千金子制霜前后提取物对TLR4/NF-κB/NLRP3信号通路的影响研究

王慧楠; 姜明瑞; 王志成; 岳珠珠; 魏晓彤; 王梦琳; 石双慧; 张婧秋; 陈梦雨; 刘芊芊; 王英姿

doi:10.3969/j.issn.1006-2157.2023.06.008

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千金子制霜前后提取物对TLR4/NF-κB/NLRP3信号通路的影响研究

实验研究 | 更新时间：2023-07-05

- 千金子制霜前后提取物对TLR4/NF-κB/NLRP3信号通路的影响研究
- Effects of extracts from Euphorbiae Semen before and after frosting on the TLR4/NF-κB/NLRP3 signaling pathway
- 北京中医药大学学报 2023年46卷第6期页码：780-789
- 作者机构：
  
  北京中医药大学中药学院　北京　102488
- 作者简介：
  
  王慧楠，女，在读博士生
  # 王英姿，女，博士，教授，博士生导师，主要研究方向：中药制剂新技术与中药炮制原理，E-mail：wangyzi@sina.com
- 基金信息：
  
  国家自然科学基金项目(82074021)
- DOI：10.3969/j.issn.1006-2157.2023.06.008
  中图分类号： R285.5
- 纸质出版日期：2023-06-30，
  
  网络出版日期：2023-04-13，
  
  收稿日期：2022-10-06，
- 稿件说明：
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王慧楠, 姜明瑞, 王志成, 等. 千金子制霜前后提取物对TLR4/NF-κB/NLRP3信号通路的影响研究[J]. 北京中医药大学学报, 2023,46(6):780-789.

WANG Huinan, JIANG Mingrui, WANG Zhicheng, et al. Effects of extracts from Euphorbiae Semen before and after frosting on the TLR4/NF-κB/NLRP3 signaling pathway[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(6):780-789.
王慧楠, 姜明瑞, 王志成, 等. 千金子制霜前后提取物对TLR4/NF-κB/NLRP3信号通路的影响研究[J]. 北京中医药大学学报, 2023,46(6):780-789. DOI： 10.3969/j.issn.1006-2157.2023.06.008.

WANG Huinan, JIANG Mingrui, WANG Zhicheng, et al. Effects of extracts from Euphorbiae Semen before and after frosting on the TLR4/NF-κB/NLRP3 signaling pathway[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023,46(6):780-789. DOI： 10.3969/j.issn.1006-2157.2023.06.008.

摘要

目的

研究千金子制霜前后提取物对Toll样受体4(TLR4)/核转录因子－κB(NF-κB)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路的影响差异。

方法

42只BALB/c小鼠按体质量随机分为空白对照组，千金子生品低、中、高剂量组(10.49、20.98、41.96 g/kg)和千金子霜品低、中、高剂量组(10.49、20.98、41.96 g/kg)，每组6只，连续灌胃7 d。酶联免疫吸附测定法检测血清白细胞介素－1β(IL-1β)和肿瘤坏死因子－α(TNF-α)的含量，蛋白质印迹法和实时荧光PCR法检测结肠组织TLR4、NF-κB p65、NLRP3蛋白和mRNA表达。30只BALB/c小鼠按体质量随机分为空白对照组、千金子生品组、千金子生品＋TAK－242组、千金子霜品组、千金子霜品＋TAK－242组，每组6只。各给药组灌胃相应药液(20.98 g/kg)，连续7 d，千金子生品＋TAK－242组和千金子霜品＋TAK－242组于给药第1、5、6、7天给药前30 min腹腔注射TAK－242溶液(3 mg/kg)。采用蛋白质印迹法和实时荧光PCR法检测结肠组织NLRP3、NF-κB p65蛋白表达，以及NLRP3、IL-1β mRNA表达。

