益气养阴逐瘀方对LPS诱导的RAW 264.7细胞炎症模型体外抗炎活性的影响

唐蕾; 吴坚; 于顾然; 储继红; 严士海; 王海丹; 戴国梁; 韩旭

doi:10.13422/j.cnki.syfjx.20190129

您当前的位置：

首页 >

文章列表页 >

益气养阴逐瘀方对LPS诱导的RAW 264.7细胞炎症模型体外抗炎活性的影响

药理 | 更新时间：2021-02-09

- 益气养阴逐瘀方对LPS诱导的RAW 264.7细胞炎症模型体外抗炎活性的影响
- Effect of Yiqi Yangyin Zhuyu Recipe in Anti-inflammation Activities in Vitro on LPS-induced RAW 264.7 Cells
- 中国实验方剂学杂志 2019年25卷第1期页码：141-148
- 作者机构：
  
  南京中医药大学　附属医院，南京　 210029
- 作者简介：
  
  唐蕾，在读硕士，从事中医内科学老年病研究，E-mail：245986178@qq.com
  韩旭，博士，主任医师，从事中医内科学老年病研究，E-mail:13057504158@126.com
- 基金信息：
  
  江苏省自然基金项目(BK20181505);江苏省卫计委科研课题(MS201506)
- DOI：10.13422/j.cnki.syfjx.20190129
  中图分类号： R289;R289.5;R364.5
- 收稿日期：2018-07-31，
  
  网络出版日期：2018-10-19，
  
  纸质出版日期：2019-01-05
- 稿件说明：
移动端阅览
唐蕾, 吴坚, 于顾然, 等. 益气养阴逐瘀方对LPS诱导的RAW 264.7细胞炎症模型体外抗炎活性的影响[J]. 中国实验方剂学杂志, 2019,25(1):141-148.

Lei TANG, Jian WU, Gu-ran YU, et al. Effect of Yiqi Yangyin Zhuyu Recipe in Anti-inflammation Activities in Vitro on LPS-induced RAW 264.7 Cells[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(1): 141-148.
唐蕾, 吴坚, 于顾然, 等. 益气养阴逐瘀方对LPS诱导的RAW 264.7细胞炎症模型体外抗炎活性的影响[J]. 中国实验方剂学杂志, 2019,25(1):141-148. DOI： 10.13422/j.cnki.syfjx.20190129.

Lei TANG, Jian WU, Gu-ran YU, et al. Effect of Yiqi Yangyin Zhuyu Recipe in Anti-inflammation Activities in Vitro on LPS-induced RAW 264.7 Cells[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(1): 141-148. DOI： 10.13422/j.cnki.syfjx.20190129.

摘要

目的：

研究益气养阴逐瘀方对脂多糖（LPS）诱导的小鼠单核巨噬细胞系RAW 264.7细胞炎症模型相关细胞因子表达及经典活化（M1）/选择性活化（M2）炎症表型转化的影响。

方法：

运用噻唑蓝（MTT）比色法检测不同给药浓度的益气养阴逐瘀方对RAW 264.7细胞增殖情况的影响；利用LPS诱导RAW 264.7细胞，构建体外炎症模型，采用格里斯试剂法（Griess）检测细胞上清液中一氧化氮（NO）的分泌量；酶联免疫吸附测定法（ELISA）检测细胞上清中M1/M2型相关炎症因子肿瘤坏死因子-

