褪黑素抑制过氧化氢诱导人髓核细胞的损伤  

Melatonin inhibits hydrogen peroxide-induced injury of human nucleus pulposus cells

作　　者：谢文冠 刘宇涛 崔文波 邝铭业 Xie Wenguan;Liu Yutao;Cui Wenbo;Kuang Mingye(Department of Orthopedics,Dongguan Eighth People’s Hospital,Dongguan Children’s Hospital Affiliated to Guangdong Medical University,Dongguan 523320,Guangdong Province,China)

机构地区：[1]东莞市第八人民医院骨科,广东医科大学附属东莞儿童医院,广东省东莞市523320

出　　处：《中国组织工程研究》2024年第14期2180-2185,共6页Chinese Journal of Tissue Engineering Research

基　　金：东莞市社会科技发展项目(202050715028791),项目负责人:邝铭业。

摘　　要：背景:椎间盘退变是造成腰痛最常见的潜在因素之一,近年来研究表明,褪黑素对缓解椎间盘退变有积极作用,然而褪黑素的潜在作用机制仍有待阐明。目的:探究褪黑素抑制过氧化氢诱导人椎间盘髓核细胞损伤的生物学作用及其潜在的作用机制。方法:体外培养人退行性椎间盘髓核细胞,通过CCK-8法检测细胞增殖情况确定褪黑素及过氧化氢的最佳干预浓度。将过氧化氢处理2 h的细胞作为模型组;经褪黑素干预24 h再用过氧化氢处理2 h的细胞作为褪黑素组;单纯培养基培养的细胞作为对照组。通过活性氧荧光探针(DCFH-DA)检测细胞内活性氧水平,免疫荧光检测BAX和Caspase3表达水平,RT-qPCR检测BAX、BCL-2、Casepase3、PI3K和AKT的mRNA表达水平。结果与结论:①CCK-8实验结果表明,过氧化氢最佳干预浓度为400μmol/L,褪黑素最佳干预浓度为5μmol/L;②与模型组比较,褪黑素组活性氧水平显著降低;③与模型组比较,褪黑素组BAX和Caspase3的平均荧光强度显著降低;④与模型组比较,褪黑素组BAX和Caspase3的mRNA表达降低,而Bcl-2 mRNA表达则升高,褪黑素组的PI3K和AKT的mRNA表达也升高;⑤结果表明,褪黑素可能通过PI3K/AKT信号通路保护退行性椎间盘髓核细胞免受过氧化氢诱导的氧化损伤。BACKGROUND:Intervertebral disc degeneration is one of the most common underlying factors causing low back pain.Recent studies have shown that melatonin has a positive effect on alleviating intervertebral disc degeneration.However,the underlying mechanism of melatonin remains to be elucidated.OBJECTIVE:To explore the biological effect and potential mechanism of melatonin in inhibiting hydrogen peroxide(H2O2)-induced injury of human nucleus pulposus cells.METHODS:Human nucleus pulposus cells insolated from degenerative intervertebral disc were cultured in vitro.Cell proliferation and the optimal intervention concentration of melatonin and H2O2 were detected by cell counting kit-8.The Human nucleus pulposus cells treated with H2O2 were used as a model group;the cells treated with H2O2 and intervened with melatonin were used as a melatonin group;the cells cultured in simple medium were used as a control group.The reactive oxygen species levels were detected by 2',7'-dichlorofluorescin diacetate(DCFH-DA),the expression levels of BAX and Caspase3 were detected by immunofluorescence,and the mRNA expression levels of BAX,BCL-2,Casepase3,PI3K and AKT were detected using the real-time fluorescent quantitative reverse transcription PCR.RESULTS AND CONCLUSION:The results of cell counting kit-8 experiment showed that the optimal intervention concentration of H2O2 was 400μmol/L and the optimal intervention concentration of melatonin was 5μmol/L.The reactive oxygen species level in the melatonin group was significantly lower than that in the model group.The average fluorescence intensity of BAX and Caspase3 in the melatonin group was significantly lower than that in the model group.The mRNA expressions of BAX and Caspase3 in the melatonin group were lower than those in the model group,while the mRNA expression of Bcl-2 was increased.In addition,the mRNA expressions of PI3K and AKT were also higher in the melatonin group compared with the model group.To conclude,melatonin may protect human nucleus pulposus cells from H2O2-indu

关 键 词：椎间盘退行性病变 褪黑素 氧化应激 凋亡 PI3K-Akt通路 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R34