(2024) 22:351
Guo et al. BMC Medicine
https://doi.org/10.1186/s12916-024-03571-0

BMC Medicine

RESEARCH

Open Access

NLRC5 exerts anti‑endometriosis effects
through inhibiting ERβ‑mediated inflammatory
response
Bao Guo2†, Haiqing Zhu2†, Chengwei Xiao3,4†, Jing Zhang2, Xiaojing Liu2, Yuan Fang2, Bing Wei2,
Junhui Zhang2*, Yunxia Cao1* and Lei Zhan1*

Abstract
Background Endometriosis is well known as a chronic inflammatory disease. The development of endometriosis
is heavily influenced by the estrogen receptor β (ERβ), while NOD-like receptors (NLRs) family CARD domain-containing 5 (NLRC5) exhibits anti-inflammatory properties during endometriosis. However, whether NLRC5-mediated antiinflammation is involved in the ERβ-mediated endometriosis is still uncertain. This study aimed to assess that relation.
Methods Nine cases of eutopic endometrial tissue and ten cases of ectopic endometrial tissue were collectedfrom patients with endometriosis, and endometrial samples from ten healthy fertile women were analyzed,
and the expression levels of ERβ were quantified using immunohistochemistry (IHC). Subsequently, we constructedmouse model of endometriosis by intraperitoneal injection. We detected the expression of ERβ, NLRC5, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-10 and measured the volume of ectopic lesions in mice with endometriosis. In vitro, human endometrial stromal cells (hESCs) were transfected respectively with ERβ-overexpressing
and NLRC5-overexpressing plasmids. We then assessed the expression of ERβ and NLRC5 using quantitative real-time
PCR (qRT-PCR) and western blot analysis. Furthermore, we measured the concentrations of TNF-α, IL-6, and IL-10
in the cell culture supernatant through enzyme-linked immunosorbent assay (ELISA). Additionally, we evaluatedthe migration and invasion ability of hESCs using transwell and wound healing assays.
Results Inhibition of NLRC5 expression promotes the development of ectopic lesions in mice with endometriosis,
upregulates the expression of pro-inflammatory factors TNF-α and IL-6, and downregulates the expression of antiinflammatory factor IL-10. The high expression of NLRC5 in endometriosis depended on the ERβ overexpression.
And ERβ promoted the migration of hESCs partially depend on inflammatory microenvironment. Lastly, NLRC5 overexpression inhibited ERβ-mediated development and inflammatory response of endometriosis.
Conclusions Our results suggest that the innate immune molecule NLRC5-mediated anti-inflammation participatesin ERβ-mediated endometriosis development, and partly clarifies the pathological mechanism of endometriosis,†

Bao Guo, Haiqing Zhu and Chengwei Xiao contributed equally to this workand share first authorship.
*Correspondence:
Junhui Zhang
huiui_zhang@foxmail.com
Yunxia Cao
caoyunxia5972@ahmu.edu.cn
Lei Zhan499329901@qq.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Guo et al. BMC Medicine(2024) 22:351

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expanding our knowledge of the specific molecules related to the inflammatory response involved in endometriosis
and potentially providing a new therapeutic target for endometriosis.
Keywords Endometriosis, ERβ, NLRC5, Inflammatory response

