Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis
Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis
复制标题
肾上腺髓质素通过 ROS-AMPK-mTOR 轴促进自噬，减轻 Leydig 细胞焦亡
DOI：
10.1038/s41419-019-1728-5
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发表时间：
2019-06-20
影响因子：
9
通讯作者：
Liu, Bo-long
中科院分区：
文献类型：
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作者：
Li, Ming-yong;Zhu, Xia-lian;Liu, Bo-long
肾上腺髓质素(adrenomedullin，ADM)在间质细胞中具有抗氧化、抗炎、抗凋亡等作用。然而，ADM在间质细胞上睑下垂中的作用和机制尚不清楚。本研究首次揭示了ADM通过促进自噬对脂多糖(LPS)所致间质细胞的热下垂和生物学功能的保护作用。用不同浓度的脂多糖和阿霉素加或不加N-乙酰-L半胱氨酸(NAC)或3-甲基腺嘌呤(3-MA)处理原代大鼠间质细胞。CCK-8和BrdU掺入法检测细胞增殖，DCFDA法检测ROS水平。采用实时定量聚合酶链式反应、免疫印迹、免疫荧光、透射电子显微镜、原位末端标记法和流式细胞术检测ADM对间质细胞的下垂、自噬、类固醇生成酶及AMPK/mTOR信号转导的影响。与NAC一样，ADM剂量依赖性地降低了脂多糖诱导的细胞毒性和ROS的过量产生。ADM还通过逆转NLRP3、Asc、caspase-1、IL-1β、IL-18、GSDMD、caspase-3、caspase-7、TUNEL阳性和PI的表达，以及活化的caspase-1双染率、DNA片段化和LDH浓度，呈剂量依赖性地改善内毒素诱导的下丘脑炎性下垂。ADM呈剂量依赖性增加脂多糖诱导的间质细胞自噬，表现为Lc3-I/II、Beclin-1和ATG-5表达增加，p62表达减少，自噬小体形成减少，Lc3-II/Lc3-I比值升高。然而，与3-MA联合处理可明显减少自噬。此外，ADM剂量依赖性地挽救了脂多糖诱导的间质细胞中类固醇生成酶的表达，包括STAR、P450scc、3β-HSD和CyP17，以及睾酮的产生。与雷帕霉素一样，ADM可剂量依赖性地增强脂多糖诱导的间质细胞AMPK的磷酸化，但降低mTOR的磷酸化，这可通过与3-MA共同孵育来挽救。此外，ADM和雷帕霉素联合作用可进一步减少脂多糖诱导的间质细胞的下垂，促进自噬。ADM可能通过ROS-AMPK-mTOR轴激活自噬，从而保护间质细胞的类固醇生成功能，使其免受热毒症的侵袭。
Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM's effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1 beta, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3 beta-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS-AMPK-mTOR axis.