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

Introduction

Endometriosis (EMS) is a common gynecological disease characterized by the growth of functional endometrial tissues in other areas outside the uterine cavity [1]. The typical symptoms in patients with EMS include chronic pelvic pain, abnormal menstruation, infertility, and dyspareunia [2]. It is extremely prevalent, occurs in 10% of women of reproductive age, and adversely affects the health and quality of life of patients [3, 4]. Furthermore, EMS is an estrogen-dependent multifactorial disease that develops in patients with risk factors and favorable genetic conditions due to hypothetical pathogenic pathways. There is not a single causative mechanism for developing EMS. For example, retrograde menstruation, the most studied pathogenic way of EMS, is common in women of reproductive age with or without EMS [5]. Still, only patients with risk factors and/or genetic conditions are highly likely to develop EMS [6]. Moreover, endometrial cells must complete a multistep process involving immune escape, adhesion, proliferation, invasion, and angiogenesis to develop and grow in ectopic sites [3, 7], which were regulated by a complex of signaling pathways and their interactions.

Studies have reported that theories on the etiology of EMS include retrograde menstruation, celomic metaplasia, altered immunity, stem cells, and genetics, which affect gametes and embryos, fallopian tube and embryo transport, and ectopic endometrium. In addition, numerous studies have shown that RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade is one of the key mitogen-activated protein kinase (MAPK) signal cascades for mediating the intracellular transmission of extracellular signals and inducing cellular processes [8]. RAS binds to RAF and then activates the downstream signal MEK1/2, followed by activation of ERK phosphorylation [8]. It has been reported that RAF/MEK/ERK plays an important regulatory role in cell growth, proliferation, migration, and apoptosis in many diseases [9], including endometrial cancer [10]. Moreover, activated RAF/MEK/ERK in the ectopic endometrium was observed [11], and was associated with the proliferation and migration of endometrial stromal cells in EMS [12]. Current evidence substantiates that RAF kinase inhibitor protein (RKIP) can combine with the RAF and interfere with the downstream RAF/MEK/ERK pathway, acting as a physiological endogenous inhibitor of the MAPK pathway [13]. However, the regulation of RAF/MEK/ERK signal in EMS is incompletely understood.

The current clinical treatment of EMS is mainly based on the elimination of lesions, reduction and elimination of clinical pain, and reduction and prevention of disease recurrence through pharmacological or surgical treatment [14]. Although conventional Western medicine (progestins, contraceptives, non-steroidal anti-inflammatory drugs, etc.) can be effective, the disease itself requires long-term treatment, and there are many side effects of Western medicine [15]. Therefore, it is especially important to choose a well-tolerated and effective treatment method. NeiyiKangfu tablet (NYKF) is a kind of traditional hospital preparation with various combinations of herbs such as Bichihua, Sanguoteng, cooked rhubarb, peach kernel, three prune, coix seed, soil turtle worm, turtle, and so on. It is used in EMS model rats because of the remarkable effect of clearing dampness and removing blood stasis [16]. Previous studies have demonstrated the efficacy of NYKF in inhibiting angiogenesis and inflammatory cytokines secretion, promoting ectopic lesion regression, and reliving pain in EMS [17–19]. However, the molecular mechanism of its therapeutic effect in EMS remains unclear. Investigating NYKF tablets may take a step forward in exploring new clinical strategies to help manage endometriosis in women. Therefore, in this study, we investigate the role of the NYKF tablet in controlling the progression of EMS by regulating the RAF/MEK/ERK signaling pathway. The RAF/MEK/ERK signaling pathway is shown in Figure 1.

Figure 1. The RAF/MEK/ERK signaling pathway.