Background
Endometriosis refers to the occurrence of endometrial glands and stroma with growth functions that aretransplanted and implanted into other parts outside theuterine cavity [1]. It can cause clinical symptoms suchas chronic pelvic pain and infertility [2]. Epidemiological studies have shown that the incidence of endometriosis in women of childbearing age is approximately 10%,and the incidence in infertile women can be as high as
30–40% [3, 4]. In addition, patients with endometriosis
have a significantly increased risk of developing tumors[5]. Notably, there is insensitivity to drug treatment inpatients with endometriosis or patients with endometriosis have an average annual recurrence rate of 10%
after treatment [6, 7]. Therefore, there is an urgent
need to alleviate the related clinical symptoms causedby endometriosis and reduce the recurrence rates after
treatment in clinical settings [8]. Exploring the pathogenesis of endometriosis has become the key approachto improving the effectiveness of clinical treatment forendometriosis. In recent years, based on related clinical symptoms, endometriosis has been demonstrated to
be strongly associated with an inflammatory response[9]. Searching for specific molecules that impact the
inflammatory response and exploring their underlying
mechanisms is important for improving treatment outcomes for patients with endometriosis.
Estrogen receptors (ERs) are key nuclear receptors
involved in the pathophysiology of endometriosis [10].
ERs include two subtypes, ERα and ERβ, which havebeen confirmed to play widely participating roles in avariety of human organ functions [11]. ERs widely participated in reproductive diseases, including endometriosis [12, 13]. While compared to ERα, ERβ has beenclearly recognized as a key molecular in the pathological process of endometriosis [14]. When compared tonormal human endometrial cells, human endometriotic
lesions—whether ovarian or peritoneal—show higher
expression of ERβ; ERα expression was not observedin these lesions. Moreover, elevated levels of ERβ contribute to the onset and progression of endometriosis
by influencing inflammatory pathways [15]. Therefore,
conducting in-depth research into the mechanism
underlying ERβ-mediated inflammation regulation in
endometriosis and identifying potential therapeutictargets will have significant clinical relevance and treatment value [16].

NOD-like receptors (NLRs), which are pattern recognition receptors (PRRs) protein family members foundin the cytoplasm, have a significant involvement in theonset and progression of inflammation and immune diseases and they play a crucial role in the immune response
and safeguarding of eukaryotic organisms by aiding in
the quick elimination of invading pathogens [17]. The
innate immune molecule, NLRs family CARD domaincontaining 5 (NLRC5), is a highly conserved memberof the newly discovered NLR-like receptor family inrecent years [18]. In the inflammatory response, NLRC5
can act as a negative regulatory factor of the inflammatory response by inhibiting the nuclear factor-kappaB(NF-κB) inflammatory signaling pathway as well as thesecretion of inflammatory factors to suppress the inflammatory response [19]. In the immune response, NLRC5
activation can suppress tumor growth by promoting
anti-tumor immune responses [20]. Research has also
revealed that NLRC5 suppresses inflammation by promoting autophagy in the secretory phase of ectopic
endometrial stromal cells [21]. According to the report,
the NLRs can be targeted by ERs to regulate the Wnt/βcatenin signaling pathway in cancer [22]. Therefore,
examining whether the NLRC5-mediated inflammatory
response is involved in ERβ-controlled endometriosis
would offer valuable understanding of the developmentof endometriosis and improve treatment outcomes.

Methods
Population sample collection

This study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Anhui Medical
University (No. SL-YX [YS] 2023-SZR010). Nine casesof secretory phase eutopic endometrium and ten casesof secretory phase ectopic endometrium were collectedfrom patients with endometriosis who underwent laparoscopic surgery at the Second Affiliated Hospital of Anhui
Medical University, and ten cases of secretory phasenormal endometrial tissue were obtained from patients
undergoing uterine resection for benign uterine diseases.
The phase of the menstrual cycle was determined basedon the endometrial histology and the expected day of themenstrual cycle provided by the women. Before surgery,none of the patients had undergone radiotherapy, chemotherapy, hormone therapy, or biological therapy. The
age (34.07 ± 5.47) and BMI (24.83 ± 2.88) of the patients
exhibited no statistically significant difference comparedGuo et al. BMC Medicine(2024) 22:351

to the age (34.23 ± 5.85) and BMI (24.76 ± 3.19) of thecontrol group (t = 0.196, P = 0.845; t =  − 0.141, P = 0.888).
Cells and cell culture

Immortalized human endometrial stromal cells (hESCs)
were purchased from FENGHUISHENGWU (Hunan,
China). The hESCs pellets were suspended again in Minimum Essential Medium (MEM) containing 10% fetal
bovine serum (FBS) (Gibco, USA) and 1% penicillin/
streptomycin (PS) (Gibco, USA), plated on a cell culture dish, and cultured in an incubator (5% C­ O2, 37 °C).
Lipopolysaccharide (LPS) (50 ng/ml) (Sigma, USA) wasused to induce an inflammatory environment in hESCs.
Adeno‑associated virus and plasmid construction

