Department of Pulmonary and Critical care Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Basic Medicine College, Chongqing Medical University,1# Medical College Road, Yuzhong District, Chongqing, China.
Bioengineered. 2022 Jan;13(1):1880-1892. doi: 10.1080/21655979.2021.2018981.
Pyroptosis has pivotal parts within disease development, rendering this attractive mechanism for novel therapeutics. This investigation aimed at analyzing melatonin roles within pyroptosis together with related mechanistics. RLE-6TN cultures were exposed to varying LPS doses for 4.5 h followed by concomitant culturing in the presence of ATP (5 mM) for 0.5 h to induce injury, and the roles of melatonin, N-Acety-L-cysteine (NAC - a ROS scavenger), ML385 (specific Nrf2 inhibitor) were examined. Apoptosis analysis was performed through lactate dehydrogenase (LDH) activity assays, together with propidium iodide (PI) stain-assay. Intracellular ROS were quantified through 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). Pyrolysis-associated proteins, such as nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific protease-1 P20 (Caspase-1 P20), gasdermin D-N (GSDMD-N), and mature interleukin-1β (IL-1β), were identified through Western blotting. Dataset outcomes demonstrated LPS/ATP induce RLE-6TN cell pyroptosis, while melatonin alleviated this phenomenon, visualized through increased cell survival rate, reduction of LDH discharge and PI cellular count. Moreover, melatonin effectively reduced NLRP3 inflammasome triggering in RLE-6TN cells. Meanwhile, this study demonstrated melatonin thwarting over NLRP3 inflammasome triggering was depending on ROS. In addition, this study found that melatonin activated Nrf2/Heme Oxygenase-1 (HO-1) pathway, with pyroptotic-inhibiting function of melatonin was reverted through a bespoke Nrf2-inhibitor and siNrf2. In summary, this study concluded that melatonin prevents RLE-6TN cellular pyroptosis through Nrf2-triggered ROS downregulation.
细胞焦亡在疾病发展中起着关键作用，使其成为新型治疗药物的有吸引力的机制。本研究旨在分析褪黑素在细胞焦亡中的作用及其相关机制。将 RLE-6TN 细胞暴露于不同剂量的 LPS 中 4.5 小时，然后同时在存在 ATP（5mM）的情况下培养 0.5 小时以诱导损伤，并检查褪黑素、N-乙酰-L-半胱氨酸（NAC-一种 ROS 清除剂）、ML385（特异性 Nrf2 抑制剂）的作用。通过乳酸脱氢酶（LDH）活性测定以及碘化丙啶（PI）染色测定来进行细胞凋亡分析。通过 2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）来定量细胞内 ROS。通过 Western blot 鉴定焦亡相关蛋白，如核苷酸结合寡聚结构域样受体含有吡喃结构域 3（NLRP3）、凋亡相关斑点样蛋白含有 CARD（ASC）、半胱氨酸天冬氨酸特异性蛋白酶-1 P20（Caspase-1 P20）、gasdermin D-N（GSDMD-N）和成熟的白细胞介素-1β（IL-1β）。数据集结果表明 LPS/ATP 诱导 RLE-6TN 细胞细胞焦亡，而褪黑素减轻了这种现象，表现在细胞存活率增加、LDH 释放减少和 PI 细胞计数减少。此外，褪黑素有效抑制了 RLE-6TN 细胞中 NLRP3 炎性小体的触发。同时，本研究表明，褪黑素对 NLRP3 炎性小体的触发的抑制作用取决于 ROS。此外，本研究发现，褪黑素激活了 Nrf2/血红素加氧酶-1（HO-1）途径，褪黑素的促焦亡作用通过一种定制的 Nrf2 抑制剂和 siNrf2 被逆转。总之，本研究得出结论，褪黑素通过 Nrf2 触发的 ROS 下调来防止 RLE-6TN 细胞发生细胞焦亡。