Materials and methods

Animals and experimental design

Forty-two healthy female C57BL/6 (10 weeks old, weighing 18–21 g) mice were used in this study. Twelve of them were randomly selected as donors for donor uterine fragments. All mice were obtained from Chengdu Dashuo Biotechnology Co., Ltd. All animals were housed in cages with free access to sterile food and water in the animal laboratory. In addition, all protocols were conducted per the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China (https://www.most.gov.cn/xxgk/xinxifenlei/fdzdgknr/fgzc/gfxwj/gfxwj2010before/201712/t20171222_137025.html). The mice were cared for in accordance with the Guide to the Care and Use of Experimental Animals (Vol. 1, 2nd ed., 1993, and Vol. 2, 1984, Canadian Council on Animal Care (CCAC), www.ccac.ca). All murine experiments were approved by the Ethics Committee of the Hospital of Chengdu University of Traditional Chinese Medicine. The donor mice were subcutaneously injected with estradiol benzoate (E2, 0.1 mg) for the first 3 days. After anesthesia with 1% sodium pentobarbital (40 mg/kg) intraperitoneally, the donor uterus was removed and cut into four 2–3 mm³ segments and placed into serum-free Dulbecco’s minimum essential medium (DMEM)/F-12 medium (DMEM/F-12 is a widely used basal medium for supporting the growth of many different mammalian cells [20]) under aseptic conditions. The recipient mice were anesthetized, intubated, and mechanically ventilated. A 5-mm incision was made on the ventral midline. Subcutaneous tissue was separated on both sides of the incision, then two prepared 2–3 mm³ endometrial tissues were implanted, respectively. Finally, the cutis was sutured. Mice in the sham group performed the same procedure as the recipient mice, except that endometrium was not implanted. On day 7 after implantation, surgery was performed on the mice to confirm the viability of the implant and to verify the establishment of an endometriosis mouse model.

After successful modeling, the recipient mice were randomly divided into four groups with five mice in each groups as follows: EMS group, was administered an equal volume of distilled water by gavage; the intervention group consisted of low-dose NYKF group (0.107 g/mL), medium-dose NYKF group (0.215 g/mL), and high-dose NYKF group (0.43 g/mL), at 0.4 mL/20 g gavage per mouse per day. After 3 weeks of consecutive treatment, mice were sacrificed and the ectopic endometrial tissues were collected for further analysis.

Preparation of the NYKF-medicated serum

Ten-week-old mice were intragastrically administered with NYKF (0.753 g/mL, 0.4 mL/20 g) once a day for consecutive five days. At 2 h after the last treatment, the serum was collected and stored at –80 °C for further cell experiments.

Hematoxylin and eosin staining

The ectopic endometrium of mice in each group were removed and fixed in 10% formalin, embedded in paraffin. The endometrium tissue section (5 μm) was stained with hematoxylin and eosin (H&E) for analysis.

TUNEL staining

TUNEL test has been designed to identify apoptotic cells by detecting DNA degradation. According to the instructions of the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (G1501-50T, Servicebio), the above paraffin sections were taken and added into xylene (2 changes, each change 10 min) and then rehydrated in graded alcohol (each 5 min). Next, the sections were incubated with 100 μL of Proteinase K at 37 °C for 20 min, PBS for three times, incubated with TUNEL working solution for 60 min, PBS for four times, and finally developed DAPI (DAPI is a highly specific DNA stain that preferentially binds to A-T regions of the DNA molecule) in dark, and dehydrated, transparent and sealed routinely. The positive apoptotic cells were green under a microscope. Apoptosis rate = the number of apoptotic positive cells/the total number of cells.

Immunohistochemistry

Immunohistochemistry (IHC) was performed to determine the expression of p-RAF, p-MEK, p-ERK1/2, and RKIP. The above paraffin sections were deparaffinized, quenched, blocked with goat serum, incubated with primary antibody against p-RAF, p-MEK, p-ERK1/2, and RKIP overnight, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody. Finally, the sections were stained with DAB and re-stained with hematoxylin. Under the light microscope, the positive cells were brown in the cytoplasm.

Cell culture

The hEM15A cell line was used to investigate whether NYKF affected ectopic endometrial cells. It was cultured in DMEM/F12 medium containing 10% fetal bovine serum (blank control), mice serum without NYKF (negative control), 5% NYKF-medicated mice serum, 10% NYKF-medicated mice serum, and 20% NYKF-medicated mice serum.

siRNA knockdowns

hEM15A cells (3 × 10⁵ cells/well) were cultured in six-well plates for 24 h and then transfected with two small-interfering RNAs (siRNA no. 1, position 599-624, 5′-CGAGTGG GATGACTATGTGCCCAAA-3′. SiRNA no. 2, position 622-646, 5′-AACTGTACGAGCAG CTGTCTGGGAA-3′) targeting human RKIP or negative control (siNC) using RiboFectTM CP Transfection Kit following the manufacturer’s instructions. The cells were analyzed 48 h after transfection.

