Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-<italic style="font-style: italic">κ</italic>B/NLRP3 Pathway

LI Yuting; LEI Zhiqiang; YOU Yu; ZHU Hongyang; RONG Ziling; CHANG Shiyao; LIU Yuhui

doi:10.13422/j.cnki.syfjx.20231013

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Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway

更新时间：2023-09-08

- Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 19, Pages: 18-25(2023)
- 作者机构：
  
  1.江西中医药大学，南昌 330004
  2.南昌大学第一附属医院，南昌 330004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20231013
  CLC： R284;R285;R289;R287;R22;R2-031;R33;R24
- Published：05 October 2023，
  
  Published Online：16 August 2023，
  
  Received：01 June 2023，
- 稿件说明：
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李玉婷,雷志强,游宇等.基于TLR4/NF-κB/NLRP3通路探讨补阳还五汤调控巨噬细胞极化的作用机制[J].中国实验方剂学杂志,2023,29(19):18-25.

LI Yuting,LEI Zhiqiang,YOU Yu,et al.Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(19):18-25.
李玉婷,雷志强,游宇等.基于TLR4/NF-κB/NLRP3通路探讨补阳还五汤调控巨噬细胞极化的作用机制[J].中国实验方剂学杂志,2023,29(19):18-25. DOI： 10.13422/j.cnki.syfjx.20231013.

LI Yuting,LEI Zhiqiang,YOU Yu,et al.Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(19):18-25. DOI： 10.13422/j.cnki.syfjx.20231013.

摘要

目的

基于Toll样受体4（TLR4）/核转录因子-

B（NF-

B）/核苷酸结合寡聚化结构域样受体3（NLRP3）通路探讨补阳还五汤调控巨噬细胞极化的作用机制。

方法

本实验通过采用不同浓度（0、1.25、2.5、5、10、20、40、80 mg·L

-1

）脂多糖（LPS）对RAW264.7巨噬细胞干预24 h，细胞增殖与活性检测（CCK-8）法检测RAW264.7巨噬细胞存活率，筛选最适浓度建立体外LPS诱导RAW246.7巨噬细胞炎症模型。将实验分为空白组（20%空白血清），模型组（20%空白血清+10 mg·L

-1

LPS），模型对照组（20%FBS+10 mg·L

-1

LPS），低、中、高（5%、10%、20%）补阳还五汤含药血清组及NLRP3抑制剂MCC950（50 μmol·L

-1

）、活性氧（ROS）抑制剂NAC（10 μmol·L

-1

）、NF-

B抑制剂PDTC（10 μmol·L

-1

）联合补阳还五汤高剂量（20%）组。酶联免疫吸附测定法（ELISA）检测RAW264.7巨噬细胞白细胞介素-1

（IL-1

）、白细胞介素-18（IL-18）、肿瘤坏死因子-

（TNF-

）的表达情况；流式细胞术检测巨噬细胞ROS水平；蛋白免疫印迹法（Western blot）检测M1型巨噬细胞相关细胞因子诱导型一氧化氮合酶（iNOS）、肿瘤坏死因子-

（TNF-

）、M2型巨噬细胞相关细胞因子精氨酸酶1（Arg-1）、白细胞介素-10（IL-10）蛋白表达，TLR4/NF-

B/NLRP3通路的相关蛋白表达。

结果

CCK-8结果显示，在10 mg·L

-1

LPS的刺激下，RAW264.7巨噬细胞的细胞活力值最高（

0.01）。与空白组比较，模型组IL-1

、IL-18、TNF-

水平显著升高（

0.01）；ROS表达量显著上升（

0.01）；M1型巨噬细胞iNOS、TNF-

蛋白表达显著升高（

0.01），M2型巨噬细胞Arg-1、IL-10蛋白表达显著降低（

0.01）；TLR4、髓样分化因子88（Myd88）、磷酸化核转录因子-

B抑制蛋白（p-I

B）/核转录因子-

B抑制蛋白（I

B）、磷酸化核转录因子-

B（p-NF-

B） p65/NF-

B p65、NLRP3、凋亡相关斑点样蛋白（ASC）、胱天蛋白酶-1前体（pro-Caspase-1）蛋白表达水平明显升高（

0.05，

0.01）。与模型组比较，补阳还五汤各给药组及抑制剂组均可明显降低IL-1

、IL-18、TNF-

水平（

0.05，

0.01），抑制RWA264.7巨噬细胞炎症因子的表达；降低细胞ROS表达水平（

0.01）；下调M1型巨噬细胞iNOS、TNF-

蛋白（

0.01），上调M2型巨噬细胞Arg-1、IL-10蛋白表达（

0.05，

0.01）；降低TLR4、Myd88、p-I

B/I

B、p-NF-

B p65/NF-

B p65、NLRP3、ASC、pro-Caspase-1蛋白表达水平（

0.05，

0.01）。

结论

补阳还五汤可改善巨噬细胞炎症反应，可能与降低巨噬细胞ROS水平，抑制RAW264.7巨噬细胞极化，下调TLR4/NF-

B/NLRP3通路蛋白表达水平有关。

Abstract

Objective

To explore the mechanism of Buyang Huanwutang in regulating macrophage polarization based on the Toll-like receptor 4 （TLR4） / nuclear factor-

B （NF-

B） / nucleotide-binding oligomerization domain-like receptor 3 （NLRP3） pathway.

