4-辛基衣康酸酯对IL-1诱导髓核细胞氧化损伤的保护作用

Protective effect of 4-octyl itaconate on IL-1-induced oxidative damage of nucleus pulposus cells

摘要:

[目的] 探讨 4-辛基衣康酸酯 （4-octyl itaconate, 4OI） 对 IL-1 诱导的小鼠髓核细胞氧化损伤的保护作用及其机制。 [方法] 分离培养小鼠髓核细胞。CCK8 法检测小鼠髓核细胞活力。荧光素酶报告基因法、免疫共沉淀、RT-qPCR 和 WB 法检测 4OI 和 IL-1 对 Nrf2 通路的影响。CellROX 探针和 DCF-DA 探针检测细胞 ROS 水平。JC-1 法、ssDNA ELISA、TBAR 检测细胞氧化损伤。LDH ELISA、台盼蓝染色法、TUNEL 法、Annexin V 流式细胞术检测细胞凋亡。通过慢病毒 shRNA 转染和 CRIS- PR/Cas9 技术研究沉默 Nrf2 和 Keap1 对 4OI 抑制细胞凋亡的影响。[结果]4OI 促进小鼠髓核细胞 Keap1-Nrf2 解偶联，Nrf2 重新进入细胞核中调控下游基因转录。4OI 显著抑制 ROS 水平，改善 IL-1 诱导的小鼠髓核细胞氧化损伤。4OI 逆转 IL-1 诱导的小鼠髓核细胞活力下降，LDH 增加和死细胞增加，4OI 显著抑制 IL-1 诱导的小鼠髓核细胞死亡。4OI 抑制 IL-1 诱导的 Caspase-3 和 Caspase-9 活性增加，以及蛋白酶 PARP1 裂解片段和细胞色素 C 含量增加，显著抑制 IL-1 诱导的细胞凋亡。沉默小鼠髓核细胞中 Nrf2 和 Keap1 的表达，4OI 对 IL-1 诱导的细胞损伤的保护作用被抵消。[结论]4OI 通过激活 Nrf2 通路保护髓核细胞免受 IL1 诱导的氧化损伤，为椎间盘退变的治疗提供了新策略。

Abstract:

[Objective] To investigate the protective effect of 4-octyl itaconate (4OI) on oxidative damage of mouse nucleus pulposus cells induced by IL-1 and its mechanism. [Methods] Mouse nucleus pulposus cells were isolated and cultured, while CCK8 method was used to detect the viability of the cells. The effects of 4OI and IL-1 on Nrf2 pathway were detected by luciferase reporter gene method, immunoprecipitation, RT-qPCR and WB method. In addition, JC-1 assay, ssDNA ELISA and TBAR were used to detect oxidative damage, LDH ELISA, Trypan blue staining, TUNEL assay and Annexin V flow cytometry were used to detect apoptosis. The effect of silencing Nrf2 and Keap1 on apoptosis inhibition by 4OI was studied by lentiviral shRNA transfection and CRISPR/Cas9 technique. [Results] The 4OI promoted the dissociation of KEap1-NRF2 in mouse nucleus pulposus cells, and Nrf2 re-entered the nucleus to regulate downstream gene transcription. The 4OI significantly inhibited ROS levels and improved IL-1-induced oxidative damage of mouse nucleus pulposus cells. 4OI reversed IL-1-induced decline in nucleus pulposus cell viability, increased LDH and increased dead cells. Furthermore, 4OI significantly inhibited IL-1-induced nucleus pulposus cell death, inhibited the increase of IL-1-induced Caspase-3 and Caspase-9 activities, as well as the increase of protease PARP1 cleavage fragment and cytochrome C content, and significantly inhibited IL1-induced apoptosis. However, silencing the expression of Nrf2 and Keap1 in mouse nucleus pulposus cells negated the protective effect of 4OI on IL-1-induced cell damage. [Conclusion] 4OI protects nucleus pulposus cells from IL-1-induced oxidative damage by activating the Nrf2 pathway, providing a new strategy for the treatment of intervertebral disc degeneration.