Effect of <italic style="font-style: italic">Liushen Capsules </italic>（六神胶囊） on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages： based on NF-κB Pathway

Qinhai MA; Ruihan CHEN; Biao LEI; Zhoulang WANG; Yuqi XIE; Bin LIU; Zifeng YANG

doi:10.13288/j.11-2166/r.2022.04.012

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Effect of Liushen Capsules （六神胶囊） on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages： based on NF-κB Pathway

更新时间：2022-02-15

- Effect of Liushen Capsules （六神胶囊） on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages： based on NF-κB Pathway
- Journal of Traditional Chinese Medicine Vol. 63, Issue 4, Pages: 362-369(2022)
- 作者机构：
  
  1.广州医科大学附属第一医院，广东省广州市越秀区东风西路195号，511436
  2.昆明理工大学医学院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2022.04.012
  CLC：
- Received：09 March 2021，
  
  Revised：2021-10-01，
  
  Published：17 February 2022
- 稿件说明：
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马钦海,陈瑞晗,雷标等.基于NF-κB通路探讨六神胶囊对脂多糖诱导巨噬细胞RAW264.7炎症反应的影响[J].中医杂志,2022,63(04):362-369.

MA Qinhai,CHEN Ruihan,LEI Biao,et al.Effect of Liushen Capsules （六神胶囊） on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages： based on NF-κB Pathway[J].Journal of Traditional Chinese Medicine,2022,63(04):362-369.
马钦海,陈瑞晗,雷标等.基于NF-κB通路探讨六神胶囊对脂多糖诱导巨噬细胞RAW264.7炎症反应的影响[J].中医杂志,2022,63(04):362-369. DOI： 10.13288/j.11-2166/r.2022.04.012.

MA Qinhai,CHEN Ruihan,LEI Biao,et al.Effect of Liushen Capsules （六神胶囊） on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages： based on NF-κB Pathway[J].Journal of Traditional Chinese Medicine,2022,63(04):362-369. DOI： 10.13288/j.11-2166/r.2022.04.012.

摘要

目的

探讨六神胶囊的抗炎作用及机制。

方法

RAW264.7细胞分为六神胶囊组和地塞米松组，六神胶囊按9.76、4.88、2.44、1.22、0.61、0.31、0.15 μg/ml梯度稀释；地塞米松按250、125、62.5、31.25、15.63、7.81、3.91 μg/ml梯度稀释，选择细胞存活率大于90%的最大3个六神胶囊浓度和最大无毒剂量的地塞米松进行后续实验。RAW264.7细胞分为空白组，地塞米松组，模型组及六神胶囊高、中、低剂量组。除空白组外，其余各组采用脂多糖诱导建立炎症模型。造模后六神胶囊高、中、低剂量组分别加入筛选浓度的高、中、低浓度六神胶囊溶液各1 ml；地塞米松组加入筛选浓度的地塞米松溶液1 ml；模型组和空白组加入1 ml培养液。24 h后收集细胞上清液检测一氧化氮（NO）的浓度，ELISA法检测细胞中白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）的含量，实时荧光定量PCR法检测细胞中IL-1β、TNF-α、IL-6和诱导型一氧化氮合酶（iNOS）mRNA表达，蛋白印迹实验检测核因子κB p65（NF-κB p65）、磷酸化核因子κB p65（p-NF-κB p65）、核因子抑制蛋白κB（IκBα）和磷酸化核因子抑制蛋白κB（p-IκBα）表达。

结果

选择六神胶囊1.22、0.61、0.31 μg/ml浓度，地塞米松31.25 μg/ml浓度进行后续实验。与空白组比较，模型组细胞中TNF-α、IL-1β、IL-6及NO水平，TNF-α、IL-1β、IL-6和iNOS mRNA表达，p-IκBα、p-NF-κB p65蛋白表达显著升高，IκBα蛋白表达显著降低（

＜0.01）；与模型组比较，地塞米松组及六神胶囊中、高剂量组细胞TNF-α、IL-1β、IL-6及NO水平，TNF-α、IL-1β、IL-6和iNOS mRNA表达，p-IκBα和p-NF-κB p65蛋白表达均显著降低，IκBα蛋白表达升高（

＜0.05或

＜0.01）；与地塞米松组比较，六神胶囊高剂量组IL-1β水平降低、IL-6水平升高，TNF-α mRNA及p-NF-κB p65蛋白表达显著降低（

＜0.01）；六神胶囊高剂量组TNF-α、IL-1β含量，IL-6、TNF-α mRNA表达，p-NF-κB p65蛋白表达低于六神胶囊中、低剂量组（

＜0.01）。

结论

六神胶囊在体外具有良好的抗炎活性，可能通过抑制NF-κB信号通路的激活，降低iNOS的表达，进一步抑制NO的合成和炎性细胞因子的产生，从而改善炎症反应，以高剂量效果较佳。

Abstract

Objective

To explore the anti-inflammatory effect of

Liushen Capsules

（六神胶囊） and the underlying mechanism.