The recombinant adeno-associated virus (AAV) vector
expressing short hairpin RNA (shRNA) against NLRC5(shNLRC5) and its negative controls (NC) were synthesized by Shanghai GenePharma Co., Ltd. and injected
into mice through the tail vein. Overexpression of ERβ(the ERβ group) and NLRC5 (the NLRC5 group) was
achieved by plasmid construction and their negative
controls (NC), which were respectively purchased from
GenePharma (Shanghai, China) and GENERAL BIOL
(Anhui, China).
Mouse model of endometriosis

From the Beijing Vital River Laboratory Animal Technology Co., Ltd., 5-week-old and 16–18 g C57BL/6 female
mice were purchased. The animals were kept in a standard laboratory environment that was free from specificpathogens. For 3–5 days, the mice were subjected to a
12–12 h cycle of light and darkness at a temperature
range of 20–24 °C and a humidity range of 60–65%.
After 3 days of acclimatization, the mice were randomly
divided into donor and recipient groups. Donor mice
were euthanized by decapitation, and their abdomens
were opened to remove the uterus. After removing theexcess tissue, the uterus was cut open along the Y-shaped

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uterine midline and placed in separate saline solution
containers. The uterine endometrium was exposed bylongitudinally cutting open each uterus and then minced
into small fragments of 1 m­ m3 using ophthalmic scissors.
The recipient mice were injected with a 1 ml saline solution containing the uterine fragments into their abdominal cavity using a sterile insulin syringe. An injectionneedle was inserted 5 mm into the left midline of thelower abdomen of the recipient mouse. Each uterus fromthe donor mouse was evenly distributed between the tworecipient mice.
Treatment

The recombinant AAV vector expressing short hairpin
RNA (shRNA) against NLRC5 (shNLRC5) and its negative controls (NC) were synthesized by Shanghai GenePharma Co., Ltd. and injected into mice through the tailvein. Overexpression of ERβ (the ERβ group) and NLRC5(the NLRC5 group) was achieved by plasmid construction and their negative controls (NC), which were respectively purchased from GenePharma (Shanghai, China)
and GENERAL BIOL (Anhui, China). The mice modelof endometriosis were randomly divided into sevengroups: the control group, the ERβ activator (ERB-041,
purchased from MedChem Express, USA) group, theERβ antagonist (PHTPP, purchased from MedChem
Express, USA) group, the NC group, the NC + ERB-041
group, the shNLRC5 group, and the shNLRC5 + ERB-041
group. Each group has 10 mice. In the ERB-041 groupand the PHTPP group (Fig. 1B), inject ERB-041 (2.5 mg/
kg in 50 µl corn oil) and PHTPP (2.5 mg/kg in 50 µl cornoil) subcutaneously into mice separately. In the control
group, an equal amount of corn oil was subcutaneously
injected. The mice were euthanized after continuous
injection (once every other day) for 3 weeks. In the shNLRC5 and shNLRC5 + ERB-041 groups (Fig. 4A), separately injected one dose of 1 × ­1010 TU/ml of shNLRC5
into the tail vein of mice, with a volume of 50 µl. In the
NC and NC + ERB-041 groups, the NC AAV was injected(See figure on next page.)
Fig. 1 ERβ promotes the development of endometriosis. A Immunohistochemical staining to detect the expression of ERβ in the epitheliumand stroma of normal endometrial tissues and endometriosis patients (both in their eutopic and ectopic endometrium). Scale bar = 20 μm.
B Experimental design flow chart for exploring the role and mechanism of ERβ in endometriosis female mice. C The gross morphologyof representative mouse endometriosis lesions and lesion sections showed glandular and interstitial tissue by H & E staining, which was consistent
with endometriosis. Scale bar = 100 μm. D–E Immunohistochemical and quantitative analyses of ERβ levels in the corn oil-treated C57BL/6 J
mice with endometriosis and the ERβ agonist (ERB-041) or ERβ antagonist (PHTPP) of C57BL/6 J mice with endometriosis (both in their eutopic
and ectopic endometrium). Scale bar = 20 μm. F Comparison of the size of ectopic lesions in the control group (n = 3), that in the ERB-041 group(n = 3), and that in the PHTPP group (n = 3). Statistical analysis was performed using a one-way ANOVA followed by Tukey’s post hoc analysis.
**P ≤ 0.01, *P < 0.05. G The relative mRNA level of ERβ in hESCs was analyzed by qRT-PCR. H The protein expression of ERβ in hESCs was analyzed
by western blotting. I–J The wound healing and transwell method was used to explore the impact of ERβ overexpression on the migrationand invasive ability of hESCs in vitro. Scale bar = 100 μm. Statistical analysis was performed using a two-tailed Student’s t-test. *P < 0.05 comparedwith NC; **P ≤ 0.01 compared with NC

