壮腰通络方含药血清对TNF-α介导的髓核细胞焦亡恶性循环的影响

冯蓬; 高春雨; 孙凯; 朱立国; 高景华; 李路广; 李建国; 车颖

doi:10.13288/j.11-2166/r.2023.21.011

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壮腰通络方含药血清对TNF-α介导的髓核细胞焦亡恶性循环的影响

实验研究 | 更新时间：2023-10-26

- 壮腰通络方含药血清对TNF-α介导的髓核细胞焦亡恶性循环的影响
- Effect of Zhuangyao Tongluo Formula（壮腰通络方） Containing Serum on TNF-α-mediated Vicious Circle of Pyroptosis of Nucleus Pulposus Cells
- 中医杂志 2023年64卷第21期页码：2224-2231
- 作者机构：
  
  1.中国中医科学院望京医院，北京市朝阳区望京中环南路6号，100102
  2.山东中医药大学
- 作者简介：
  
  gaochunyu8526@sina.com
- 基金信息：
  
  国家自然科学基金(82205152);中国中医科学院优秀青年科技人才(ZZ16-YQ-023);中国中医科学院望京医院院级科研课题苗圃培育项目(WJYY-YJKT-2022-03)
- DOI：10.13288/j.11-2166/r.2023.21.011
  中图分类号：
- 收稿：2023-05-22，
  
  修回：2023-08-11，
  
  纸质出版：2023-11-02
- 稿件说明：
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冯蓬,高春雨,孙凯等.壮腰通络方含药血清对TNF-α介导的髓核细胞焦亡恶性循环的影响[J].中医杂志,2023,64(21):2224-2231.

FENG Peng,GAO Chunyu,SUN Kai,et al.Effect of Zhuangyao Tongluo Formula（壮腰通络方） Containing Serum on TNF-α-mediated Vicious Circle of Pyroptosis of Nucleus Pulposus Cells[J].Journal of Traditional Chinese Medicine,2023,64(21):2224-2231.
冯蓬,高春雨,孙凯等.壮腰通络方含药血清对TNF-α介导的髓核细胞焦亡恶性循环的影响[J].中医杂志,2023,64(21):2224-2231. DOI： 10.13288/j.11-2166/r.2023.21.011.

FENG Peng,GAO Chunyu,SUN Kai,et al.Effect of Zhuangyao Tongluo Formula（壮腰通络方） Containing Serum on TNF-α-mediated Vicious Circle of Pyroptosis of Nucleus Pulposus Cells[J].Journal of Traditional Chinese Medicine,2023,64(21):2224-2231. DOI： 10.13288/j.11-2166/r.2023.21.011.

摘要

目的

探讨壮腰通络方延缓腰椎间盘退变的可能机制。

方法

利用佛波酯、干扰素γ和脂多糖将THP-1细胞诱导为M1巨噬细胞，与髓核细胞建立Transwell共培养体系。首先以胎牛血清作为对照血清，采用MTT法分析不同体积分数（5%、10%、15%、20%）壮腰通络方大鼠含药血清对M1和髓核细胞活性的影响，筛选后续实验药物浓度。随后建立实验一，分为髓核细胞对照组、M1巨噬细胞对照组、髓核细胞+壮腰方组、髓核细胞+肿瘤坏死因子α（TNF-α）组、髓核细胞+TNF-α+壮腰方组、共培组、共培+壮腰方组，每组3个复孔。利用ELISA法检测各组培养上清白细胞介素1β（IL-1β）和白细胞介素18（IL-18）含量，实时荧光定量PCR检测髓核细胞中IL-1β和IL-18 mRNA表达，细胞免疫荧光法检测髓核细胞中Gasdermin D（GSDMD）蛋白表达。实验二利用TNF-α过表达质粒或TNF-α空载体质粒干预M1巨噬细胞，并在共培养条件下分为共培组、空载体质粒+共培组、空载体质粒共培+壮腰方组、共培+壮腰方组、过表达质粒共培+壮腰方组、过表达质粒+共培组，每组3个复孔。利用实时荧光定量PCR和蛋白免疫印迹法检测各组M1巨噬细胞中TNF-α mRNA和蛋白表达。

结果

最终筛选出10%的壮腰通络方含药血清进行后续实验。实验一结果显示，与髓核细胞对照组比较，髓核细胞+壮腰方组IL-1β、IL-18含量及mRNA、GSDMD表达差异无统计学意义（

