特女贞苷对脂多糖诱导的巨噬细胞促炎反应的抑制作用及机制

doi:10.3969/j.issn.1004-616x.2023.04.001

癌变·畸变·突变 ›› 2023, Vol. 35 ›› Issue (4): 245-252.doi: 10.3969/j.issn.1004-616x.2023.04.001

• 论著 • 下一篇

特女贞苷对脂多糖诱导的巨噬细胞促炎反应的抑制作用及机制

姜晓旭¹, 孙慕涵², 张渤旭³, 王钰越³, 马加骏³, 师敏捷², 于卫华², 郭显⁴

1. 空军军医大学基础医学院计算机基础教研室, 陕西西安 710032;
2. 空军军医大学军事预防医学系军事毒理学教研室, 陕西西安 710032;
3. 空军军医大学学员旅, 陕西西安 710032;
4. 空军军医大学航空航天医学系航空航天医学教育部重点实验室, 陕西西安 710032

收稿日期:2023-04-17 修回日期:2023-06-14 出版日期:2023-07-30 发布日期:2023-08-04
通讯作者: 于卫华;郭显
作者简介:姜晓旭，E-mail：511390237@qq.com。
基金资助:
国家自然科学基金(32171231)；陕西省重点研发计划(2022SF-276)；航空航天临床医学双一流建设引导专项后备人才扶持基金(2022HBRC07)

Protection against LPS-induced pro-inflammatory response by nuezhenide in macrophages

JIANG Xiaoxu¹, SUN Muhan², ZHANG Boxu³, WANG Yuyue³, MA Jiajun³, SHI Minjie², YU Weihua², GUO Xian⁴

1. Department of Basic Computer Science, School of Basic Medicine, Air Force Medical University, Xi'an 710032;
2. Department of Military Toxicology, School of Preventive Medicine, Air Force Medical University, Xi'an 710032;
3. Cadet Brigade, Air Force Medical University, Xi'an 710032;
4. Key Laboratory of Aerospace Medicine, Ministry of Education, Department of Aerospace Medicine, Air Force Medical University, Xi'an 710032, Shaanxi, China

Received:2023-04-17 Revised:2023-06-14 Online:2023-07-30 Published:2023-08-04

摘要/Abstract

摘要： 目的：探讨特女贞苷(NED)抑制细菌脂多糖(LPS)诱导巨噬细胞促炎反应的机制。方法：用50 μmol/L NED处理RAW264.7巨噬细胞12 h后，采用CCK-8法测定细胞活力，Annexin V-FITC/PI法测定细胞凋亡率，转录组测序分析异常基因表达，免疫荧光检测Nrf2蛋白表达定位。进一步使用100 ng/mL LPS(模型组)与不同浓度(0、12.5、25和50 μmol/L)NED联合处理RAW264.7细胞干预12 h后，采用ELISA测定细胞上清液中TNF-α和IL-6含量；实时荧光定量PCR(qPCR)检测细胞TNF-α和IL-6 mRNA的表达；分别采用DCFH-DA、MitoTracker-Green和JC-1染色测定细胞中活性氧、线粒体含量和膜电位；试剂盒测定细胞内抗氧化酶活力、线粒体复合物I和III活性以及ATP合成的变化。结果：与空白对照组相比，50 μmol/L NED对巨噬细胞存活和凋亡无明显影响(P>0.05)，但可抑制模型组LPS诱导促炎因子TNF-α、IL-6的表达和分泌(P<0.05)，并增强抗炎因子转录表达(P<0.05)，呈现明显抗炎效应。与模型组比较，联合NED干预组增强Nrf2核转位及下游抗氧化因子转录表达(P<0.05)，提高细胞还原型谷胱甘肽(GSH)含量(P<0.05)，并降低活性氧(ROS)和氧化型谷胱甘肽(GSSG)水平(P<0.05)，减轻LPS刺激巨噬细胞氧化应激(P<0.05)。此外，NED刺激线粒体生物合成基因表达升高(P<0.05)，逆转LPS诱导线粒体膜电位、复合物I和III活性下降(P<0.05)，促进线粒体数量增多和ATP合成(P<0.05)。结论：特女贞苷能够激活Nrf2抗氧化系统，增强线粒体生物合成和代谢功能，抑制LPS诱导巨噬细胞促炎分化，具有成为抗炎症疾病候选药物的潜力。

关键词: 特女贞苷, 巨噬细胞, 炎症反应, 抗氧化, 线粒体生物合成

Abstract: OBJECTIVE： This study aimed to investigate effects of nuezhenide (NED) on LPS-induced proinflammatory response in macrophages. METHODS： RAW264.7 cells were treated with 50 μmol/L NED for 12 hours. Differential gene expressions were detected via transcriptome，cell viability using the CCK8 kit，and apoptosis rates measured using Annexin V-FITC/PI staining，and expression and localization of Nrf2 protein were detected by immunofluorescence. The cells were treated with LPS (100 ng/mL) and NED (12.5，25 and 50 μmol/L) for 12 hours. Then，TNF-α and IL-6 contents in the supernatant of cultured cells were determined by ELISA，and expressions of TNF-α and IL-6 by RT-PCR. Moreover，ROS，mitochondrial mass，and membrane potential were measured by flow cytometry after staining with DCFH-DA，MitoTracker-Green and JC-1，respectively. Enzyme activities of SOD and CAT，activities of mitochondrial complexes I and Ⅲ，as well as ATP contents were determined using commercial kits. RESULTS： Our results show that 50 μmol/L NED had no significant effects on survival and apoptosis of the RAW264.7 macrophages (P>0.05). However，NED treatments blunted LPS-induced expression of TNF-α and IL-6 (P<0.05)，and enhanced expression of anti-inflammatory cytokines (P<0.05). Meanwhile，NED significantly promoteed Nrf2 nuclear translocation and downstream antioxidant transcription (P<0.05)，which contributed to inhibition of LPS-induced accumulation of ROS and GSSG (P<0.05). In addition，NED reversed the LPS-induced decreased of mitochondrial membrane potential，mitochondrial complexes I and III activities，as well as ATP contents through promoting mitochondrial biogenesis (P<0.05). CONCLUSION： NED inhibited LPS-induced macrophages pro-inflammatory differentiation through enhancing Nrf2 antioxidant system and mitochondrial biogenesis. Application of NED may be a potential strategy for treatment of inflammatory diseases.

Key words: nuezhenide, macrophages, inflammatory response, antioxidant, mitochondrial biogenesis

中图分类号:

R392.11

姜晓旭, 孙慕涵, 张渤旭, 王钰越, 马加骏, 师敏捷, 于卫华, 郭显. 特女贞苷对脂多糖诱导的巨噬细胞促炎反应的抑制作用及机制[J]. 癌变·畸变·突变, 2023, 35(4): 245-252.

JIANG Xiaoxu, SUN Muhan, ZHANG Boxu, WANG Yuyue, MA Jiajun, SHI Minjie, YU Weihua, GUO Xian. Protection against LPS-induced pro-inflammatory response by nuezhenide in macrophages[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2023, 35(4): 245-252.

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特女贞苷对脂多糖诱导的巨噬细胞促炎反应的抑制作用及机制

Protection against LPS-induced pro-inflammatory response by nuezhenide in macrophages

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