Guo et al. BMC Medicine(2024) 22:351

Fig. 1 (See legend on previous page.)

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Guo et al. BMC Medicine(2024) 22:351

into the mouse tail vein at a concentration of 1 × ­1010 TU/
ml. After 3 weeks, mice in the NC + ERB-041 and shNLRC5 + ERB-041 groups were subcutaneously injectedwith 2.5 mg/kg ERB-041. The NC and shNLRC5 groups
were subcutaneously injected with the same amount ofcorn oil on alternate days. After continuous injection for
3 weeks, the mice were euthanized. Open the abdominal cavity of the mice, eutopic and ectopic endometrial
tissue were collected respectively. Then, we determined
the size of ectopic lesions using the following method:V ­(mm3) = 0.52 × length × width × height. Animal experiments were approved by the Ethics Review Committee of
the Department of Laboratory Animal Science of Anhui
Medical University (No. LLSC20231700). Anhui Medical University’s animal experimental guidelines were followed when conducting animal experiments and nursing
procedures.
Hematoxylin and eosin staining

The lesions collected from mice with endometriosis wereembedded in paraffin wax and prepared into multiple
5 mm paraffin sections. The paraffin sections were thendewaxed under gradient ethanol, followed by hematoxylin and eosin (H & E) staining, dehydration, penetration,and sealing. Images were collected using a 3D digital slicing scanner (Pannoramic MIDI, 3Dhistech, Hungary).
Immunohistochemical staining

Dewaxed and dehydrated paraffin-embedded sections ofeutopic and ectopic endometria from mice with endometriosis underwent antigen retrieval, endogenous peroxidase blocking, and application of the primary antibody.
The specific primary antibodies used in immunohistochemistry were as follows: NLRC5 (1:100, Affinity), ERβ(1:100, Affinity), tumor necrosis factor-alpha (TNF-α)(1:500, Abcam), interleukin (IL)-6 (1:1000, Abcam), andIL-10 (1:1000, Abcam). Next, an enzyme-labeled goatanti-mouse/rabbit IgG polymer was added, and the slices
were dehydrated with alcohol, made transparent withxylene, and sealed with neutral gum. Images were collected and analyzed using a 3D digital slicing scanner(Pannoramic MIDI, 3Dhistech, Hungary).
Western blotting

The tissue proteins of mouse eutopic and ectopic endometria and cellular proteins of hESCs were extracted
and lysed in radioimmunoprecipitation assay (RIPA)
buffer (Beyotime, China). The proteins in these extracts
were separated using 6% and 10% SDS-PAGE and thentransferred to a 0.45-µm polyvinylidene fluoride (PVDF)
membrane (Millipore, USA). After blocking, the membranes were sequentially incubated with the corresponding primary antibodies overnight at 4 °C and secondary

Page 5 of 15

antibodies for 1 h at room temperature. The primary
antibodies used were ERβ (1:1000, Affinity) and NLRC5(1:500, Bioworld). The membranes were exposed anddeveloped after immersion in ECL reagent (Biosharp,
China). The relative target protein levels were equal to
the ratios of their gray values to GAPDH (1:10,000, Elabscience), which served as an internal reference.
Quantitative real‑time PCR

