Effect of Medicated Serum Prepared with Chang'an Ⅰ Prescription on Sensitized Mast Cell Degranulation

Xiang-xue MA; Hai-jie JI; Lin LYU; Hao-meng WU; Feng-yun WANG; Xu-dong TANG

doi:10.13422/j.cnki.syfjx.20202136

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Effect of Medicated Serum Prepared with Chang'an Ⅰ Prescription on Sensitized Mast Cell Degranulation

Pharmacology | 更新时间：2020-12-10

- Effect of Medicated Serum Prepared with Chang'an Ⅰ Prescription on Sensitized Mast Cell Degranulation
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 21, Pages: 48-54(2020)
- 作者机构：
  
  1.中国中医科学院西苑医院，北京 100091
  2.山西省中医药研究院，太原 030012
  3.中国中医科学院，北京 100700
  4.广州中医药大学第二附属医院，广州 510120
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20202136
  CLC： R2-0;R289;R57
- Received：06 April 2020，
  
  Published Online：25 August 2020，
  
  Published：05 November 2020
- 稿件说明：
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马祥雪,吉海杰,吕林等.肠安Ⅰ号方含药血清对致敏肥大细胞活化脱颗粒的影响[J].中国实验方剂学杂志,2020,26(21):48-54.

MA Xiang-xue,JI Hai-jie,LYU Lin,et al.Effect of Medicated Serum Prepared with Chang'an Ⅰ Prescription on Sensitized Mast Cell Degranulation[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(21):48-54.
马祥雪,吉海杰,吕林等.肠安Ⅰ号方含药血清对致敏肥大细胞活化脱颗粒的影响[J].中国实验方剂学杂志,2020,26(21):48-54. DOI： 10.13422/j.cnki.syfjx.20202136.

MA Xiang-xue,JI Hai-jie,LYU Lin,et al.Effect of Medicated Serum Prepared with Chang'an Ⅰ Prescription on Sensitized Mast Cell Degranulation[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(21):48-54. DOI： 10.13422/j.cnki.syfjx.20202136.

摘要

目的

探讨肠安Ⅰ号方含药血清对免疫球蛋白E（IgE）介导的大鼠嗜碱性粒细胞（RBL-2H3）活化脱颗粒的影响及对非受体型酪氨酸蛋白激酶/脾酪氨酸蛋白激酶/丝裂原活化蛋白激酶（Lyn/Syk/MAPK）信号通路的调节作用。

方法

制备肠安Ⅰ号方含药血清，将SD雄性大鼠随机分为肠安Ⅰ号方高、中、低剂量组及空白组，每组10只，给药剂量：空白组给予10 mL·kg

-1

蒸馏水灌胃，肠安Ⅰ号方低、中、高剂量组分别予1.15，2.30，4.60 g·kg

-1

药液灌胃，给药体积10 mL·kg

-1

，1次/d，连续灌胃7 d。细胞分组：空白组，予正常大鼠血清；模型组，予正常大鼠血清；酮替芬组，予正常大鼠血清+30 μmol·L

-1

酮替芬。肠安Ⅰ号方低、中、高剂量组分别为予肠安Ⅰ号方低、中、高剂量含药血清。建立IgE介导的RBL-2H3细胞活化、脱颗粒模型，采用甲苯胺蓝染色进行肥大细胞计数；采用比色法检测细胞脱颗粒

-氨基己糖释放率；采用酶联免疫吸附测定（ELISA）检测细胞上清中肥大细胞类胰蛋白酶（MCT），肿瘤坏死因子-

（TNF-

），单核细胞趋化蛋白-1（MCP-1）及组胺含量；采用蛋白免疫印迹法（Western blot）检测Lyn/Syk/MAPK通路蛋白表达。

结果

对于细胞活化脱颗粒，与空白组比较，模型组细胞

-氨基己糖释放率显著升高（

0.01），脱颗粒率明显升高（

0.05）；与模型组比较，肠安Ⅰ号方含药血清各剂量组细胞

-氨基己糖释放率显著下降（

0.01），细胞脱颗粒率明显下降（

0.05）。对于活性介质释放，与空白组比较，模型组细胞上清组胺，MCT，TNF-

及MCP-1含量均显著增高（

0.01）；与模型组比较，肠安Ⅰ号方各剂量组细胞上清组胺，MCT，TNF-

及MCP-1含量均显著减低（

0.01）。与正常组比较，模型组细胞Lyn和Syk及细胞外调节蛋白激酶1/2（ERK1/2），c-Jun氨基末端激酶（JNK），p38 MAPK磷酸化水平明显升高（