＞0.05），而髓核细胞+TNF-α组和共培组IL-1β、IL-18含量及mRNA、GSDMD表达均显著增加（

＜0.01）。与共培组比较，共培+壮腰方组IL-1β、IL-18含量及mRNA、GSDMD表达均显著降低（

＜0.01）。实验二结果显示，与共培组比较，空载体质粒+共培组TNF-α mRNA和蛋白表达差异无统计学意义（

＞0.05）。与空载体质粒+共培组比较，空载体质粒共培+壮腰方组TNF-α mRNA和蛋白表达均显著降低（

＜0.01）。与共培组和空载体质粒+共培组比较，共培+壮腰方组TNF-α mRNA和蛋白表达显著降低（

＜0.01）；与共培+壮腰方组比较，过表达质粒共培+壮腰方组TNF-α mRNA和蛋白表达均显著增加（

＜0.01）；与过表达质粒共培+壮腰方组比较，过表达质粒+共培组TNF-α mRNA和蛋白表达均显著增加（

＜0.01）。

结论

壮腰通络方含药血清能够通过降低M1巨噬细胞中TNF-α的表达，抑制由TNF-α诱导的髓核细胞焦亡恶性循环，延缓腰椎间盘退变。

Abstract

Objective

To explore the mechanism of

Zhuangyao Tongluo Formula

（壮腰通络方，ZTF） in delaying intervertebral disc degeneration

Methods

M1 macrophages were induced from THP-1 cells using LPS， IFN-γ and PMA. The induced M1 macrophages were then co-cultured with nucleus pulposus cells in a transwell system. Fetal bovine serum was used as the control serum， and the effects of different concentrations （5%， 10%， 15%， 20%） of serum from rats treated with ZTF on the activity of M1 macrophages and nucleus pulposus cells were analyzed using MTT assay. Experiment 1 was established， including the nucleus pulposus cell control group， M1 macrophage control group， nucleus pulposus cell + ZTF

group， nucleus pulposus cell + TNF control group， nucleus pulposus cell + TNF + ZTF

group， co-culture group， and co-culture + ZTF

group. The levels of IL-1β， and IL-18 in the culture supernatant were detected using ELISA. The mRNA expression of IL-1β and IL-18 in nucleus pulposus cells was detected using qPCR. Additionally， the expression of GSDMD protein in nucleus pulposus cells was detected using cell immunofluorescence. In experiment 2， co-culture groups were constructed using TNF-α overexpression （OE） or empty vector （EV） plasmids， including co-culture group， TNF-EV + co-culture group， TNF-EV co-culture group + ZTF， co-culture + ZTF

group， TNF-OE co-culture group + ZTF， and TNF-OE + co-culture group. The mRNA and protein expression of TNF-α in M1 cells in each group were detected using qPCR and WB.

Results

The ZTF with 10% serum was selected for subsequent experiments. The results of experiment 1 showed that compared to the control group of nucleus pulposus cells， there was no statistically significant difference in the levels of IL-1β， IL-18， mRNA， and GSDMD expression in the nucleus pulposus cells + ZTF

group （

＞0.05）. However， the TNF-α + co-culture group showed a significant increase in IL-1β， IL-18 levels， mRNA， and GSDMD expression （

＜0.01）. When compared to the co-culture group， the ZTF+ co-culture group showed a significant decrease in IL-1β， IL-18 levels， mRNA， and GSDMD expression （

＜0.01）. The results of experiment 2 showed that there was no statistically significant difference in TNF-α mRNA and protein expression between the empty vector plasmids + co-culture group and the co-culture group （

＞0.05）. Compared to the empty vector + co-culture group， the expression of TNF-α mRNA and protein was significantly reduced in the empty vector co-culture + ZTF

group （

＜0.01）. Compared to the co-culture group and the empty vector + co-culture group， the expression of TNF-α mRNA and protein was significantly reduced in the co-culture + ZTF group （

＜0.01）. Compared to the co-culture + ZTF

group， the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture + ZTF group （

＜0.01）. Compared to the overexpression vector co-culture + ZTF

group， the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture group （

＜0.01）.

Conclusion

ZTF

serum can inhibit the TNF-α-induced apoptosis of nucleus pulposus cells and delay lumbar disc degeneration by reducing the expression of TNF-α in M1 macrophages.

关键词

Keywords

references

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