The quality of total RNA was confirmed by Nanodrop
2000 (Thermo Fisher Scientific, USA), followed byextraction from eutopic endometrial tissue, ectopic
endometrial tissue, and treated cells using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme,
China). Total RNA (1000 ng) was reverse transcribedusing HyperScriptTM III RT SuperMix for quantitative
real-time PCR (qRT-PCR) with gDNA Remover (EnzyArtisan, China). qRT-PCR was conducted to quantify
gene expression using the LightCycler 480 SYBR GreenI Master (Roche, Switzerland). The primer sequences foreach gene were designed and validated for specificity. The
relative expression of genes of interest was calculated andnormalized to that of β-actin using the ­2−ΔΔCt method[23]. The PCR primers used are shown in Additional
file 1: Table S1.
Transfection

hESCs were seeded at 1 × ­105 cells/well into six-wellplates and were transfected, respectively, with ERβoverexpressing and NLRC5-overexpressing plasmids atapproximately 80% confluence. The jetPRIME Transfection Reagent Kit (Polyplus-Transfection, France) wasused to transfect the plasmid. Initially, a 2 µg DNA sample was diluted in 200 µl of jetPRIME buffer. Following a
10-s vortexing, 4 µl of jetPRIME reagent were introduced
to the transfection mixture and vortexed for 1 s. Next,
the cells were left to incubate at room temperature for10 min. Ultimately, the mixture was incorporated into
the culture medium.
Transwell migration and invasion assay

Chambers, either with or without Matrigel (BD Biosciences, USA), from Corning (USA), were utilized toassess cell migration and invasion, correspondingly. The
transfected cells were seeded in the upper compartment
in 100 µl serum-free medium. Afterwards, 600 µl of complete medium was added to the lower compartment.
Cells on the upper side of the membranes were removed.
Cells on the lower side of slide were stained with 0.1%
crystal violet solution and then photographed under amicroscope. hESCs that had migrated or invaded thelower surface of the membranes were counted using theImageJ software (version 1.46, USA).

Guo et al. BMC Medicine(2024) 22:351

Wound healing assay

We conducted a comparative study of cell migration
using a wound healing assay. First, the cells were seeded
into a 12-well plate and cultured in a complete medium.
When cell confluence reached 90–95%, we scratcheda straight line using a 10-µl pipette tip. Subsequently,
the original medium was discarded, and the cells were
washed with PBS to remove the detached cells. Serumfree MEM medium was added to the wells. An inverted
fluorescence microscope (IX73, Olympus, Japan) wasused to photograph the scratched area at 0, 24, and 48 h
after scratching. The wound width and migration area
were analyzed using the ImageJ software.
Enzyme‑linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) kit
from China (ELISA LAB) was utilized to determine theconcentrations of TNF-α, IL-6, and IL-10 by utilizing
cell culture supernatant collected from control, control + LPS, NC, NC + LPS, ERβ, ERβ + LPS, NLRC5,
NLRC5 + LPS, and ERβ + NLRC5 + LPS groups. A standard curve was used to determine the concentrations of
TNF-α, IL-6, and IL-10.
Statistical analysis

The results are presented as the mean ± S.D. for continuous variables. Statistical data were analyzed by two-tailed
Student’s t-test and one-way ANOVA, followed by Tukey’s post hoc analysis, using GraphPad Prism software(version 8.0, USA). The P value was set at less than 0.05 todetermine statistical significance.

