Effects and mechanisms of higenamine on polarization and pyroptosis of M1 macrophages

WAN Zijian; FAN Tianyou; CHEN Deta; LI Yumei

doi:10.16025/j.1674-1307.2024.12.009

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Effects and mechanisms of higenamine on polarization and pyroptosis of M1 macrophages

更新时间：2025-01-08

- Effects and mechanisms of higenamine on polarization and pyroptosis of M1 macrophages
- Beijing Journal of Traditional Chinese Medicine Vol. 43, Issue 12, Pages: 1366-1377(2024)
- 作者机构：
  
  上海中医药大学附属市中医医院，上海 200040
- 作者简介：
- 基金信息：
- DOI：10.16025/j.1674-1307.2024.12.009
  CLC：
- Published：25 December 2024，
  
  Received：19 March 2024，
- 稿件说明：
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万梓健，樊天佑，陈德塔，等.去甲乌药碱对M1巨噬细胞极化和焦亡的影响及机制研究［J］.北京中医药，2024，43（12）：1366-1377.

WAN Zijian,FAN Tianyou,CHEN Deta,et al.Effects and mechanisms of higenamine on polarization and pyroptosis of M1 macrophages[J]. Beijing Journal of Traditional Chinese Medicine,2024,43(12):1366-1377.
万梓健，樊天佑，陈德塔，等.去甲乌药碱对M1巨噬细胞极化和焦亡的影响及机制研究［J］.北京中医药，2024，43（12）：1366-1377. DOI： 10.16025/j.1674-1307.2024.12.009.

WAN Zijian,FAN Tianyou,CHEN Deta,et al.Effects and mechanisms of higenamine on polarization and pyroptosis of M1 macrophages[J]. Beijing Journal of Traditional Chinese Medicine,2024,43(12):1366-1377. DOI： 10.16025/j.1674-1307.2024.12.009.

摘要

目的

探讨去甲乌药碱（HG）对M1巨噬细胞（RAW264.7细胞诱导）极化和焦亡的影响及机制。

方法

采用CCK-8法测定RAW264.7细胞的增殖抑制率，筛选HG最佳实验浓度。将细胞随机分为空白组、白细胞介素（IL）-4+IL-13组、脂多糖（LPS）组、LPS+三磷酸腺苷（ATP）组、IL-4+IL-13+HG组、LPS+HG组、LPS+ATP+HG组。空白组、IL-4+IL-13组、LPS组、LPS+ATP组分别给予等量PBS（IL-4和IL-1320 ng/mL）、1 μg/mL LPS、1 μg/mL LPS+5 mmol/L ATP干预；各给药组在以上干预基础上均给予50 μmol/L HG处理。Real-time PCR检测巨噬细胞极化和焦亡相关mRNA的表达，Western blotting法检测巨噬细胞极化和焦亡相关蛋白的表达，流式细胞术检测巨噬细胞表面标记物，进行M1巨噬细胞全基因转录组测序分析、基因本体（GO）富集分析、基因组百科全书（KEGG）通路富集分析。显微镜下观察细胞形态，酶联免疫吸附试验（ELISA）检测转化生长因子（TGF）-β信号通路相关蛋白TGF-β1和血小板反应蛋白（THBS）-1。

结果

与空白组比较，LPS组IL-1β、诱导型一氧化氮合酶（iNOS）、肿瘤坏死因子（TNF）-α mRNA和iNOS、TNF-α蛋白相对表达量均高（均

0.01）；与LPS组比较，LPS+HG组IL-1β、iNOS、TNF-α mRNA和iNOS、TNF-α蛋白相对表达量均低（均

0.01）。与空白组比较，LPS组CD206、精氨酸酶（Arg）-1蛋白相对表达量低（均

0.05），CD86蛋白相对表达量及CD86/CD206高（均

0.01）；与LPS组比较，LPS+HG组CD206、Arg-1蛋白相对表达量高（均

0.01），CD86蛋白相对表达量及CD86/CD206低（均

0.01）。与空白组比较，IL-4+IL-13组Arg-1、IL-10 mRNA和CD206、Arg-1蛋白相对表达量高（

0.05，

0.01）；与IL-4+IL-13组比较，IL-4+IL-13+HG组Arg-1、IL-10 mRNA和CD206、Arg-1蛋白相对表达量高（

0.05，

0.01）。HG导致M1巨噬细胞337个基因表达下调、148个基因表达上调，在十大核心基因中选择相关性最大的THBS-1基因进行KEGG富集分析，结果显示THBS-1/TGF-β1信号通路在HG调控M1巨噬细胞中具有重要作用。与空白组比较，LPS组THBS-1 mRNA相对表达量高、TGF-β1 mRNA相对表达量低（均

0.01）；与LPS组比较，LPS+HG组THBS-1 mRNA相对表达量低、TGF-β1 mRNA相对表达量高（均

0.01）。用HG处理LPS+ATP诱导的巨噬细胞焦亡后，显微镜观察发现HG显著改善了焦亡巨噬细胞的细胞肿胀和破裂。与空白组比较，LPS+ATP组NOD样受体蛋白

（NLRP）3、半胱天冬氨酸蛋白酶（Casp）-1、IL-1β mRNA和NLRP3、Gasdermins-D Full体（GSDMD-F）、Gasdermins-D N端体（GSDMD-N）、Casp-1蛋白相对表达量高（

0.05，

0.01）；与LPS+ATP组比较，LPS+ATP+HG组NLRP3、Casp-1、IL-1β mRNA和NLRP3、GSDMD-F、GSDMD-N、Casp-1蛋白相对表达量低（

0.05，

0.01）。与空白组比较，LPS+ATP组TGF-β1 mRNA和蛋白相对表达量均低（均

0.01），THBS-1 mRNA和蛋白相对表达量均高（均

0.01）；与LPS+ATP组比较，LPS+ATP+HG组TGF-β1 mRNA和蛋白相对表达量均高（

0.01，

0.05），THBS-1 mRNA和蛋白相对表达量均低（均

0.01）。

结论

HG可以抑制M1巨噬细胞极化和焦亡并促进其由M1向M2转化，该作用与THBS-1/TGF-β1通路有关。

Abstract

Objective

To investigate the effects and mechanisms of higenamine （HG） on polarization and pyroptosis of M1 macrophages （induced by RAW264.7 cells）.