Cell proliferation assay

Proliferation experiments were performed with CCK-8 according to the manufacturer’s protocol. hEM15A cells (3 × 10³ cells/well) were seeded in the 96-well plate at 37 °C and 5% CO₂ overnight. After discarding the supernatant and washing it with PBS, the cells were cultured with different concentrations of drug-containing serum for 48 h. CCK-8 solution was added to the cells (10 μL/well), and then the 96-well plate was incubated at 37 °C and 5% CO₂ for 1 h. After that, the OD₄₅₀ values (optical density at 450 nm wavelength) were measured using the microplate reader (SpectraMAX Plus384).

Flow cytometry

hEM15A cells (3 × 10⁵ cells/well) were seeded in the six-well plate at 37 °C and 5% CO₂ overnight. After further incubation with different concentrations of drug-containing serum for 48 h, the cells were collected and incubated with 5 μL AnnexinV-FITC for 15 min and 5 μL propidium iodide (PI) for 5 min at 4 °C. The cell apoptosis was assayed on a flow cytometer.

Transwell assay

hEM15A cells were cultured with different drug-containing serum for 48 h, and then resuspended with a serum-free medium. Cell suspension (200 μL) containing 3 × 10⁵ cells was added to each insert. Complete DMEM (500 μL) was added to the lower chamber. After culture at 37 °C and 5% CO₂ for 24 h, the chambers were removed, fixed with 4%paraformaldehyde, and stained with 0.1% crystal violet solution. Cells were counted using an inverting microscope (×200).

Enzyme-linked immune-sorbent assay

Secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum were measured by enzyme-linked immune-sorbent assay (ELISA) kit according to the manufacturer’s protocol.

Real-time quantitative PCR

Total RNA was extracted from cultured cells or endometrial tissues using RNA Trizol Reagent (Hefei Bomei Biotechnology Co. Ltd., vs18061730) according to the manufacturer’s protocol. Reversed transcription was performed using PrimeScript™ RT reagent kit (cat. no. RR047A; Takara Bio, Inc.). mRNA expression was measured using TB Green TM Premix Ex TaqTM II (cat. no. RR820A; Takara Bio, Inc.) in the Applied Biosystems Prism 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.), using β-actin as an internal normalized reference. The primers were as follows: β-actin-F: 5′-AGCCTCGCCTTTGCCG A-3′, β-actin-R: 5′-CTGGTGCCTGGGGCG-3′; RKIP-1-F: 5′-CGGGATCCATGCCGGTGG ACCTCAGCAA-3′, RKIP-1-R: 5′-CCCTCGAGCTTCCCAGACAGCTGCTCGT-3′. Relative expression was calculated using the 2^–ΔΔCT method.

Western bolt analysis

Total protein was extracted from ectopic endometrial tissues and cells of each group using Radio Immunoprecipitation Assay (RIPA) lysis buffer (P0013, Beyotime), and the concentration of protein was measured using a bicinchoninic acid (BCA) protein assay kit (P0009, Beyotime). Protein samples of equal quantity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the PVDF membrane (ISEQ00010, Sigma-Aldrich) on ice. After blocking with 5% skimmed milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) buffer for 1 h at room temperature, the membranes were incubated with the primary antibodies against Bax (A19684, abclonal), Bcl-2 (A19693, abclonal), cleaved-caspase3 (A2156, abclonal), ERK1/2(A4782, abclonal), p-ERK1/2 (AP0472, abclonal), MEK (A19565, abclonal), p-MEK (AP1021, abclonal), RAF (ab200653, abcam), p-RAF (no. 9427, abclonal), and RKIP (ab76582, abcam) overnight at 4 °C. Next, the membranes were incubated with HRP-conjugated secondary antibody (ab6721, abcam) for 1 h at room temperature and then washed with TBST for 30 min. The enhanced electrochemiluminescence (ECL) method was conducted to visualize the protein bands, with β-actin (AC026, abclonal) as a loading control.

Statistical analysis

In this work, the mean ± SD was used to express all obtained results. The data were analyzed by one-way ANOVA followed by Dunnett’s post hoc test using GraphPad Prism 5.0 (GraphPad Software, La Jolla, California, USA). A p value less than .05 was considered statistically significant.