0.05）；与模型组比较，肠安Ⅰ号方各剂量Lyn，Syk及ERK1/2，JNK及p38蛋白磷酸化水平明显降低（

0.05）。

结论

肠安Ⅰ号方含药血清通过下调RBL-2H3细胞活化上游信号通路关键蛋白Lyn，Syk和下游ERK1/2，JNK及p38蛋白的磷酸化水平，抑制肥大细胞活化脱颗粒，减少组胺，MCT，TNF-

及MCP-1等活性介质释放，这可能是其抑制肥大细胞活化，治疗腹泻型肠易激综合征（IBS-D）内脏高敏感的机制之一。

Abstract

Objective

To observe the influence of Chang'an Ⅰ prescription drug-containing serum on IgE-mediated RBL-2H3 cell degranulation model， and explore the mechanism of Chang'an Ⅰ prescription in inhibiting RBL-2H3 activation degranulation and releasing inflammatory mediators with v-yes-1 Yanaguchi sarcoma viral related oncogene homolog （Lyn）/spleen tyrosine protein kinase （Syk）/mitogen-activated protein kinase （MAPK） signal pathway.

Method

Preparation for Chang'an Ⅰ prescription serum. Animal group， SD male rats were randomly divided into Chang'an Ⅰ prescription serum high， medium， low dose， and blank control groups with 10 rats in each group. Dosage： 10 mL·kg

-1

distilled water was given to blank control group， while Chang'an Ⅰ prescription serum high， medium and low dose groups were respectively given to the Chang'an Ⅰ prescription concentrated crude drug with concentration of 1.15，2.30，4.60 g·kg

-1

， respectively once a day for 7 days continuously and then blood was taken from aorta ventralis and centrifuged. Ketotifen as the positive control drug. Mast cells are counted with toluidine blue staining. Cellular release of

-aminohexose was detected by colorimetric method. Contents of MCT， TNF-

， MCP-1 and histamine were measured by enzyme-linked immunosorbent assay （ELISA） kits， Lyn/Syk/MAPK protein levels were detected by immunoblotting.

Result

For cell activation and degranulation， compared with the blank control group， the model group had more cell degranulation （

0.05）， compared with model group， the cell degranulation rate of each dose group of Chang'an Ⅰ prescription decreased （

0.05）. The release rate of

-hexosamine in each dose group of Chang'an Ⅰ prescription decreased significantly （

0.01）. For the release of active mediators， compared with the blank control group， the contents of histamine， MCT， TNF-

and MCP-1 all increased in the model group （

0.01）， compared with the model group， the contents in each dose group of Chang'an Ⅰ prescription all decreased significantly （

0.01）. Compared with the normal group， the phosphorylation levels of Lyn and Syk， extracellular regulatory protein kinase 1/2（ERK1/2）， c-Jun N-terminal kinase （JNK）， and mitogen-activated protein kinase p38 increased in the model group （

0.05）. Compared with the model group， the Lyn， Syk and ERK1/2， JNK and p38 protein phosphorylation levels reduced in Chang'an Ⅰ prescription group （

0.05）.

Conclusion

Chang'an Ⅰ prescription drug-containing serum down-regulates the phosphorylation levels of proteins Lyn， Syk， and ERK1/2， JNK， and p38， inhibits RBL-2H3 cell activation and degranulation， reduces the release of cytokines and chemokines， such as histamine， MCT， TNF-

and MCP-1， it may be one of its mechanisms for treating IBS-D visceral hypersensitivity.

关键词

Keywords

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