Biejiajian Wan Regulates Polarization of Macrophages via HIF-1<italic style="font-style: italic">α</italic>/NF-<italic style="font-style: italic">κ</italic>B Signaling Pathway

Yang LIU; Chunyu HE; Tong LI; Weiguang CHEN; Yanhao MA; Ying KUANG; Songqi HE; Haitao SUN

doi:10.13422/j.cnki.syfjx.20221201

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Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway

更新时间：2022-08-30

- Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 19, Pages: 9-16(2022)
- 作者机构：
  
  1.南方医科大学，广州 510515
  2.南方医科大学中西医结合医院，广州 510315
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20221201
  CLC： R2-0;R22;R285.5;R289;R33
- Published：05 October 2022，
  
  Published Online：20 April 2022，
  
  Received：13 February 2022，
- 稿件说明：
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刘洋,何春雨,李彤等.鳖甲煎丸通过HIF-1α/NF-κB信号通路调控巨噬细胞极化的机制探讨[J].中国实验方剂学杂志,2022,28(19):9-16.

LIU Yang,HE Chunyu,LI Tong,et al.Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(19):9-16.
刘洋,何春雨,李彤等.鳖甲煎丸通过HIF-1α/NF-κB信号通路调控巨噬细胞极化的机制探讨[J].中国实验方剂学杂志,2022,28(19):9-16. DOI： 10.13422/j.cnki.syfjx.20221201.

LIU Yang,HE Chunyu,LI Tong,et al.Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(19):9-16. DOI： 10.13422/j.cnki.syfjx.20221201.