Methods

The proliferation inhibition rate of RAW264.7 cells was determined by the CCK-8 method to screen the optimal experimental concentration of HG. The cells were randomly divided into blank group， interleukin （IL）-4+interleukin （IL）-13 （IL-4+IL-13） group， lipopolysaccharide （LPS） group， LPS+adenosine triphosphate （ATP）（LPS+ATP） group， IL-4+IL-13+HG group， LPS+HG group， and LPS+ATP+HG group. Equal amounts of PBS （IL-4 20 ng/mL，IL-13 20 ng/mL），1 μg/mL LPS， 1 μg/mL LPS+5 mmol/L ATP interventions were given to the blank， IL-4+IL-13， LPS， and LPS+ATP groups， respectively； 50 μmol/L HG treatment was given to each dosing group based on the above interventions. The expression of mRNA related to macrophage polarization and pyroptosis was detected by Real-time PCR， and that of macrophage polarization and pyroptosis-related proteins was detected by Western blotting. Macrophage surface markers were detected by flow cytometry. Whole gene transcriptome sequencing analysis， Gene Ontology （GO） enrichment analysis， and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis of M1 macrophages were performed. Cell morphology was observed under a microscope. Enzyme-linked immunosorbent assay （ELISA） was performed to detect transforming growth factor （TGF）-β signaling pathway-related proteins TGF-β1 and platelet-responsive protein （THBS）-1.

Results

Compared with the blank group， the relative expression of interleukin （IL）-1β， inducible nitric oxide synthase （iNOS）， tumor necrosis factor （TNF）-α mRNA， and iNOS and TNF-α proteins was higher in the LPS group （all

0.01）.Compared with the LPS group，the relative expression of interleukin （IL）-1β， iNOS， TNF-α mRNA， and iNOS and TNF-α proteins was lower in the LPS+HG group （all

0.01）. Compared with the blank group， the relative expression of CD206 and arginase （Arg）-1 proteins in the LPS group was lower （both

0.05）. Compared with the LPS group， the relative expression of the above indexes in the LPS+HG group was higher （both

0.01）. Compared with the blank group， the relative expression of CD86 protein and CD86/CD206 in the LPS group was higher （both

0.01）. Compared with the LPS group， the above indexes in the LPS+HG group were lower （

0.01） and higher （

0.01）， respectively. Compared with the blank group， the relative expression of Arg-1， interleukin （IL）-10 mRNA， CD206 and Arg-1 proteins in the IL-4+IL-13 group was higher （

0.05，

0.01）. Compared with the IL-4+IL-13 group， the relative expression of the above indexes in the IL-4+IL-13+HG group was higher （

0.05，

0.01）. HG led to the down-regulation of 337 genes and up-regulation of 148 genes expression in M1 macrophages. Among the ten core genes， the THBS-1 gene with the greatest correlation was selected for KEGG enrichment analysis， and the results showed that the THBS-1/TGF-β1 signaling pathway had an important role in the regulation of M

1 macrophages by HG. Compared with the blank group， the relative expression of THBS-1 and TGF-β1 mRNA in the LPS group was higher and lower， respectively （both

0.01）. Compared with the LPS group， the relative expression of the above indexes in the LPS+HG group was lower and higher， respectively （both

0.01）. After treating LPS+ATP-induced macrophage pyroptosis with HG， microscopic observation revealed that HG significantly improved cell swelling and rupture of pyrogenic macrophages. Compared with the blank group， NOD-like receptor protein （NLRP） 3， cysteine aspartate protease （Casp）-1 and IL-1β mRNA and NLRP3， Gasdermins-D （GSDMD） Full and N-terminal bodies （GSDMD-F and GSDMD-N）， and Casp-1 protein relative expression was higher （

0.05，

0.01）. Compared with the LPS+ATP group， the relative expression of the above indexes was lower in the LPS+ATP+HG group （

0.05，

0.01）. Compared with the blank group， the relative expression and protein level of TGF-β1 and THBS-1 mRNA in the LPS+ATP group were lower and higher， respectively （both

0.01）. The relative expression and protein level of the above indexes in the LPS+ATP+HG group were higher and lower， respectively， compared with those in the LPS+ATP group （

0.05，

0.01）.

Conclusion

HG inhibits M1 macrophage polarization and pyroptosis and promotes M1 to M2 conversion， and the HG inhibition of M1 macrophage polarization and pyroptosis is associated with the THBS-1/TGF-β1 signaling pathway.

关键词

去甲乌药碱M1巨噬细胞细胞极化细胞焦亡血小板反应蛋白1/转化生长因子β1信号通路

Keywords

higenamineM1 macrophagecell polarizationcell pyroptosisTHBS-1/TGF-β1 signaling pathway

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