Mitochondrial Pathway Is Involved in Advanced Glycation End Products-Induced Apoptosis of Rabbit Annulus Fibrosus Cells.
Mitochondrial Pathway Is Involved in Advanced Glycation End Products-Induced Apoptosis of Rabbit Annulus Fibrosus Cells.
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线粒体途径参与晚期糖基化终产物诱导的兔纤维环细胞凋亡
DOI：
10.1097/brs.0000000000002930
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发表时间：
2019-05-15
期刊：
影响因子：
3
通讯作者：
Xiong L
中科院分区：
文献类型：
--
作者：
Hu Y;Shao Z;Cai X;Liu Y;Shen M;Yao Y;Yuan T;Wang W;Ding F;Xiong L
晚期糖基化终产物（AGEs）可诱导兔纤维环（AF）细胞凋亡，线粒体途径可能参与AGEs介导的细胞凋亡。抗氧化剂N-乙酰-L-半胱氨酸可显著逆转AGE诱导的AF细胞凋亡。本研究可为糖尿病椎间盘退变提供理论依据。实验研究。本研究旨在探讨晚期糖基化终产物（AGEs）是否诱导纤维环（AF）细胞凋亡，并进一步探讨其发生机制。近年来的研究表明，AGEs的蓄积是糖尿病椎间盘退变的重要因素。然而，AGEs对椎间盘的影响尚不清楚。用不同浓度的AGEs处理AF细胞3天。分别采用CCK-8和EdU掺入法检测细胞活力和细胞增殖。Annexin V/PI凋亡检测试剂盒和Hoechst 33342检测细胞凋亡。Western blotting检测凋亡相关蛋白Bax、Bcl-2、细胞色素c、caspase-3、caspase-9的表达。实时荧光定量PCR（RT-PCR）检测Bax和Bcl-2 mRNA表达水平。应用5，5 ′，6，6 ′ -四氯-1，1 ′，3，3 ′-四乙基碘化咪唑碳菁（JC-1）染色和2′，7 ′-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探针分别检测AF细胞线粒体膜电位（MMP）和细胞内活性氧（ROS）的产生。结果表明AGEs对AF细胞增殖有抑制作用，并诱导AF细胞凋亡。分子生物学数据显示，AGEs可显著上调Bax的表达，抑制Bcl-2的表达。此外，AGEs增加细胞色素c释放到胞质溶胶中，并增强caspase-9和caspase-3的激活。此外，AGEs处理导致AF细胞MMP降低和细胞内ROS积累。抗氧化剂N-乙酰-L-半胱氨酸（NAC）可显著逆转AGE诱导的MMP降低和AF细胞凋亡。以上结果提示AGEs可诱导AF细胞凋亡，线粒体途径可能参与了AGEs介导的细胞凋亡，为糖尿病IVD退变提供了理论依据。证据等级：不适用
Advanced glycation end-products (AGEs) can induce rabbit annulus fibrosus (AF) cell apoptosis and mitochondrial pathway may be involved in AGEs-mediated cell apoptosis. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced AF cell apoptosis. This research may provide a theoretical basis for diabetic intervertebral disc degeneration. Experimental study. The purposes of this study were to evaluate whether advanced glycation end-products (AGEs) induce annulus fibrosus (AF) cell apoptosis and further to explore the mechanism by which this process occurs. Recent studies revealed that AGEs accumulation is considered an important factor in diabetic intervertebral disc (IVD) degeneration. However, the effect of AGEs on intervertebral disc remains unclear. AF cells were treated with various concentrations of AGEs for 3 days. Cell viability and cell proliferation were measured by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays, respectively. Cell apoptosis was examined by Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3, and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by real-time PCR (RT-PCR). Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by 5,5′,6,6′ -Tetrachloro-1,1′,3,3′- tetraethyl-imidacarbocyanine iodide (JC-1) staining and 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine (NAC) significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggested that AGEs induce rabbit AF cell apoptosis and mitochondrial pathway may be involved in AGEs-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration. Level of Evidence: N/A