摘要

目的

探讨鳖甲煎丸通过调控巨噬细胞极化防治肝纤维化的作用和机制。

方法

运用血清药理学方法，体外培养小鼠巨噬细胞RAW264.7，通过氯化钴（CoCl

）刺激RAW264.7细胞建立体外缺氧模型，将细胞随机分为空白组、CoCl

模型组（200 mmol·L

-1

）、鳖甲煎丸低（0.55 g·kg

-1

）、中（1.1 g·kg

-1

）、高（2.2 g·kg

-1

）和扶正祛瘀胶囊组（0.56 g·kg

-1

扶正祛瘀胶囊）。采用细胞增殖与活性检测（CCK-8）法检测各组细胞增殖水平；实时荧光定量聚合酶链式反应（Real-time PCR）检测巨噬细胞缺氧诱导因子-1

（HIF-1

）、白细胞介素-1

（IL-1

）、IL-6 mRNA表达水平；蛋白免疫印迹法（Western blot）检测巨噬细胞极化相关蛋白与HIF-1

/核转录因子-

B（NF-

B）信号通路相关蛋白表达水平；细胞免疫荧光检测NF-

B信号通路相关蛋白、HIF-1

的分布及表达。

结果

与空白组比较，CoCl

刺激24 h后，RAW264.7细胞增殖受抑制明显（

0.05）；与模型组比较，用相应含药血清处理24 h后，各用药组能促进RAW264.7细胞增殖（

0.05）。与空白组比较，模型组HIF-1

、IL-1

、IL-6 mRNA表达均有明显升高（

0.05）；与模型组比较，鳖甲煎丸与扶正祛瘀胶囊处理后能不同程度的降低巨噬细胞中HIF-1

、IL-1

、IL-6 mRNA的表达水平（

0.05）。与空白组比较，模型组HIF-1

及M1巨噬细胞表型蛋白IL-6、肿瘤坏死因子-

（TNF-

）表达明显升高（

0.05），M2巨噬细胞表型蛋白IL-10表达明显降低（

0.05）。与模型组比较，鳖甲煎丸与扶正祛瘀胶囊组HIF-1

、IL-6、TNF-

蛋白表达降低（

0.05），CD163、IL-10蛋白表达升高（

0.05）。与空白组比较，模型组NF-

B p65、磷酸化（p）-NF-

B p65、磷酸化NF-

B抑制蛋白激酶

（p-IKK

）、磷酸化NF-

B抑制蛋白

（p-I

）蛋白表达明显升高（

0.05）；与模型组比较，鳖甲煎丸与扶正祛瘀胶囊组NF-

B p65、p-NF-

B p65、p-IKK

、p-I

蛋白表达明显降低（

0.05）。与空白组比较，模型组促进HIF-1

和NF-

B p65的入核表达；与模型组比较，鳖甲煎丸和扶正祛瘀胶囊组能抑制HIF-1

和NF-

B p65的入核表达。

结论

鳖甲煎丸可调控巨噬细胞极化，减轻缺氧环境下巨噬细胞相关炎症反应损伤，从而延缓肝纤维化进展，其机制可能与其调控HIF-1

/NF-

B信号通路有关。

Abstract

Objective

To investigate the effect and mechanism of Biejiajian Wan on liver fibrosis by regulating the polarization of macrophages.

Method

Raw264.7 cells were cultured

in vitro

by serum pharmacological method， and the hypoxia model of RAW264.7 cells was established by stimulating RAW264.7 cells with cobalt chloride （CoCl

）. The cells were randomly divided into blank group， CoCl

hypoxia model group （200 mmol·L

-1

）， Biejiajian Wan low-dose group （200 mmol·L

-1

+0.55 g·kg

-1

Fuzheng Quyu capsules）， medium-dose group （200 mmol·L

-1

+1.1 g·kg

-1

Biejiajian Wan）， and high-dose group （200 mmol·L

-1

+2.2 g·kg

-1

Biejiajian Wan） and Fuzheng Quyu capsule group （200 mmol·L

-1

+0.56 g·kg

-1

Biejiajian Wan）. Cell proliferation was detected by cell counting kit-8 （CCK-8）， and the gene expression of hypoxia inducible factor-1

（HIF-1

）， interleukin-1

（IL-1

） and interleukin-6 （IL-6） in macrophages was detected by real time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expression of macrophage polarization-related protein and HIF-1

/nuclear factor-kappa B （NF-

B） signaling pathway-related protein was tested by Western blot， and the distribution and expression of NF-

B signaling pathway-related protein and HIF-1

were determined by cell immunofluorescence.

Result

Compared with the conditions in the blank group， the proliferation of RAW264.7 cells was inhibited after CoCl

stimulation for 24 hours （

0.05）， the mRNA expression of HIF-1

， IL-1

and IL-6 in the model group were increased （

0.05）， the protein expression of HIF-1

and M1 macrophage phenotypic proteins IL-6 and tumor necrosis factor-

（TNF-

） was boosted while that of M2 macrophage phenotypic protein interleukin-10 （IL-10） was reduced （

0.05）， the protein expression of NF-

B p65， phosphorylation （p）-NF-

B p65， phosphorylated NF-

B inhibits protein kinase

（p-IKK

） and phosphorylated NF-

B inhibits protein

(p-I

） was elevated （

0.05）， the nuclear expression of HIF-1

and NF-

B p65 was promoted. Compared with the conditions in the model group， after 24 hours of treatment with corresponding drug-containing serum， each treatment group promoted the proliferation of RAW264.7 cells （

0.05）， the mRNA expression levels of HIF-1

， IL-1

and IL-6 in macrophages were reduced （

0.05）， the protein expression of HIF-1

， IL-6 and TNF-

was decreased， while that of CD163 and IL-10 was increased （

0.05）， the protein expression of NF-

B p65， p-NF-

B p65， p-IKK

and p-I

was lowered （

0.05）， the nuclear expression of HIF-1

and NF-

B p65 was inhibited.

Conclusion

Biejiajian Wan could modulate the polarization of macrophages， attenuate the injury of macrophage-associated inflammatory response under hypoxia， and thus delay the progression of liver fibrosis， which might be related to its regulation of HIF-1

/NF-

B signaling pathway.

关键词

鳖甲煎丸巨噬细胞缺氧诱导因子-1α （HIF-1α）核转录因子-κB（NF-κB）炎症因子

Keywords

Biejiajian Wanmacrophageshypoxia inducible factor-1α （HIF-1α）nuclear factor-kappa B （NF-κB）inflammatory factors

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Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway

DOI：10.13422/j.cnki.syfjx.20221201

摘要

Abstract

关键词

Keywords

references