结果

千金子生品可导致IL-1β和TNF-α大量释放，上调TLR4、NF-κB p65、NLRP3蛋白和mRNA表达(

0.05，

0.01)，千金子制霜后对IL-1β、TNF-α分泌，以及TLR4、NF-κB p65、NLRP3蛋白和mRNA表达的促进作用减弱(

0.05，

0.01)。加入TLR4的阻断剂TAK－242后，可抑制NLRP3、NF-κB p65蛋白表达，以及NLRP3、IL-1β mRNA表达(

0.01)。

结论

千金子生品可能通过激活TLR4介导的NF-κB/NLRP3通路，促进TNF-α和IL-1β释放，诱导结肠组织产生炎性反应，而制霜后对TLR4/NF-κB/NLRP3通路传导及促炎因子分泌的上调作用明显减弱，提示其制霜减毒的作用机制可能与致炎能力下降及干预TLR4/NF-κB/NLRP3信号通路有关。

Abstract

Objective

To investigate the effects of the extracts from Euphorbiae Semen before and after frosting on the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB)/NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling pathway.

Methods

According to the body weight

42 BALB/c mice were randomly divided into a blank control group

Euphorbiae Semen low-

medium-

and high- dose groups (10.49

20.98

and 41.96 g/kg

respectively) and Euphorbiae Semen Pulveratum low-

medium-

and high- dose groups (10.49

20.98

and 41.96 g/kg

respectively)

with six mice in each group. Mice received continuous gavage for 7 d. The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum of mice were determined by enzyme linked immune sorbent assay. The protein and the mRNA expression of TLR4

NF-κB p65

and NLRP3 in colon tissues of mice were detected by Western blotting and real-time PCR. According to the body weight

30 BALB/c mice were randomly divided into a blank control group

Euphorbiae Semen group

Euphorbiae Semen + TAK-242 group

Euphorbiae Semen Pulveratum group

and Euphorbiae Semen Pulveratum+ TAK-242 group

with six mice in each group. The administration groups were respectively given 20.96 g/kg of Euphorbiae Semen or Euphorbiae Semen Pulveratum

and the continuous gavage time was 7 d. The Euphorbiae Semen + TAK-242 group and Euphorbiae Semen Pulveratum+ TAK-242 group were intraperitoneally injected with TAK242 (3 mg/kg) 30 min before and on Day 1

and 7 of administration. The protein expression of NLRP3 and NF-κB p65

and the mRNA expression of NLRP3 and IL-1β in colon tissues of mice were assessed by Western blotting and real-time PCR.

Results

Euphorbiae Semen not only significantly promoted the release of IL-1β and TNF-α

but it also significantly increased the protein and the mRNA expression of TLR4

NF-κB p65 and NLRP3 (

0.05

0.01). However

upregulation of IL-1β and TNF-α secretion

and the protein and mRNA expressions of TLR4

NF-κB p65

NLRP3 were decreased by Euphorbiae Semen Pulveratum (

0.05

0.01). NLRP3 and NF-κB p65 protein expression and NLRP3 and IL-1β mRNA expression were inhibited after the intervention with the TLR4 inhibitor TAK-242 (

0.01).

Conclusion

Euphorbiae Semen could induce an inflammatory response of the colon tissue by activating the TLR4-mediated NF-κB/NLRP3 pathway

thus promoting the release of TNF-α and IL-1β. However

upregulation of the TLR4/NF-κB/NLRP3 signaling pathway and pro-inflammatory factor secretion were significantly alleviated by Euphorbiae Semen Pulveratum

indicating that the detoxification mechanism of Euphorbiae Semen frosting may be related to the decreased ability to cause inflammation and the intervention of TLR4/NF-κB/NLRP3 signaling pathway.

关键词

千金子制霜减毒TLR4/NF-κB/NLRP3信号通路炎性反应小鼠

Keywords

Euphorbiae Semendetoxification by frostingTLR4/NF-κB/NLRP3 signaling pathwayinflammatory responsemice

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