球状脂联素通过AMPK/mTOR途径诱导自噬减轻H2O2诱导的大鼠软骨细胞凋亡。
Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway.
作者信息
Hu Junzheng, Cui Weiding, Ding Wenxiao, Gu Yanqing, Wang Zhen, Fan Weimin
机构信息
Department of Orthopedics, the First Affiliated Hospital with Nanjing Medical University, Nanjing, China.
Department of Respiratory Medicine, Zhongda Hospital, Southeast University, Nanjing, China.
出版信息
Cell Physiol Biochem. 2017;43(1):367-382. doi: 10.1159/000480416. Epub 2017 Aug 31.
BACKGROUND/AIMS: Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN), secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H2O2-induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes.
METHODS
H2O2 was used to induce apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H2O2. Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosis-related proteins were identified by Western blot analysis.
RESULTS
H2O2 (400 µM)-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 µg/mL). gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H2O2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H2O2. Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role in the induction of autophagy and protection of H2O2-induced chondrocytes apoptosis by gAPN.
CONCLUSIONS
gAPN protected chondrocytes from H2O2-induced apoptosis by inducing autophagy possibly associated with AMPK/mTOR signal-pathway activation.
背景/目的：软骨细胞凋亡与骨关节炎的发生和发展密切相关。脂肪组织分泌的全身性脂联素（gAPN）在多种细胞类型中具有强大的抗炎和抗凋亡特性。本研究旨在探讨gAPN诱导的自噬在抑制H2O2诱导的细胞凋亡中的作用以及gAPN诱导软骨细胞自噬的潜在机制。
方法
用H2O2诱导大鼠软骨细胞凋亡损伤。采用CCK-8法检测不同浓度gAPN处理的细胞在有无H2O2情况下的活力。通过流式细胞术和TUNEL染色检测细胞凋亡。用JC-1荧光染色法检测线粒体膜电位。使用自噬抑制剂3-MA和巴弗洛霉素A1处理细胞，然后评估gAPN诱导自噬的效果。为确定下游途径，用AMPK抑制剂Compound C预孵育软骨细胞。通过蛋白质免疫印迹分析鉴定Beclin-1、LC3B、P62和凋亡相关蛋白。
结果
gAPN（0.5μg/mL）减轻了H2O2（400μM）诱导的软骨细胞凋亡和caspase-3激活。gAPN增加了Bcl-2表达并降低了Bax表达。gAPN还消除了H2O2诱导的线粒体膜电位丧失。此外，gAPN的抗凋亡作用与gAPN诱导的自噬有关，表现为Beclin-1和LC3B形成增加以及P62降解。特别是，3-MA抑制gAPN诱导的自噬可阻止gAPN对H2O2诱导的细胞凋亡的保护作用。此外，gAPN增加了p-AMPK表达并降低了p-mTOR表达。Compound C部分抑制了自噬相关蛋白的表达，并恢复了被gAPN抑制的p-mTOR表达。因此，AMPK/mTOR途径在gAPN诱导自噬和保护H2O2诱导的软骨细胞凋亡中起重要作用。
结论
gAPN可能通过激活AMPK/mTOR信号通路诱导自噬，从而保护软骨细胞免受H2O2诱导的凋亡。
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