Results

NYKF treatment controls the progression of endometrial lesions in EMS mice

EMS model was developed surgically and verified by HE staining, which exhibited inflammatory cell infiltration, endometrial epithelial cells proliferating, endometrial tissue necrosis, and fibrosis compared with the sham group (Figure 2A). To investigate the role of NYKF in EMS, intragastric administration of the low dose of NYKF, medium dose of NYKF, and high dose of NYKF was performed for 21 days. After the treatment, the histopathological examination of the ectopic endometrium was performed. Results showed lesions in the surgically induced EMS group were characterized by intense inflammatory cell infiltration, proliferation, and necrosis. NYKF treatment reduced the histological severity of endometrium, especially in the high-dose NYKF group, which is characterized by reduced epithelial cell necrosis and hyperplasia (Figure 2B). Next, we measured the production of proinflammatory factors in response to EMS or NYKF. The production of IL-6 and TNF-α was significantly increased in the EMS group, which was reversed by treatment of high-dose NYKF. The low dose of NYKF did not markedly reduce the secretion of IL-6 and TNF-α (Figure 2C,D). These results showed that NYKF may improve the development of EMS and inhibit EMS-induced inflammatory response.

Figure 2. NYKF controls the progression of endometriosis in EMS mice. A: HE staining proved the establishment of EMS model. B: Endometrial lesions and pathologic changes in the EMS mice (n = 6), low-dose NYKF group (n = 6), medium-dose NYKF group (n = 6), and high-dose NYKF group (n = 6) were observed by HE staining (×400). Scale bar: 50 μm. ELISA was performed to measure the protein levels of IL-6 (C) and TNF-α (D). Data are shown as means ± SD (n = 6), and data between multiple groups were compared by one-way ANOVA. **p < .01 compared with Sham group; #p < .05, ##p < .01 compared with EMS group.

NYKF may inhibit RAF/MEK/ERK signal via mediating RKIP in EMS mice

To investigate the role of NYKF on this signaling pathway, relative protein expression was detected. As shown in Figure 3, expression of p-RAF, p-MEK, and p-ERK1/2 were significantly decreased after NYKF treatment compared with the model group. The expression of p-ERK1/2 was markedly reduced even in the low dose of the NYKF group, while the expression of p-RAF and p-MEK was not significantly changed at this dose (Figure 3B–D). To explore whether NYKF could regulate RKIP, we detected the protein expression of RKIP in ectopic endometrial tissue. Results showed that the protein expression of RKIP was significantly increased, and this effect was dose-dependent in the NYKF group compared with the model group (Figure 3E). Taken together, these results indicated that NYKF may inhibit RAF/MEK/ERK pathway through upregulating RKIP expression.

Figure 3. Effects of NYKF on protein expression in ectopic endometrium by IHC. A: The expression of p-RAF, p-MEK, p-ERK1/2, and RKIP in ectopic endometrium (×400). OD values were calculated for p-RAF (B), p-MEK (C), p-ERK1/2 (D), and RKIP (E). Data are shown as means ± SD (n = 6), and data between multiple groups were compared by one-way ANOVA. **p < .01 compared with EMS group.

NYKF induced apoptosis in ectopic endometrium tissue of EMS mice

Furthermore, the apoptosis-related protein in ectopic endometrium was also evaluated. Bax and cleaved caspase-3 protein expression were significantly increased and Bcl-2 protein expression was decreased in the NYKF group relative to the EMS group (Figure 4A–D). And this effect was dose-dependent. In addition, the TUNEL assay revealed that the proportion of TUNEL-positive cells was higher in a dose-dependent manner by NYKF treatment, which indicated that NYFK may promote the apoptosis of endometrial cells (Figure 4E,F). These results confirmed that NYKF treatment promotes apoptosis in ectopic endometrium tissue of EMS mice.

Figure 4. NYKF induced apoptosis in the ectopic endometrium of EMS mice. After establishing the EMS model surgically, the mice were gavaged by low-dose, medium-dose, and high-dose of NYKF once a day for 21 days. A: The image of western blot band. The expression of apoptosis-related protein, including Bax (B), Bcl-2 (C), and cleaved caspase-3 (D) was measured by Western Blot. E: Apoptosis rate in each groups. F: Cell apoptosis in endometrium tissues of each experimental group measured by TUNEL staining (×400). Data are shown as means ± SD (n = 6), and data between multiple groups were compared by one-way ANOVA. *p < .05, **p < .01, ****p < .0001 compared with EMS group; ## p < .01, ####p < .0001 compared with low-dose NYKF group.

NYKF inhibits proliferation and migration and promotes apoptosis of hEM15A cells

To further examine the effect of NYKF on EMS, the hEM15A cells were treated with 5% (low), 10% (medium), or 20% (high) NYKF-medicated mice serum, and the proliferation, apoptosis, and migration of the cells were measured. CCK-8 assay found that NYKF treatment significantly inhibited hEM15A cell proliferation in a dose-dependent manner compared with the negative control group (Figure 5A). Flow cytometry analysis found that different doses of NYKF can significantly induce the apoptosis of hEM15A cells, and the efficacy reached its highest at high-dose NYKF (Figure 5B,D). Transwell analysis showed that NYKF treatment significantly inhibited hEM15A cell migration at medium and high-dose of medicated serum compared with negative control. 5% NYKF-medicated mice serum did not significantly affect the migration of hEM15A cells (Figure 5C,E).