ResultsERβ promoted the development of endometriosis

In order to investigate the role of ERβ in the pathogenesisof endometriosis, we first detected the expression of ERβ
in patients with endometriosis. Immunohistochemical
results revealed that the expression of ERβ was significantly higher in the epithelium and stroma of endometriosis patients than in the epithelium and stroma of normal
endometrium, and the expression of ERβ was also significantly higher in the epithelium and stroma of ectopic
endometrium than in the epithelium and stroma of
eutopic endometrium among women with endometriosis(Fig. 1A). We established an endometriosis mouse model
to explore the role of ERβ in the development of endometriosis (Fig. 1B). The lesions were confirmed to be endometriosis using H & E staining that demonstrated growthof glandular and stromal endometrial tissue (Fig. 1C).
Both in eutopic and ectopic endometrial tissue, immunohistochemical results revealed that ERB-041 could
significantly increase ERβ expression in the epitheliumand stroma of the endometrium in the ERB-041 group

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compared to the endometrium epithelium and stromaof the control group, and that PHTPP could significantly
decrease ERβ expression in the epithelium and stromaof the endometrium in the PHTPP group compared tothe endometrium epithelium and stroma of the control
group (Fig. 1D, E). Additionally, we noted that the volume of ectopic lesions in the ERB-041 group was significantly larger than that in the control group, whereas thevolume of ectopic lesions in the PHTPP group was significantly smaller than that in the control group, indicatingthat ERβ could promote the development of endometriosis (Fig. 1F). The ERβ overexpression in hESCs was confirmed through qRT-PCR and western blotting (Fig. 1G,
H). Lastly, in in vitro, we noted that the overexpression ofERβ could promote the migration and invasion of hESCs(Fig. 1I, J).
ERβ promoted the development of endometriosis
by activating inflammatory response

Inflammatory response is one of the most importantfactors contributing to the pathogenesis of endometriosis. We speculated whether ERβ contributed tothe development of endometriosis by regulating the
inflammatory response. In the endometriosis mousemodel, both in the eutopic and ectopic endometrial
tissue, immunohistochemical results that ERB-041
could increase the expression levels of pro-inflammatory factors TNF-α (Fig. 2A, B) and IL-6 (Fig. 2C, D)
and decrease the expression level of anti-inflammatory
factor IL-10 (Fig. 2E, F). Accordingly, PHTPP had the
opposite effect (Fig. 2A–F). We observed that TNF-α
was present in both the epithelium and stroma of
eutopic and ectopic endometrial tissues in C57BL/6 J
mice suffering from endometriosis (Fig. 2A, B). In thestroma of eutopic endometrial tissues, there is expression of IL-6 (Fig. 2C) and IL-10 (Fig. 2E), while the epithelium shows minimal expression of these cytokines.
However, IL-6 (Fig. 2D) and IL-10 (Fig. 2F) are predominantly observed in the epithelium of ectopic endometrial tissues, with almost no expression in the stroma.
The induction of inflammatory responses is a crucial
aspect of Gram-negative bacteria’s virulence, and thisis mainly carried out by LPS. Cell scratch test showedthat LPS concentration of 50 ng/ml and 500 ng/ml did
not affect the migration ability of hESCs. However,
the migration ability of hESCs was inhibited when theconcentration of LPS was 1000 ng/ml, suggesting thatthe concentration of LPS at 1000 ng/ml would impair
cell function (Fig. 2G). Furthermore, the ELISA results
demonstrated that 50 ng/ml LPS effectively created an
inflammatory environment in hESCs (Fig. 2H). As aresult, hESCs were exposed to 50 ng/ml LPS for futureGuo et al. BMC Medicine(2024) 22:351

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Fig. 2 ERβ promotes the development of endometriosis by activating inflammatory response. A–F Immunohistochemical and quantitative
analyses of TNF-α, IL-6, and IL-10 levels in the corn oil-treated C57BL/6 J mice with endometriosis and ERB-041 or PHTPP of C57BL/6 J micewith endometriosis (both in their eutopic and ectopic endometrium). Scale bar = 20 μm. G A wound healing assay was used to explore
the impact of LPS concentration on the migration ability of hESCs in vitro. Scale bar = 100 μm. Statistical analysis was performed using a one-way
analysis of variance (ANOVA), followed by Tukey’s post hoc analysis. **P ≤ 0.01, ns, non-significant. H ELISA analysis of TNF-α, IL-6, and IL-10 levelsin the conditioned media harvested 48 h after plating hESCs with LPS. *P < 0.05 compared with control; **P ≤ 0.01 compared with control. I ELISA
analysis of TNF-α, IL-6, and IL-10 levels in the conditioned media harvested 48 h after plating hESCs. J–K Migration and invasive capabilities of hESCs
in different groups, assessed by wound healing and transwell assays. Scale bar = 100 μm. Statistical analysis was performed using a one-way analysis
of variance (ANOVA), followed by Tukey’s post hoc analysis. **P ≤ 0.01, *P < 0.05, ns, non-significant

