NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP

Abstract

Background

NeiyiKangfu tablets (NYKF) are widely used clinically for the treatment of endometriosis (EMS), whose mechanism of action has been extensively studied. Researchers have found that NYKF may control the development of ectopic lesions by inhibiting angiogenesis and inflammatory cytokine secretion. Nevertheless, NYKF’s mechanism of action remains unclear.

Methods

In the present study, the function of NYKF in the progression of EMS and the associated underlying mechanism was investigated by in vivo and in vitro experiments. EMS model mice were treated with NYKF and the pro-inflammatory factors and apoptosis of ectopic endometrium as well as RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling activation were assessed. In addition, human endometriosis-derived immortalized entopic stromal (hEM15A) cells transfected with or without RAF kinase inhibitor protein (RKIP)-small-interfering RNA (siRNA) were also treated with NYKF and the proliferation, migration, apoptosis, and RAF/MEK/ERK signaling activation were measured by Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, and western blot, respectively.

Results

Results showed that NYKF increased the expression of RKIP, inhibited RAF/MEK/ERK signaling activation, and induced apoptosis while inhibiting proliferation and migration both in EMS mice and hEM15A cells. RKIP knockdown could inhibit the effect of NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and the proliferation and migration of hEM15A cells.

Conclusions

In conclusion, these results suggest that NYKF treatment promotes apoptosis and inhibits proliferation and migration in EMS by inhibiting the RAF/MEK/ERK signaling pathway by targeting RKIP.

摘要

背景

内异康复片（NYKF）在临床上被广泛用于治疗子宫内膜异位症（EMS）, 其作用机制已被广泛研究。研究人员发现, NYKF可能通过抑制血管生成和炎性细胞因子分泌来控制异位病变的发展。然而, NYKF的作用机制仍不清楚。

方法

本研究通过体内和体外实验研究NYKF在EMS发生发展中的作用及其相关机制。观察NYKF对EMS模型小鼠促炎症因子、异位内膜细胞凋亡及RAF/MEK/ERK信号通路的影响。此外, 还对转染或不转染RAF激酶抑制蛋白(RKIP)-小干扰RNA(SiRNA)的人子宫内膜异位症永生化内膜基质(HEM15A)细胞进行NYKF治疗, 分别用细胞计数试剂盒(CCK-8)、流式细胞仪、Transwell和Western印迹检测细胞的增殖、迁移、凋亡和RAF/MEK/ERK信号的激活。

结果

NYKF可上调EMS小鼠和hEM15A细胞RKIP的表达, 抑制RAF/MEK/ERK信号通路的激活, 诱导细胞凋亡, 抑制细胞增殖和迁移。RKIP基因敲除可抑制NYKF的作用, 激活RAF/MEK/ERK信号通路, 促进hEM15A细胞的增殖和迁移。

结论

NYKF通过RKIP抑制RAF/MEK/ERK信号通路, 从而促进EMS细胞的凋亡, 抑制EMS的增殖和迁移。

Keywords:

• NeiyiKangfu tablets
• endometriosis
• RAF/MEK/ERK signaling pathway
• RKIP
• apoptosis

关键词:

• 内异康复片
• 子宫内膜异位症
• RAF/MEK/ERK信号通路
• RKIP
• 细胞凋亡

Acknowledgments

Not applicable.

Author contributions

Data curation, W.Y., L.F. and R.L.; Formal analysis, W.Y. and Y.Z.; Funding acquisition, W.Y.; Investigation, L.P.; Software, T.Z. and L.Z.; Writing – original draft, W.Y.; Writing – review & editing, W.Y. All authors have read and agreed to the published version of the manuscript.

Institutional review board statement

All murine experiments were approved by the Ethics Committee of Hospital of Chengdu University of Traditional Chinese Medicine.

Informed consent statement

Not applicable.

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This research was funded by Chengdu University of Traditional Chinese Medicine “Xinglin Scholars” talent research promotion program, grant number XSGG2019018.