Tetramethylpyrazine inhibits NETs formation induced by high glucose by regulating NRF2/HO-1 pathway

Jinquan CHEN; Long ZHAO; Xuanheng DING; Lingda WANG; Yan WEN; Hao XIE; Hui PENG

doi:10.16016/j.2097-0927.202204038

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Tetramethylpyrazine inhibits NETs formation induced by high glucose by regulating NRF2/HO-1 pathway

Basic Medicine | 更新时间：2026-01-09

- Tetramethylpyrazine inhibits NETs formation induced by high glucose by regulating NRF2/HO-1 pathway
- Journal of Army Medical University Vol. 44, Issue 21, Pages: 2157-2164(2022)
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院眼科，眼科学重庆市市级重点实验室，重庆市眼科研究所
- 作者简介：
- 基金信息：
- DOI：10.16016/j.2097-0927.202204038
  CLC： R285.5;R331.144;R392.12
- Received：05 April 2022，
  
  Revised：2022-06-13，
  
  Published：15 November 2022
- 稿件说明：
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Jinquan CHEN, Long ZHAO, Xuanheng DING, et al. Tetramethylpyrazine inhibits NETs formation induced by high glucose by regulating NRF2/HO-1 pathway[J]. Journal of Army Medical University, 2022, 44(21): 2157-2164.
DOI：

Jinquan CHEN, Long ZHAO, Xuanheng DING, et al. Tetramethylpyrazine inhibits NETs formation induced by high glucose by regulating NRF2/HO-1 pathway[J]. Journal of Army Medical University, 2022, 44(21): 2157-2164. DOI： 10.16016/j.2097-0927.202204038.

摘要

目的

探讨川芎嗪(tetramethylpyrazine

TMP)对高糖诱导的中性粒细胞外捕获网(neutrophil extracellular traps

NETs)形成的影响及机制。

方法

运用CCK-8检测不同浓度(0、5、10、20、40、80 μmol/L)的TMP处理4 h后中性粒细胞的活性。将中性粒细胞分为7组：Control组(1.5 g/L葡萄糖)、TMP组(40 μmol/L TMP)、HG组(4.5 g/L葡萄糖)、HG+TMP组(40 μmol/L TMP+4.5 g/L葡萄糖)、HG+TMP+ML385组(4.5 g/L葡萄糖+40 μmol/L TMP+10 μmol/L ML385)、HG+ML385组(4.5 g/L葡萄糖+10 μmol/L ML385)和HG+DPI组(4.5 g/L葡萄糖+10 μmol/L DPI)。通过免疫荧光观察NETs的结构；运用DHE和DCFH-DA检测中性粒细胞内ROS和超氧化物阴离子的表达量；采用ELISA试剂盒检测培养基中MPO-DNA的含量，Pico-Green kit试剂盒检测培养基中dsDNA含量，Western blot检测细胞内H3-cit、NRF2、HO-1、NOX2等的表达，评估TMP对NRF2信号级联的调控作用。

结果

80 μmol/L的TMP会降低中性

粒细胞活性(

0.01)

其余浓度的TMP对中性粒细胞活性无显著影响。与Control组比较，TMP组中性粒细胞NRF2、HO-1表达明显上升(

0.01)。与HG组比较，HG+TMP组中性粒细胞产生的H3-cit表达显著降低(

0.05)，并且培养基中dsDNA和MPO-DNA含量明显下降(

0.01)。免疫荧光未观察到HG+TMP组中NETs形成；HG+TMP组中性粒细胞产生的ROS和超氧化物阴离子较HG组中性粒细胞显著降低(

0.01)。使用NRF2抑制剂ML385处理后，TMP对H3-cit、NRF2和HO-1的作用被逆转；培养基中dsDNA和MPO-DNA含量明显上升(

0.01)，并且胞内ROS和超氧化物阴离子相较HG+TMP组的中性粒细胞显著升高(

0.01)。

结论

TMP可以激活NRF2/HO-1通路并抑制ROS的形成，从而抑制高糖诱导的中性粒细胞外捕获网形成。

Abstract

Objective

To investigate the effect and mechanism of tetramethylpyrazine (TMP) on the formation of neutrophil extracellular traps (NETs) induced by high glucose.

Methods

The viability of neutrophils treated with TMP of 0

and 80 μmol/L for 4 h was measured by CCK-8 assay. Neutrophils were divided into Control group (1.5 g/L glucose)

TMP group (40 μmol/L TMP)

HG group (4.5 g/L glucose)

HG+TMP group (40 μmol/L TMP+4.5 g/L glucose)

HG+TMP+ML385 group (4.5 g/L glucose+40 μmol/L TMP+10 μmol/L ML385)

HG+ML385 group (4.5 g/L glucose+10 μmol/L ML385) and HG+DPI group (4.5 g/L glucose+10 μmol/L DPI). The structure of NETs was observed with immunofluorescence assay. The contents of reactive oxygen species (ROS) and superoxide anions were detected using dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay and dihydroethidine (DHE) method. MPO-DNA and dsDNA in the culture medium was measured with ELISA and Pico-Green kit

respectively. Western blotting was used to detect the intracellular expression of H3-cit

NRF2

HO-1

and NOX2 proteins.

Results

TMP of 80 μmol/L had significant effect on neutrophil (

0.01)

but no such effects were seen in TMP at other doses viability. Compared with the Control group

the expression levels of NRF2 a

nd HO-1 were increased significantly in TMP group (

0.01)

while that of H3-cit was obviously decreased after TMP treatment (

0.05)

and the contents of dsDNA and MPO-DNA in the medium were decreased notably (

0.01). NETs formation was not observed by immunofluorescence assay in the HG+TMP group. The productions of ROS and superoxide anions in the HG+TMP group were significantly lower than those produced in the HG group (

0.01). The effects of TMP on the expression of H3-cit

NRF2

and HO-1 were reversed after treatment with NRF2 inhibitor ML385. Compared with the HG+TMP group

the contents of dsDNA and MPO-DNA were increased significantly (

0.01)

and the contents of intracellular ROS and superoxide anions were elevated remarkably in HG+TMP+ML385 group (

0.01).

Conclusion

TMP can activate the NRF2/HO-1 pathway and then suppress the production of ROS

and thus inhibit the NETs formation induced by high glucose.

关键词

Keywords

references

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