experiments. Importantly, we noted that overexpressing ERβ was unable to alter the levels of TNF-α, IL-6,
and IL-10, whereas the addition of LPS to the hESCs
to construct an inflammatory environment resulted inoverexpressing ERβ being able to promote the secretion of pro-inflammatory factors TNF-α and IL-6 andinhibit the secretion of anti-inflammatory factor IL-10(Fig. 2I). Furthermore, only LPS could not promote
the migration of hESCs, whereas in the case of overexpression of ERβ, the migration and invasion abilities of
hESCs significantly increased (Fig. 2J, K). These results
indicated that in endometriosis, ERβ-mediated endometriosis development is partially dependent on the
inflammatory environment, and that ERβ promotes thedevelopment of endometriosis by activating the inflammatory response.
ERβ promoted NLRC5 expression in endometriosis

Our previous study indicated that the innate immune
molecule NLRC5 is upregulated in patients with endometriosis, and we believe that the inflammatory environment contributed to NLRC5 upregulation in
endometriosis [24]. In view of the decisive role of ERβ
in the pathogenesis of endometriosis, we were curious about the role of ERβ in NLRC5 expression. In both
eutopic and ectopic endometrial tissues of the endometriosis mouse model, the ERB-041 group showed a significant increase in the expression levels of NLRC5 mRNAand protein in the epithelium and stroma compared toGuo et al. BMC Medicine(2024) 22:351

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Fig. 3 ERβ promoted NLRC5 expression in endometriosis. A–D Comparison of the expression levels of NLRC5 mRNA and protein in the eutopic
and ectopic endometrial tissue of C57BL/6 J mice with endometriosis in the control group (n = 3), that in the ERB-041 group (n = 3), and that in
the PHTPP group (n = 3). E–F The relative mRNA and protein levels of NLRC5 in hESCs were analyzed by qRT-PCR and western blotting. Statisticalanalysis was performed using a one-way ANOVA followed by Tukey’s post hoc analysis. **P ≤ 0.01, *P < 0.05, ns, non-significant

the control group (Fig. 3A–D). Conversely, the PHTPP
group displayed a significant decrease in the expression
levels of NLRC5 mRNA and protein in the epitheliumand stroma compared to the control group (Fig. 3A–D).
In in vitro, we noted that the expression levels of NLRC5mRNA and protein in the hESCs were significantly
increased due to ERβ overexpression (Fig. 3E, F). These
results indicate that ERβ could promote NLRC5 expression in endometriosis.

(Fig. 4B, C). Furthermore, the volume of ectopic lesions
in the shNLRC5 group was significantly larger than thatin the NC group (Fig. 4D). The overexpression of NLRC5
in hESCs was confirmed through qRT-PCR and western
blotting (Fig. 4E–F). Moreover, NLRC5 overexpression
was observed to suppress the migration and invasion of
hESCs in vitro (Fig. 4G–H).

