Trigonochinene E promotes lysosomal biogenesis and enhances autophagy via TFEB/TFE3 in human degenerative NP cells against oxidative stress
Trigonochinene E promotes lysosomal biogenesis and enhances autophagy via TFEB/TFE3 in human degenerative NP cells against oxidative stress
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DOI：
10.1016/j.phymed.2023.154720
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发表时间：
2023-03-01
期刊：
影响因子：
7.9
通讯作者：
Hao, Xiaojiang
中科院分区：
文献类型：
--
作者：
Niu, Zhenpeng;Tang, Guihua;Hao, Xiaojiang
背景：巨自噬（Macroautophagy，以下简称自噬）是自噬的主要形式，其功能是将细胞内的物质转运至溶酶体进行降解。大量研究表明，溶酶体生物合成和自噬通量的受损加剧了自噬相关疾病的发展。因此，恢复溶酶体生物合成和细胞自噬通量的修复药物可能对这些疾病的日益增加的患病率具有治疗潜力。目的：本研究的目的是探索从Trigonostemon flavidus中分离的芳香族四降二萜--胡芦巴烯E（TE）对溶酶体生物合成和自噬的影响，并阐明潜在的潜在机制。本研究应用了四种人细胞系，HepG 2，髓核（NP），HeLa和HEK 293细胞。MTT法检测TE的细胞毒性。采用基因转移技术、蛋白质印迹法、实时荧光定量PCR和共聚焦显微镜技术对40 μ M TE诱导的溶酶体生物发生和自噬通量进行了分析。结果：TE通过激活溶酶体转录因子EB（TFEB）和转录因子E3（TFE 3）促进溶酶体生物合成和自噬流。从机制上讲，TE通过mTOR/PKC/ROS非依赖性和内质网（ER）应激介导的途径诱导TFEB和TFE 3核转位。ER应激的PERK和IRE 1 α分支对于TE诱导的自噬和溶酶体生物发生至关重要。TE激活PERK，PERK介导TFEB/TFE 3的钙调神经磷酸酶去磷酸化，而IRE 1 α被激活并导致STAT 3失活，这进一步增强了自噬和溶酶体生物合成。在功能上，TFEB或TFE 3的敲低损害TE诱导的溶酶体生物发生和自噬通量。此外，TE诱导的自噬保护NP细胞免受氧化应激，以改善椎间盘退变（IVDD）。结论：在这里，我们的研究表明，TE可以通过PERK-钙调磷酸酶轴和IRE 1 α-STAT 3轴诱导TFEB/TFE 3依赖性溶酶体生物发生和自噬。与其他调节溶酶体生物发生和自噬的药物不同，TE显示出有限的细胞毒性，从而为使用TE治疗自噬-溶酶体途径受损疾病（包括IVDD）的治疗机会提供了新的方向。
Background: Macroautophagy (henceforth autophagy) is the major form of autophagy, which delivers intracel-lular cargo to lysosomes for degradation. Considerable research has revealed that the impairment of lysosomal biogenesis and autophagic flux exacerbates the development of autophagy-related diseases. Therefore, reparative medicines restoring lysosomal biogenesis and autophagic flux in cells may have therapeutic potential against the increasing prevalence of these diseases.Purpose: The aim of the present study was thus to explore the effect of trigonochinene E (TE), an aromatic tet-ranorditerpene isolated from Trigonostemon flavidus, on lysosomal biogenesis and autophagy and to elucidate the potential underlying mechanism.Methods: Four human cell lines, HepG2, nucleus pulposus (NP), HeLa and HEK293 cells were applied in this study. The cytotoxicity of TE was evaluated by MTT assay. Lysosomal biogenesis and autophagic flux induced by 40 mu M TE were analyzed using gene transfer techniques, western blotting, real-time PCR and confocal micro-scopy. Immunofluorescence, immunoblotting and pharmacological inhibitors/activators were applied to deter-mine the changes in the protein expression levels in mTOR, PKC, PERK, and IRE1 alpha signaling pathways.Results: Our results showed that TE promotes lysosomal biogenesis and autophagic flux by activating the tran-scription factors of lysosomes, transcription factor EB (TFEB) and transcription factor E3 (TFE3). Mechanistically, TE induces TFEB and TFE3 nuclear translocation through an mTOR/PKC/ROS-independent and endoplasmic reticulum (ER) stress-mediated pathway. The PERK and IRE1 alpha branches of ER stress are crucial for TE-induced autophagy and lysosomal biogenesis. Whereas TE activated PERK, which mediated calcineurin dephosphoryla-tion of TFEB/TFE3, IRE1 alpha was activated and led to inactivation of STAT3, which further enhanced autophagy and lysosomal biogenesis. Functionally, knockdown of TFEB or TFE3 impairs TE-induced lysosomal biogenesis and autophagic flux. Furthermore, TE-induced autophagy protects NP cells from oxidative stress to ameliorate intervertebral disc degeneration (IVDD). Conclusions: Here, our study showed that TE can induce TFEB/TFE3-dependent lysosomal biogenesis and auto-phagy via the PERK-calcineurin axis and IRE1 alpha-STAT3 axis. Unlike other agents regulating lysosomal biogenesis and autophagy, TE showed limited cytotoxicity, thereby providing a new direction for therapeutic opportunities to use TE to treat diseases with impaired autophagy-lysosomal pathways, including IVDD.