（TNF-

），白细胞介素-6（IL-6），白细胞介素-1

（IL-1

），一氧化氮合酶（iNOS），白细胞介素-10（IL-10），转化生长因子-

（TGF-

），精氨酸-1（Arg-1）的表达量；实时荧光定量PCR（Real-time PCR）检测M1型巨噬细胞标志基因TNF-

，IL-6，IL-1

，iNOS和M2型巨噬细胞标志基因IL-10，TGF-

，Arg-1 mRNA的表达；蛋白免疫印迹法（Western blot）检测细胞内相关蛋白TNF-

，IL-6，IL-1

，iNOS的表达。

结果：

MTT比色法显示，与空白组比较，益气养阴逐瘀方2.0 g·L

－1

及以下时对细胞增殖无明显影响。Griess法显示，与空白组相比，LPS组NO释放量显著上调（

0.01）；与LPS组相比，各给药组NO释放量均显著下调（

0.01）。ELISA显示，与空白组相比，LPS组M1/M2型巨噬细胞相关炎症因子表达均显著上调（

0.01）；与LPS组相比，益气养阴逐瘀方各给药组可明显下调M1型巨噬细胞促炎症因子表达（

0.01），对M2型巨噬细胞抗炎症因子无影响。Real-time PCR显示，与空白组相比，LPS组M1/M2型巨噬细胞相关mRNA表达显著上调（

0.01）；与LPS组相比，益气养阴逐瘀方各给药组可明显下调M1型巨噬细胞促炎症因子基因表达（

0.05，

0.01），对M2型mRNA表达无影响。Western blot显示，与空白组比较，LPS组TNF-

，IL-6，IL-1

，iNOS蛋白的表达显著上调（

0.01）；与LPS组相比，益气养阴逐瘀方各给药组TNF-

，IL-6，IL-1

，iNOS蛋白的表达均下调，且于2.0 g·L

－1

下调最显著（

0.01）。

结论：

益气养阴逐瘀方可以有效抑制LPS诱导的RAW 264.7细胞炎症。其诱导已经分化的促炎亚型M1型巨噬细胞向抗炎亚型M2型巨噬细胞转化不明显，其抗炎机制可能与通过抑制巨噬细胞向M1亚型方向极化从而发挥免疫调节作用，以减少NO

TNF-

，IL-6等相关炎症因子分泌相关。

Abstract

Objective:

To investigate the effect of Yiqi Yangyin Zhuyu recipe on expressions of inflammatory factor and transformation of classically activated macrophages(M1)/alternatively activated macrophages (M2) inflammatory phenotype in lipopolysaccharide (LPS)-induced RAW 264.7 cells.

Method:

Methyl-thiazdyl-tetrazolium(MTT) reduction assay was used to detect the effect of different concentrations of Yiqi Yangyin Zhuyu recipe on the proliferation of the cells. The release of nitric oxide was detected by the Griess method. Enzyme linked immunosorbent assay(ELISA)was used to detect the release of M1/M2 inflammatory cytokines in cell supernatant. The expressions of the pro-inflammatory factor genes of M1-macrophages and the anti-inflammatory factor genes of M2-macrophages were detected by Real-time PCR. The protein expression levels of tumor necrosis factor-

(TNF-

)

interleukin(IL)-6

IL-1

nitric oxide synthase(iNOS) were detected by Western blot.

Result:

Results of MTT showed that Yiqi Yangyin Zhuyu recipe with the concentration of 2.0 g·L

-1

and below had no effect on the cell proliferation. Results of Griess indicated that compared with blank group

the release of nitric oxide of LPS-induced group was increased (

0.01); compared with LPS-induced group

Yiqi Yangyin Zhuyu recipe group could reduce the release of NO (

0.01). Results of ELISA showed that compared with blank group

the release of M1/M2 inflammatory cytokines of LPS-induced group were both up-regulated (

0.01); compared with LPS-induced group

the secretion of M1-macrophages inflammatory cytokines was down-regulated in Yiqi Yangyin Zhuyu recipe group (

0.01)

but with no change in M2-macrophages. Results of Real-time PCR indicated that compared with blank group

the expressions of M1/M2 were both increased (

0.01

0.05); compared with LPS-induced group

the expression levels of M1-macrophages were down-regulated in traditional Chinese medicine group (

0.01); anti-inflammatory factor genes of M2-macrophages had no significant change. Results of Western blot showed that compared with blank group

expression levels of TNF-

IL-6

IL-1

iNOS were up-regulated (

0.01); compared with LPS-induced group

the expressions of TNF-

IL-6

IL-1

iNOS were down-regulated in Yiqi Yangyin Zhuyu recipe group

especially at the concentration at 2.0 g·L

-1

(

0.01).

Conclusion:

Yiqi Yangyin Zhuyu recipe could effectively inhibit the inflammatory reaction induced by LPS. The anti-inflammatory mechanism of Yiqi Yangyin Zhuyu recipe may be related to inhibition of macrophages to M1 phenotype polarization

so as to play the role of regulating immune and reducing the release of inflammatory cytokines

like NO

TNF-

IL-6

IL-1

关键词

Keywords

references

Galkina E , Ley K. Immune and inflammatory mechanisms of atherosclerosis [J]. Annu Rev Immunol , 2008 , 27 ( 1 ): 165 - 197 .

王冬菊．心脑血管疾病流行概况及主要影响因素 [J]．预防医学论坛， 2016 ， 12 ( 1 ): 71 - 75 .

YE L X , YU J , LIANG Y X ,et al . Beclin 1 knockdown retards re-endothelialization and exacerbates neointimal formation via a crosstalk between autophagy and apoptosis [J]. Atherosclerosis , 2014 , 237 ( 1 ): 146 - 154 .

YU Y , GUO S , FENG Y , et al . Phospholipid transfer protein deficiency decreases the content of S1P in HDL via the loss of its transfer capability [J]. Lipids , 2014 , 49 ( 2 ): 183 - 190 .

