紫铆花素抑制脂多糖诱导的骨髓源性巨噬细胞炎症反应的研究

doi:10.3969/j.issn.1004-616x.2020.03.003

癌变·畸变·突变 ›› 2020, Vol. 32 ›› Issue (3): 171-176.doi: 10.3969/j.issn.1004-616x.2020.03.003

紫铆花素抑制脂多糖诱导的骨髓源性巨噬细胞炎症反应的研究

陈子卓¹, 徐宇航¹, 姜晓旭², 赵九洲¹, 朱敬朴¹, 郑义鹏¹, 吴浩芝¹, 李文丽³, 王欣³, 海春旭³, 于卫华³

1. 空军军医大学基础医学院, 陕西西安 710032;
2. 空军军医大学第一附属医院综合诊疗科, 陕西西安 710032;
3. 空军军医大学军事预防医学院军事毒理学与防化医学教研室, 陕西省自由基生物学与医学重点实验室, 特殊作业环境危害评估与防治教育部重点实验室, 陕西西安 710032

收稿日期:2020-03-03 修回日期:2020-04-21 出版日期:2020-05-31 发布日期:2020-06-03
通讯作者: 王欣,E-mail:wangxin@fmmu.edu.cn;海春旭,E-mail:cx-hai@fmmu.edu.cn;于卫华,E-mail:yuweihua1@fmmu.edu.cn E-mail:wangxin@fmmu.edu.cn;cx-hai@fmmu.edu.cn;yuweihua1@fmmu.edu.cn
作者简介:陈子卓,E-mail:761795560@qq.com;徐宇航,E-mail:635427714@qq.com;姜晓旭,E-mail:511390237@qq.com。
基金资助:
国家自然科学基金（NSFC-31800706）

Inhibition of LPS-induced inflammatory response by butein in bone marrow primary macrophages

CHEN Zizhuo¹, XU Yuhang¹, JIANG Xiaoxu², ZHAO Jiuzhou¹, ZHU Jingpu¹, ZHENG Yipeng¹, WU Haozhi¹, LI Wenli³, WANG Xin³, HAI Chunxu³, YU Weihua³

1. School of Basic Medical Sciences, Air Force Medical University, Xi'an 710032;
2. Department of Comprehensive Diagnosis and Treatment, First Affiliated Hospital of Air Force Medical University, Xi'an 710032;
3. Department of Toxicology, Key Lab of Hazard Assessment and Control in Special Operational Environment of Ministry of Education, Shaanxi Provincial Key Lab of Free Radical Biology and Medicine, School of Public Health, Air Force Medical University, Xi'an 710032, Shaanxi, China

Received:2020-03-03 Revised:2020-04-21 Online:2020-05-31 Published:2020-06-03

摘要/Abstract

摘要： 目的：研究紫铆花素（butein）对脂多糖（LPS）诱导小鼠骨髓源性巨噬细胞炎症反应的抑制效应，并探讨JAK2-STAT3通路在其中的作用。方法：分离C57BL/6小鼠骨髓，用40 ng/mL的巨噬细胞集落刺激因子刺激7 d，诱导为骨髓源性巨噬细胞（BMDM）。采用500 ng/mL的LPS刺激BMDM 12 h建立炎症模型，butein干预组采用5、10、20 μmol/L butein与LPS共处理，butein单独处理组为20 μmol/L的butein处理12 h，并设立空白对照组。ELISA法检测BMDM培养液中TNF-α、IL-6和NO的水平；流式细胞术检测细胞内ROS水平；Western blot法检测细胞内iNOS、p-JAK2、JAK2、p-STAT3和STAT3蛋白表达水平，并分析P-JAK2/JAK2和P-STAT3/STAT3蛋白表达水平的比值变化。结果：ELISA实验结果显示，LPS刺激BMDM后，培养液中的TNF-α、IL-6和NO含量显著升高，而5、10、20 μmol/L的butein干预可抑制上述促炎因子的分泌，且呈现剂量-效应关系。流式细胞术检测结果显示，butein抑制了LPS活化BMDM的ROS水平升高。Western blot检测结果表明，LPS刺激后BMDM内iNOS蛋白表达水平升高，JAK2和STAT3蛋白表达水平虽未明显变化，但磷酸化水平显著增加。而butein干预可有效抑制JAK2和STAT3蛋白磷酸化。结论：Butein可抑制LPS诱导的巨噬细胞炎症反应，JAK2-STAT3信号通路可能参与调控这一效应，提示butein是一种炎症相关疾病的候选药物。

关键词: 紫铆花素, Janus激酶2, 信号转导子和转录激活子3, 巨噬细胞, 炎症反应

Abstract: OBJECTIVE: To investigate whether butein protects against LPS-induced inflammation in primary bone marrow macrophages,and to analyze the role of JAK2-STAT3 in this process. METHODS: In order to obtain bone marrow-derived macrophages (BMDM),bone marrows samples from C57BL/6 mice were treated with 40 ng/mL GM-CSF for 7 d. BMDM cells were stimulated with 500 ng/mL LPS for 12 h in the presence or absence of 5,10,20 μmol/L butein. Levels of TNF-α,IL-6 and NO in the culture medium were assessed by ELISA specific kits. To detect intracellular levels of reactive oxygen species (ROS),BMDM were stained with DCFH-DA and analyzed by flow-cytometry. Moreover,Western blot assay was used to detect protein expression of iNOS,p-JAK2,JAK2,p-STAT3 and STAT3. Ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were determined. RESULTS: Exposure to LPS significantly increased the levels of TNF-α,IL-6 and NO in the culture medium of BMDM cells. However,administration of 5,10 and 20 μmol/L butein inhibited the production of these proinflammatory factors in a dose-dependent manner. Flow cytometry assays indicate that butein attenuated ROS generation in LPS-activated BMDM macrophages. In addition,Western blot results suggest that LPS promoted iNOS,p-JAK2 and p-STAT3 protein expression,and butein blunted this process. CONCLUSION: Butein inhibited LPS-induced inflammation in BMDM cells by regulating expressions in the JAK2-STAT3 pathway. Therefore,butein may be useful as a drug for control of inflammatory diseases.

Key words: butein, JAK2, STAT3, macrophages, inflammatory response

中图分类号:

R114

陈子卓, 徐宇航, 姜晓旭, 赵九洲, 朱敬朴, 郑义鹏, 吴浩芝, 李文丽, 王欣, 海春旭, 于卫华. 紫铆花素抑制脂多糖诱导的骨髓源性巨噬细胞炎症反应的研究[J]. 癌变·畸变·突变, 2020, 32(3): 171-176.

CHEN Zizhuo, XU Yuhang, JIANG Xiaoxu, ZHAO Jiuzhou, ZHU Jingpu, ZHENG Yipeng, WU Haozhi, LI Wenli, WANG Xin, HAI Chunxu, YU Weihua. Inhibition of LPS-induced inflammatory response by butein in bone marrow primary macrophages[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(3): 171-176.

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紫铆花素抑制脂多糖诱导的骨髓源性巨噬细胞炎症反应的研究

Inhibition of LPS-induced inflammatory response by butein in bone marrow primary macrophages

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