Figure 5. NYKF treatment inhibited proliferation and migration and promoted apoptosis of hEM15A cells. hEM15A cells were cultured with 5%, 10%, 20% NYKF-medicated mice serum, mice serum without NYKF (NC), or without mice serum (BC). Cell proliferation was measured by CCK-8 (A). Apoptosis was measured by flow cytometry (B and D). Migration was measured by Transwell assay (C and E). BC: blank control; NC: negative control. Data are shown as means ± SD (n = 3), and data between multiple groups were compared by one-way ANOVA. **p < .01, ***p < .001, ****p < .0001.

NYKF inhibits RAF/MEK/ERK1/2 activation and promotes RKIP expression in hEM15A cells

Furthermore, we assessed by western blot the p-RAF/RAF, p-MEK/MEK, and p/ERK/ERK ratio 24 h after incubation of hEM15A cells with different cultures. Our results showed that NYKF-medicated serum treatment significantly inhibited the expression of p-ERK1/2, p-MEK, and p-RAF compared with the negative control group (Figure 6A–D). More importantly, NYKF treatment significantly induced RKIP expression in hEM15A cells in a dose-dependent manner (Figure 6A,E), which further confirmed the role of NYKF in regulating RAF/MEK/ERK1/2 signaling pathway and RKIP expression no matter in vivo or in vitro.

Figure 6. NYKF treatments regulate RAF/MEK/ERK signaling pathway in hEM15A cells. hEM15A cells were cultured with 5%, 10%, 20% NYKF-medicated mice serum, mice serum without NYKF (NC), or without mice serum (BC). A: The image of western bolt band. The ratio of p-RAF/RAF (B), p-MEK/MEK (C), p-ERK/ERK (D), and relative protein expression of RKIP (E) were measured by western blotting. Data are shown as means ± SD (n = 3), and data between multiple groups were compared by one-way ANOVA. *p < .01, **p < .01 compared with NC group.

RKIP knockdown inhibits NYKF-mediated regulation in hEM15A cells

To clarify if RKIP plays a key role in NYKF-mediated RAF/MEK/ERK activation and progression in EMS, hEM15A cells were transfected with either si-RKIP or si-NC. Western blot assay and real-time PCR revealed that RKIP protein and mRNA expression was significantly suppressed in hEM15A cells following si-RKIP-1 (Figure 7A–C). Subsequent CCK-8 assay and flow cytometry analysis results showed that RKIP knockdown did not significantly affect the proliferation and apoptosis of hEM15A cells in the negative control. However, it can remarkably reduce the efficacy of NYKF-medicated serum on the proliferation (inhibition rate from 21% in the 10%NYKF group to 11.6% in the si-RKIP + 10%NYKF group) and apoptosis (apoptosis rate from 24% to 11%) of hEM15A cells (Figure 7D,E,G). Transwell assay revealed that RKIP knockdown significantly improved the migration of hEM15A cells. In addition, it also impaired the ability of NYKF to inhibit migration of the cells compared to si-RKIP + 10%NYKF group with the 10%NYKF group (Figure 7F,H).

Figure 7. NYKF treatment inhibits hEM15A cells proliferation and migration and induces apoptosis by targeting RKIP. hEM15A cells were transfected with either si-RKIP or si-NC for 24 h, and then cultured with 10% NYKF-medicated mice serum, following which cells were harvested for subsequent experiments. Transection efficiency of si-RKIP was determined by western blot (A and B) and real-time PCR (C) in hEM15A cells. Cell proliferation was measured by CCK-8 (D). Apoptosis was measured by flow cytometry (E and G). Migration was measured by Transwell assay (F and H). NC: negative control. Data are shown as means ± SD (n = 3), and data between multiple groups were compared by one-way ANOVA. *p < .05, **p < .01, ***p < .001, ****p < .0001.

Previous studies have shown that RKIP regulates the RAF/MEK/ERK pathway, which is directly related to cell proliferation and migration. To clarify whether NYKF-mediated RKIP expression directly affected the activation of the signaling, the expression and phosphorylation levels of some components, including RAF, MEK, and ERK1/2, were measured by western blot. Results showed that RKIP knockdown significantly increased RAF and MEK phosphorylation compared with those in the control group, which was reversed significantly by NYKF treatment (Figure 8).