ZHANG T , ZHANG X , YU W , et al . Effects of chemokine-like factor 1 on vascular smooth muscle cell migration and proliferation in vascular inflammation [J]. Atherosclerosis , 2013 , 226 ( 1 ): 49 - 57 .

Cheang W S , TIAN X Y , Wong W T ,et al . Metformin protects endothelial function in diet-induced obese mice by inhibition of endoplasmic reticulum stress through 5′adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor δ pathway [J]. Arterioscl Throm Vas , 2014 , 34 ( 4 ): 830 - 836 .

LIU X , ZHAO G , YAN Y ,et al . Ginkgolide B reduces atherogenesis and vascular inflammation in ApoE －/－ mice [J]. PLoS One , 2012 , 7 ( 5 ): 36237 .

任建勋，李磊，林成仁，等．痰瘀同治方对小型猪冠状动脉粥样硬化炎症反应的影响 [J]．中国中药杂志， 2014 ， 39 ( 2 ): 285 - 290 .

李明明，黄芳，韩林涛，等．瓜蒌薤白白酒汤对大鼠心肌缺血再灌注损伤的保护作用 [J]．中国实验方剂学杂志， 2013 ， 19 ( 16 ): 188 - 192 .

Legein B , Temmerman L , Biessen E A ,et al . Inflammation and immune system interactions in atherosclerosis [J]. Cell Mol Life Sci , 2013 , 70 ( 20 ): 3847 - 3869 .

JU Y L , MIN H K , Choi D ,et al . Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry [J]. J Chromatogr A , 2010 , 1217 ( 10 ): 1660 - 1666 .

WU D , LIU J , PANG X ,et al . Palmitic acid exerts pro-inflammatory effects on vascular smooth muscle cells by inducing the expression of C-reactive protein, inducible nitric oxide synthase and tumor necrosis factor- α [J]. Int J Mol Med , 2014 , 34 ( 6 ): 1706 - 1712 .

姜红菊，李润智，杜修文，等．宁心解毒汤、血塞通、瑞舒伐他汀对ApoE －/－小鼠炎症因子和As斑块的影响 [J]．中国动脉硬化杂志， 2016 ， 24 ( 12 ): 1214 - 1218 .

刘桂林，窦迎春，刘粉叶，等．三七总皂苷对ox-LDL诱导的人脐静脉内皮细胞CD40，VCAM-1表达的影响 [J]．山东大学学报：医学版， 2010 , 48 ( 10 ): 14 - 19 .

王令谆，李七一．益气养阴活血化痰法治疗冠心病心绞痛160例临床观察 [J]．山西医药杂志， 2013 ， 42 ( 7 ): 733 - 735 .

刘福明，李七一，刘健，等 . 益气养阴活血化痰法对冠心病患者抵抗素及IL-1、TNF- α 、Hs-CRP的影响 [J]．吉林中医药， 2016 ， 36 ( 12 ): 1214 - 1216 .

张洪斌，李七一．益气养阴活血化痰法治疗冠心病心绞痛对冠状动脉内皮功能的影响及其临床研究 [J]．中华中医药学刊， 2008 ， 26 ( 12 ): 2646 - 2647 .

严士海，李七一．冠心平对ApoE －/－小鼠动脉粥样硬化氧化与抗氧化指标的影响 [J]．中成药， 2014 ， 36 ( 6 ): 1285 - 1287 .

Swirski F K , Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure [J]. Science , 2013 , 339 ( 6116 ): 161 - 166 .

Hoebe K , Janssen E , Beutler B. The interface between innate and adaptive immunity [J]. Nat Immunol , 2004 , 5 ( 10 ): 971 - 974 .

Gordon S , Martinez F O. Alternative activation of macrophages:mechanism and functions [J]. Immunity , 2010 , 32 ( 5 ): 593 - 604 .

Dill B D , Gierlinski M , Härtlova A ,et al . Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors [J]. Mol Cell Proteomics , 2015 , 14 ( 5 ): 1334 - 1349 .

Woollard K J , Geissmann F. Monocytes in atherosclerosis: subsets and functions [J]. Nat Rev Cardiol , 2010 , 7 ( 2 ): 77 - 86 .

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

基于PI3K/Akt/NF-κB信号通路探讨阳和汤调控巨噬细胞极化减轻骨关节炎的作用机制

基于NLRP3炎症小体介导的巨噬细胞焦亡探究黄芩-黄连对动脉粥样硬化的干预机制

基于巨噬细胞和胶质细胞探讨明睛颗粒对湿性年龄相关性黄斑变性纤维血管膜的影响

艳山姜挥发油通过调控巨噬细胞CD36和ABCA1表达抑制泡沫细胞的形成

月华胶囊对耐多药结核分支杆菌感染自噬的影响