桃核承气汤不同有效部位对TGF-<italic style="font-style: italic">β</italic><sub>1</sub>诱导的HK-2细胞分泌与降解细胞外基质的影响

周珊珊; 艾中柱; 李伟男; 巴元明

doi:10.13422/j.cnki.syfjx.20201904

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桃核承气汤不同有效部位对TGF-β₁诱导的HK-2细胞分泌与降解细胞外基质的影响

经典名方 | 更新时间：2020-09-16

- 桃核承气汤不同有效部位对TGF-β₁诱导的HK-2细胞分泌与降解细胞外基质的影响
- Effect of Different Effective Parts of Taohe Chengqitang on Synthesis and Degradation of Extracellular Matrix in HK-2 Cells Induced by TGF-β₁
- 中国实验方剂学杂志 2020年26卷第19期页码：127-134
- 作者机构：
  
  1.湖北中医药大学，武汉 430065
  2.湖北省中医院，武汉 430061
  3.湖北省中医药研究院，武汉 430074
- 作者简介：
  
  周珊珊，博士，从事中医药防治慢性肾脏疾病研究，E-mail：zhouss2009@126.com
  * 巴元明，教授，博士生导师，从事中医药防治慢性肾脏疾病研究，E-mail：bayuanming@126.com
- 基金信息：
  
  武汉市科技计划项目(2017060201010223)
- DOI：10.13422/j.cnki.syfjx.20201904
  中图分类号： R2-0;R22;R285.5;R289
- 收稿日期：2020-01-13，
  
  网络出版日期：2020-07-21，
  
  纸质出版日期：2020-10-05
- 稿件说明：
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周珊珊,艾中柱,李伟男等.桃核承气汤不同有效部位对TGF-β₁诱导的HK-2细胞分泌与降解细胞外基质的影响[J].中国实验方剂学杂志,2020,26(19):127-134.

ZHOU Shan-shan,AI Zhong-zhu,LI Wei-nan,et al.Effect of Different Effective Parts of Taohe Chengqitang on Synthesis and Degradation of Extracellular Matrix in HK-2 Cells Induced by TGF-β₁[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(19):127-134.
周珊珊,艾中柱,李伟男等.桃核承气汤不同有效部位对TGF-β₁诱导的HK-2细胞分泌与降解细胞外基质的影响[J].中国实验方剂学杂志,2020,26(19):127-134. DOI： 10.13422/j.cnki.syfjx.20201904.

ZHOU Shan-shan,AI Zhong-zhu,LI Wei-nan,et al.Effect of Different Effective Parts of Taohe Chengqitang on Synthesis and Degradation of Extracellular Matrix in HK-2 Cells Induced by TGF-β₁[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(19):127-134. DOI： 10.13422/j.cnki.syfjx.20201904.

摘要

目的

探讨桃核承气汤不同有效部位对转化生长因子-

（TGF-

）诱导的人肾小管上皮细胞（HK-2）中细胞外基质合成与降解的影响。

方法

采用系统溶剂法萃取70%乙醇提取的桃核承气汤

得到石油醚萃取物质部位，三氯甲烷萃取物质部位，乙酸乙酯萃取物质部位，正丁醇萃取物质部位，萃余水相物质部位，多糖物质部位和芒硝物质部位。利用TGF-

诱导HK-2细胞构建纤维化模型，以不同质量浓度（0，50，100，200，400，800 mg·L

-1

）桃核承气汤各部位干预细胞，酶联免疫吸附测定（ELISA）检测细胞上清液胶原蛋白Ⅰ

1（Col-Ⅰ

1），纤维粘连蛋白（FN）含量，初步筛选得到主要有效物质部位进行后续实验验证。细胞增殖与毒性检测试剂盒（CCK-8）确定筛选出的有效部位的最佳浓度；蛋白免疫印迹法（Western blot）检测胶原蛋白-Ⅰ(Col-Ⅰ)，胶原蛋白-Ⅲ(Col-Ⅲ)，基质金属蛋白酶-2（MMP-2），基质金属蛋白酶抑制因子2（TIMP2），结缔组织生长因子（CTGF）蛋白的表达水平；免疫荧光检测

-平滑肌肌动蛋白（

-SMA）的表达；实时荧光定量聚合酶链式反应（Real-time PCR）检测纤溶酶原激活抑制剂-1（PAI-1） mRNA的表达。

结果

ELISA检测结果显示，与模型组比较，乙酸乙酯、正丁醇、三氯甲烷萃取部位质量浓度为200，400 mg·L

-1

时能显著降低Col-Ⅰ

1和FN含量（

0.05，

0.01）。CCK-8检测结果显示，各组药物干预质量浓度为400，800 mg·L

-1

时显著抑制细胞活性(

0.01)。Western blot检测结果显示，与模型组比较，乙酸乙酯、三氯甲烷萃取部位均能降低Col-Ⅰ，Col-Ⅲ，TIMP2和CTGF蛋白的表达，上调MMP-2的表达（