Figure 8. NYKF blocks the activation of RAF/MEK/ERK signaling pathway by enhancing RKIP. hEM15A cells were transfected with si-RKIP for 24 h, and then cultured with 10% NYKF-medicated mice serum, following which cells were harvested for subsequent experiments. A: The image of western bolt band. The relative protein expression of RKIP, (B) ratio of p-RAF/RAF (D), p-MEK/MEK (C), p-ERK/ERK (E) were measured by Western blot. Data are shown as means ± SD (n = 3), and data between multiple groups were compared by one-way ANOVA. *p < .05, **p < .01 compared with NC group; ##p < .01 compared with 10%NYKF group.

Taken together, these findings indicated that RKIP is a key intermediary in the regulation of hEM15A cells induced by NYKF treatment.

Discussion

EMS is a common disease in reproductive women, causing abnormal menstruation and infertility, with no curative treatment up to now. The traditional therapeutic methods for EMS include drugs and surgery. Surgery removes the EMS lesions but may damage ovaries or other organs and is prone to recurrence [21, 22]. Current evidence suggests that Traditional Chinese medicine is becoming a routine treatment for EMS because of its effectiveness in lessening chronic pain, preventing relapse, and promoting fertility [23–25]. In the study, we proved through in vivo and in vitro experiments that NYKF can inhibit the activation of RAF/MEK/ERK signaling pathway by promoting RKIP expression, thereby inducing apoptosis, while inhibiting proliferation and migration of ectopic endometrium tissue or cells to treat EMS.

In this study, we showed NYKF treatment reduced the severity of the histological index and proinflammatory cytokines secretion in EMS mice, indicating its function of inhibiting inflammation and alleviating the progression of EMS, which is consistent with previously reported therapeutic effects of NYKF on EMS [19]. In addition, NYKF treatment also increased the expression of Bax and cleaved caspase-3 and decreased the expression of Bcl-2 in the ectopic endometrium. Studies have shown that Bax, Bcl-2, and cleaved caspases-3 are related to cell apoptosis [26, 27]. It means that NYKF induces endometrium apoptosis and TUNEL staining confirms our results for inducing apoptosis in NYKF groups. Furthermore, in vitro experiments also found that NYKF-medicated serum could promote apoptosis, but inhibit the proliferation and migration of hEM15A cells.

Given the up-stream regulator of RAF/MEK/ERK signaling in the expression of Bax, Bcl-2, and cleaved caspase-3 [28], we, therefore, suggested that NYKF could induce cell apoptosis and inhibit inflammation through RAF/MEK/ERK signaling to control the progression of EMS. Previous studies reported that this pathway plays a key role in the initiation and progression of EMS [29, 30]. It contributed to the increased invasiveness of ectopic endometrium stroma cells and was also involved in the migration of endometriotic cells induced by peritoneal fluid from patients with EMS [31]. So that inhibiting this signaling pathway may be a benefit for EMS. Our results showed that NYFK treatment could inhibit the activation of RAF/MEK/ERK signaling in EMS mice or hEM15A cells, which suggested that NYKF may play a therapeutic role by inhibiting this pathway.

More importantly, the expression of RKIP was significantly increased after NYKF treatment. RKIP is an important physiological endogenous inhibitor of RAF/MEK/ERK signaling by combining with RAF [13], and has been previously implicated in many cancers (e.g. prostate cancer, non-small cell lung cancer) [32, 33], where high RKIP expression is associated with small tumor size, while low RKIP expression is associated with poor prognosis [34, 35]. The present study found that RKIP knockdown conferred an inhibitory effect on NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and proliferation and migration of hEM15A cells, suggesting that NYKF treatment blocked the RAF/MEK/ERK signaling by targeting RKIP.

Conclusions

In conclusion, the present study demonstrated the molecular mechanism of action of NYFK in treating EMS, by which NYKF treatment inhibited proliferation, and migration, and promoted apoptosis in EMS via inhibiting the activation of RAF/MEK/ERK pathway by targeting RKIP. This provides experimental research evidence for the clinical efficacy of NYKF in treating EMS. However, due to the complexity of EMS development, the mechanism by which NYKF inhibits the progression of EMS needs to be studied in further expanded samples and clinical trials.

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.

Acknowledgments

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.