞培养物来测试自噬抑制或激活对椎间盘细胞命运和基质平衡的影响。具体来说,研究人员使用不同的化学抑制剂(包括雷帕霉素、3-甲基腺嘌呤、MK-2206和PP242)来调节PI3K/Akt/mTOR信号通路中不同蛋白的活性,以评估IL-1β诱导的细胞衰老、凋亡和在营养缺乏培养条件下生长的rAF细胞的基质稳态。雷帕霉素是mTOR复合体1(mTORC1)的抑制剂,它能减少rAF细胞培养物中mTOR及其效应因子p70/S6K的磷酸化。雷帕霉素还能诱导自噬通量,其测量方法是增加关键自噬标记物的表达,包括 LC3 点的数量、LC3-II 的表达和细胞质 HMGB1 的强度,以及降低 p62/SQSTM1 的表达。正如预期的那样,IL-1β的刺激促进了rAF细胞的衰老、凋亡和基质平衡失调,增强了凝集素溶解、MMP-3和MMP-13的表达。这些研究结果表明,雷帕霉素通过抑制mTORC1,通过依赖于Akt和mTORC2活性的PI3K/Akt/mTOR途径诱导自噬,从而减轻炎症对椎间盘细胞的不利影响。因此,我们的研究结果将自噬、雷帕霉素和PI3K/Akt/mTOR信号转导确定为治疗IDD的潜在治疗靶点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
JOR Spine
JOR Spine ORTHOPEDICS-
CiteScore
6.40
自引率
18.90%
发文量
42
审稿时长
10 weeks
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