Methods

The RAW264.7 cells were divided into

Liushen Capsules

group and dexamethasone group.

Liushen Capsules

solution

at the concentration of 9.76， 4.88， 2.44， 1.22， 0.61， 0.31， and 0.15 μg/ml were used for gradient dilution， and dexamethasone solution at the concentration of 250， 125， 62.5， 31.25， 15.63， 7.81， and 3.91 μg/ml were administered for gradient dilution. The top three maximum concentrations of

Liushen Capsules

solution with cell viability over 90%， as well as the maximum non-toxic concentration of dexamethasone solution were selected for subsequent experiments. RAW264.7 cells were divided into blank group， dexamethasone group， model group and high-， medium- and low dose

Liushen Capsules

groups. Except for the blank group， the rats were induced by lipopolysaccharide to establish the inflammation model. After modeling， 1 ml of the selected concentrations of

Liushen Capsules

solutions were added into the corresponding high-， medium- and low-dose

Liushen Capsules

groups， while 1 ml of the dexamethasone solution with selected concentration was added in the dexamethasone group； and 1 ml of culture medium was given in the model group and the blank group. After 24 hours， the cell supernatant was collected to detect the concentration of nitric oxide （NO）， and the ELISA method was used to detect the levels of interleukin-1β （IL-1β）， tumor necrosis factor α （TNF-α）， and interleukin-6 （IL-6） in the cells. The mRNA expressions of IL-1β， TNF-α， IL-6 and inducible nitric oxide synthase （iNOS） in the cells were detected by real-time fluorescence quantitative PCR， while the expressions of nuclear factor κB p65 （NF-κB p65）， phosphorylated NF-κB p65 （p-NF-κB p65）， inhibitor of nuclear factor κB α （IκBα） and phosphorylated IκBα （p-IκBα） were detected through western blotting.

Results

The selected concentrations of

Liushen Capsules

solution were 1.22， 0.61， and 0.31 μg/ml for subsequent experiments， and the selected concentration of dexamethasone solution was 31.25 μg/ml. Compared to those in the blank group， the levels of TNF-α， IL-1β， IL-6 and NO in the cells， the mRNA expressions of TNF-α， IL-1β， IL-6 and iNOS， and the protein expressions of p-IκBα and p-NF-κB p65 significantly increased in the model group， and the protein expression of IκBα was significantly reduced （

＜0.01）. Compared to those in the model group， the levels of TNF-α， IL-1β， IL-6 and NO， the mRNA expressions of TNF-α， IL-1β， IL-6 and iNOS， the protein expre-ssions of IκBα and p-NF-κB p65 were significantly reduced， while the protein expression of IκBα was increased （

＜0.05 or

＜0.01）. Compared to the dexamethasone group， the high-dose

Liushen Capsules

group had lower level of IL-1β， higher level of IL-6， and decreased expressions of TNF-α mRNA and p-NF-κB p65 protein （

＜0.01）. The levels of TNF-α and IL-1β， the mRNA expressions of IL-6 and TNF-α， and the protein expression of p-NF-κB p65 in the high-dose

Liushen Capsules

group were significantly lower than those in the medium- and low-dose groups （

＜ 0.01）.

Conclusion

Liushen Capsules

has good anti-inflammatory activity in vitro， which may improve the inflammatory responses by inhibiting the activation of NF-κB signaling pathway， reducing the expression of iNOS， further inhibiting NO synthesis and inflammatory cytokines productions， and higher dose shows better effect.

关键词

Keywords

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Effect of Liushen Capsules （六神胶囊） on Lipopolysaccharide-induced Inflammatory Response in RAW264.7 Macrophages： based on NF-κB Pathway

DOI：10.13288/j.11-2166/r.2022.04.012

摘要

Abstract

关键词

Keywords

references