Method

RAW264.7 macrophages were intervened with lipopolysaccharide （LPS） of different concentrations （0， 1.25， 2.5， 5， 10， 20， 40， and 80 mg·L

-1

） for 24 hours. Cell Counting Kit-8 （CCK-8） assay was used to determine the cell viability of RAW264.7 macrophages. The optimal concentration was chosen to establish an

in vitro

inflammation model induced by LPS. Cells were divided into a blank group （20% blank serum）， a model group （20% blank serum + 10 mg·L

-1

LPS）， a model control group （20% FBS + 10 mg·L

-1

LPS）， low-， medium-， and high-dose （5%， 10%， and 20%） Buyang Huanwutang-containing serum groups， a high-dose （20%） Buyang Huanwutang combined with NLRP3 inhibitor MCC950 （50 μmol·L

-1

） group， a high-dose （20%） Buyang Huanwutang combined with reactive oxygen species （ROS） inhibitor NAC （10 μmol·L

-1

） group， and a high-dose （20%） Buyang Huanwutang combined with NF-

B inhibitor PDTC （10 μmol·L

-1

） group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of interleukin-1

（IL-1

）， interleukin-18 （IL-18）， and tumor necrosis factor-

（TNF-

） in RAW264.7 macrophages. Flow cytometry was employed to measure ROS levels in macrophages. Western blot was used to determine the protein expression of M1-type macrophage-related factors inducible nitric oxide synthase （iNOS） and TNF-

， M2-type macrophage-related factors arginase-1 （Arg-1） and interleukin-10 （IL-10）， as well as the proteins in the TLR4/NF-

B/NLRP3 pathway.

Result

CCK-8 results indicated that under 10 mg·L

-1

LPS stimulation， RAW264.7 macrophages exhibited the highest cell viability （

0.01）. Compared with the blank group， the model group showed significantly increased levels of IL-1

， IL-18， and TNF-

（

0.05，

0.01）， increased ROS expression （

0.05，

0.01）， increased protein expression of M1-type macrophage factors iNOS and TNF-

（

0.01）， decreased protein expression of M2-type macrophage factors Arg-1 and IL-10 （

0.05，

0.01）， and upregulated expression levels of TLR4， myeloid differentiation factor 88 （MyD88）， phosphorylated inhibitor of NF-

B （p-I

B）/NF-

B inhibitor （I

B）， phosphorylated NF-

B （p-NF-

B） p65/NF-

B p65， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and pro-Caspase-1 （

0.05，

0.01）. Compared with the model group， all Buyang Huanwutang-treated groups and inhibitor groups significantly reduced levels of IL-1

， IL-18， and TNF-

（

0.01）， suppressed the expression of inflammatory factors in RAW264.7 macrophages， decreased cellular ROS expression levels （

0.01）， downregulated M1-type macrophages iNOS and TNF-

protein expression （

0.01）， upregulated M2-type macrophages Arg-1 and IL-10 protein expression （

0.01）， and lowered protein expression levels of TLR4， MyD88， p-I

B/I

B， p-NF-

B p65/NF-

B p65， NLRP3， ASC， and pro-Caspase-1 （

0.05，

0.01）.

Conclusion

Buyang Huanwutang can improve macrophage inflammation， potentially by reducing macrophage ROS levels， inhibiting RAW264.7 macrophage polarization， and downregulating the protein expression levels of the TLR4/NF-

B/NLRP3 pathway.

关键词

补阳还五汤RAW264.7巨噬细胞巨噬细胞极化Toll样受体4（TLR4）/核转录因子-κB（NF-κB）/核苷酸结合寡聚化结构域样受体3（NLRP3）通路

Keywords

Buyang HuanwutangRAW264.7 macrophage cellsmacrophage cells polarizationToll-like receptor 4 （TLR4）/ nuclear factor-κB （NF-κB） nucleotide-binding oligomerization domain-like receptors 3（NLRP3） signaling pathway

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Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway

DOI：10.13422/j.cnki.syfjx.20231013

摘要

Abstract

关键词

Keywords

references