0.05），正丁醇萃取部位作用效果更显著（

0.01）。免疫荧光结果显示，乙酸乙酯、三氯甲烷萃取部位均能抑制

-SMA的表达（

0.05），正丁醇萃取部位抑制作用更显著（

0.01）。Real-time PCR检测发现，乙酸乙酯、三氯甲烷萃取部位均能抑制PAI-1 mRNA的表达（

0.05），正丁醇部位更显著（

0.01）。

结论

本研究初步筛选出桃核承气汤抗肾脏纤维化的主要有效物质部位为乙酸乙酯萃取物质部位、正丁醇萃取物质部位和三氯甲烷萃取物质部位，其中正丁醇萃取物质部位活性最强，其作用机制可能与调节TGF-

诱导的HK-2细胞分泌细胞外基质（ECM）的合成与降解有关。

Abstract

Objective

To explore the effect of different effective parts of Taohe Chengqitang on the synthesis and degradation of extracellular matrix in human kideny-2（HK-2） cells induced by transforming growth factor-

（TGF-

）.

Method

Petroleum ether extract

ethyl acetate extract

n-butanol extract

raffinate and polysaccharide extract

mirabilite extract were extracted with 70% ethanol by systematic solvent method. The HK-2 cell fibrosis model induced by TGF-

was built to intervene the cells in different parts of Taohe Chengqitang with different concentrations (0

100

200

400

800 mg·L

-1

). Enzyme-linked immunosorbent assay（ELISA）kit assay was used to detect collagen（Col）-Ⅰ

1 and fibronectin （FN）in supernatant to screen out the main active parts. Cell counting kit-8 （CCK-8）method was used to determine the best concentration of intervention site of bioactive components. Western blot analysis was used to detect the expression levels of Col-Ⅰ

Col-Ⅲ

matrix metalloproteinase-2 (MMP-2)

matrix metalloproteinase inhibitor2 (TIMP2)

and connective tissue growth factor (CTGF). Immunofluorescence assay was used to detect the expression of

-smooth muscle actin（

-SMA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） analysis was used to detect the mRNA expression of plasminogen activator inhibitor-1（PAI-1）.

Result

ELISA kit assay demonstrated that compared with the model group

ethyl acetate extract

n-butanol extract and chloroform extract significantly reduced the Col-Ⅰ

1 and FN content at the concentrations of 200 and 400 mg·L

-1

(

0.05

0.01). CCK-8 assay showed that the cells viability was significantly inhibited with drug intervention at the concentrations of 400 and 800 mg·L

-1

(

0.01). Western blot demonstrated that compared with the model group

ethyl acetate extract

n-butanol extract and chloroform extract decreased the expression levels of Col-Ⅰ

Col-Ⅲ

TIMP2 and CTGF in HK-2 cells induced by TGF-

and increased the expression of MMP-2 (

0.05)

with more significant effect in n-butanol extract (

0.01). The results of immunofluorescence showed that ethyl acetate extract

n-butanol extract and chloroform extract could inhibit the expression of

-SMA (

0.05)

with more significant effect in n-butanol extract (

0.01). The results of Real-time PCR showed that ethyl acetate extract and chloroform extract inhibited mRNA expression of PAI-1 (

0.05)

with more significant effect in n-butanol extract (

0.01).

Conclusion

The extracts of ethyl acetate

n-butanol and chloroform are the active parts of Taohe Chengqitang with the anti-renal fibrosis effect

with n-butanol extract as the most active part. The mechanism on anti-renal fibrosis may be related to its regulation of extracellular matrix (ECM) synthesis and degradation.

关键词

Keywords

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桃核承气汤不同有效部位对TGF-β1诱导的HK-2细胞分泌与降解细胞外基质的影响

Effect of Different Effective Parts of Taohe Chengqitang on Synthesis and Degradation of Extracellular Matrix in HK-2 Cells Induced by TGF-β1

DOI：10.13422/j.cnki.syfjx.20201904

摘要

Abstract

关键词

Keywords

references

桃核承气汤不同有效部位对TGF-β₁诱导的HK-2细胞分泌与降解细胞外基质的影响

Effect of Different Effective Parts of Taohe Chengqitang on Synthesis and Degradation of Extracellular Matrix in HK-2 Cells Induced by TGF-β₁