旋覆代赭汤含药血清对食管平滑肌细胞5-羟色胺4受体、环磷酸腺苷及钙离子浓度的影响

黄棪; 鲁军; 王霞; 杨东升; 张俊杰

doi:10.13288/j.11-2166/r.2019.19.013

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旋覆代赭汤含药血清对食管平滑肌细胞5-羟色胺4受体、环磷酸腺苷及钙离子浓度的影响

实验研究 | 更新时间：2021-11-10

- 旋覆代赭汤含药血清对食管平滑肌细胞5-羟色胺4受体、环磷酸腺苷及钙离子浓度的影响
- Effects of Xuanfu Daizhe Decoction（旋覆代赭汤） Containing Serum on 5-HT₄R,cAMP and Ca²⁺ in Esophageal Smooth Muscle Cells
- 中医杂志 2019年60卷第19期页码：1679-1683
- 作者机构：
  
  浙江中医药大学
- 作者简介：
- 基金信息：
  
  浙江省自然科学基金（LY17H270017）;浙江省中医药科技计划项目（2016ZA046）
- DOI：10.13288/j.11-2166/r.2019.19.013
  中图分类号： R285.5
- 纸质出版：2019
- 稿件说明：
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[1]黄棪,鲁军,王霞,杨东升,张俊杰.旋覆代赭汤含药血清对食管平滑肌细胞5-羟色胺4受体、环磷酸腺苷及钙离子浓度的影响[J].中医杂志,2019,60(19):1679-1683.

HUANG Yan, LU Jun, WANG Xia, et al. Effects of Xuanfu Daizhe Decoction（旋覆代赭汤） Containing Serum on 5-HT₄R,cAMP and Ca²⁺ in Esophageal Smooth Muscle Cells[J]. Journal of Traditional Chinese Medicine2019, 60(19): 1679-1683. DOI: 10.13288/j.11-2166/r.2019.19.013.
[1]黄棪,鲁军,王霞,杨东升,张俊杰.旋覆代赭汤含药血清对食管平滑肌细胞5-羟色胺4受体、环磷酸腺苷及钙离子浓度的影响[J].中医杂志,2019,60(19):1679-1683. DOI： 10.13288/j.11-2166/r.2019.19.013.

HUANG Yan, LU Jun, WANG Xia, et al. Effects of Xuanfu Daizhe Decoction（旋覆代赭汤） Containing Serum on 5-HT₄R,cAMP and Ca²⁺ in Esophageal Smooth Muscle Cells[J]. Journal of Traditional Chinese Medicine2019, 60(19): 1679-1683. DOI: 10.13288/j.11-2166/r.2019.19.013. DOI：

摘要

目的探讨旋覆代赭汤治疗反流性食管炎的可能作用机制。方法 40只大鼠随机分为旋覆代赭汤低、中、高剂量组及空白组

每组10只

旋覆代赭汤低中高剂量组给药量分别为4. 71、9. 42、18. 84g/（kg·d）

每日1次

连续灌胃3天

空白组给予10 ml/（kg·d）生理盐水灌胃。末次灌胃后腹主动脉取血

分离血清。旋覆代赭汤各剂量组分别设5%、10%、20%、30%共4个浓度

采用CCK-8法检测各浓度对食管平滑肌细胞（ESMC）增殖的影响

并筛选最佳给药浓度。原代培养ESMC

在运用5-羟色胺4受体（5-HT4R）激动剂（马来酸替加色罗）和抑制剂（GR113808）

及腺苷酸环化酶（AC）的激动剂（Forskolin）和抑制剂（SQ22536）干预5羟色胺（5-HT）通路的信号传导

再用筛选出的旋覆代赭汤含药血清孵育

ELISA法检测胞内环磷酸腺苷（cAMP）含量

免疫荧光法检测5-HT4R荧光强度

Ca2+探针检测细胞内Ca2+浓度。结果 5%旋覆代赭汤低剂量组对ESMC的增殖作用最强

故选取用于后续实验。与5-HT4R激动剂组比较

5-HT4R激动剂加中药组Ca2+荧光增强比值增加（P <0. 01）。与AC激动剂组比较

AC激动剂加中药组5-HT4R荧光强度、Ca2+荧光强度显著增加（P <0. 01）。与5-HT4R抑制剂组比较

5-HT4R抑制剂加中药组5-HT4R荧光强度、Ca2+荧光增强比值、c AMP浓度均增加（P <0. 05或P <0. 01）。与AC抑制剂组比较

AC抑制剂加中药组5-HT4R荧光增强比值、Ca2+荧光强度、cAMP浓度均增加（P <0. 05或P <0. 01）。结论旋覆代赭汤可能是通过增加5-HT4R表达

继而激活AC

促进cAMP释放

使胞内Ca2+浓度升高

引起食管平滑肌收缩

从而减轻反流。

Abstract

Objective To investigate the possible action mechanism of Xuanfu Daizhe Decoction（旋覆代赭汤） in the treatment of reflux esophagitis. Methods A total of 40 rats were randomly divided into a blank group and low-

medium-

and high-dose groups of Xuanfu Daizhe Decoction

with 10 rats in each group. The low-

medium-

and high-dose groups of Xuanfu Daiyu Decoction were given 4. 71

9. 42

18. 84 g/（ kg·d） respectively once a day

continuously for 3 days. The blank group was given 10 ml/（ kg·d） normal saline. Blood was taken from the abdominal aorta after the last gavage

and serum was separated. Four concentrations of 5%

10%

20% and 30% were set in each dose group of Xuanfu Daiyu Decoction. CCK-8 method was used to determine the effects of various concentrations on the proliferation of esophageal smooth muscle cells（ ESMC）

and to screen the optimal concentration. Primary culture of ESMC

and 5-hydroxytryptamine 4 receptor（ 5-HT

R） agonist（ tegaserod maleate） and inhibitor（ GR113808）

and adenylate cyclase（ AC） agonist( Forskolin and inhibitor（ SQ22536） were used to interfere the signal transduction of 5-hydroxytryptamine（ 5-HT） pathway

and then incubated with the selected Xuanfu Daizhe Decoction serum. The intracellular cyclic adenosine monophosphate（ cAMP） content was detected by ELISA. The fluorescence intensity of 5-HT

R was detected by immumofluorescence method

and the intracellular Ca

concentration was detected by Ca

probe. Results The low-dose group of 5% Xuanfu Daizhe Decoction had the strongest proliferation effect on ESMC

so it was selected for subsequent experiments. Compared with the 5-HT

R agonist group

the Ca

fluorescence enhancement ratio of the 5-HT

R agonist plus Chinese medicine group increased（ P

0. 01）.Compared with the AC agonist group

the 5-HT

R fluorescence intensity and Ca

fluorescence intensity of the AC agonist plus Chinese medicine group increased significantly（ P

0. 01）. Compared with the 5-HT

R inhibitor group

5-HT

R fluorescence intensity

fluorescence enhancement ratio

and cAMP concentration in the 5-HT

R inhibitor plus Chinese medicine group increased（ P

0. 05 or P

0. 01）. Compared with the AC inhibitor group

the5-HT

R fluorescence enhancement ratio

fluorescence intensity

and cAMP concentration of the AC inhibitor plus Chinese medicine group increased（ P

0. 05 or P

0. 01）. Conclusion Xuanfu Daizhe Decoction may increase the expression of 5-HT

then activate AC

promote the release of c AMP

increase the intracellular Ca

concentration

cause contraction of esophageal smooth muscle

and reduce reflux.

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