NLRC5 affects the development of endometriosis

These results indicated that NLRC5 inhibits the development of endometriosis. We further investigatedwhether NLRC5 inhibited the development of endometriosis by regulating the inflammatory response.
Immunohistochemical findings showed that there wasan elevated expression of the pro-inflammatory factors TNF-α and IL-6 in both the eutopic and ectopic
endometrial tissue of the shNLRC5 group as compared to the endometrial epithelium and stroma of theNC group (Fig. 5A–D). Additionally, a decrease in theexpression of the anti-inflammatory factor IL-10 was
observed in the endometrial epithelium and stroma of
the shNLRC5 group in comparison to the NC group(Fig. 5E, F). The ELISA results showed that comparedIn our previous study, we found evidence suggestingthat NLRC5 has the ability to suppress inflammation in
endometriosis. Nevertheless, we still lack understanding regarding the involvement of NLRC5 in the development of endometriosis. To address this, we conducted aninvestigation to determine the contribution of NLRC5 in
the progression of endometriosis in a mouse model. Dueto the excessive length of the NLRC5 overexpressed transcript from the mouse source, it is difficult to construct an
NLRC5 overexpression AAV. Therefore, we constructed
an shNLRC5 AAV vector (Fig. 4A). Immunohistochemistry results revealed that the shNLRC5 AAV inhibited theexpression of NLRC5 in a mouse endometriosis model

NLRC5 inhibited the development of endometriosis
by attenuating inflammatory response

Guo et al. BMC Medicine(2024) 22:351

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Fig. 4 NLRC5 affects the development of endometriosis. A Experimental design flow chart for exploring the effect of shNLRC5 on the ERB-041
in endometriosis female mice. B–C Immunohistochemical and quantitative analyses of NLRC5 levels in the saline-treated C57BL/6 J micewith endometriosis or the recombinant AAV vector that expresses the short hairpin RNA (shRNA) against NLRC5 (shNLRC5) and its negative controls(NC) of C57BL/6 J mice with endometriosis (both in their eutopic and ectopic endometrium). Scale bar = 20 μm. D Comparison of the size of ectopic
lesions in the NC group (n = 3) and that in the shNLRC5 group (n = 3). Statistical analysis was performed using a two-tailed Student’s t-test. *P < 0.05
compared with NC. E–F The relative mRNA and protein levels of NLRC5 in hESCs were analyzed by qRT-PCR and western blotting. Statistical analysis
was performed using a one-way ANOVA followed by Tukey’s post hoc analysis. **P ≤ 0.01, *P < 0.05, ns, non-significant. G–H Migration and invasive
capability of hESCs in different groups assessed by wound healing assay and transwell assay. Scale bar = 100 μm

Guo et al. BMC Medicine(2024) 22:351

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Fig. 5 NLRC5 inhibits the development of endometriosis by attenuating inflammatory response. A–F Immunohistochemical and quantitative
analyses of TNF-α, IL-6, and IL-10 levels in the eutopic and ectopic endometrial tissue of C57BL/6 J mice with endometriosis in the NC groupand that in the shNLRC5 group. Scale bar = 20 μm. G ELISA analysis of TNF-α, IL-6, and IL-10 levels in the conditioned media harvested 48 h
after plating hESCs. H–I Migration and invasive capability of hESCs in different groups assessed by wound healing assay and transwell assay.
Scale bar = 100 μm. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s post hoc analysis. **P ≤ 0.01, *P < 0.05, ns,
non-significant

to its NC group, the NLRC5 group did not cause any
significant changes in the levels of inflammatory factors(Fig. 5G). However, when LPS was added to the endometrial stromal cells to create an inflammatory environment, NLRC5 was able to suppress the release of

pro-inflammatory factors TNF-α and IL-6 and stimulate the secretion of the anti-inflammatory factor IL-10(Fig. 5G). Importantly, the inhibitory effect of NLRC5
overexpression on the migration and invasion of hESCs
in an inflammatory environment was stronger than thatof NLRC5 overexpression alone (Fig. 5H, I).

Guo et al. BMC Medicine(2024) 22:351

NLRC5 inhibited ERβ‑mediated developmentof endometriosis by attenuating inflammatory response

The findings suggest that NLRC5 plays a role in suppressing the inflammatory response, thereby impeding
the progression of endometriosis. We further detected
whether NLRC5 could inhibit the ERβ-mediateddevelopment of endometriosis. In the mouse modelof endometriosis, the shNLRC5 + ERB-041 group had
significantly bigger volumes of ectopic lesions compared to the NC + ERB-041 group (Fig. 6A). Additionally, the ERB-041 group showed significantly larger
volumes of ectopic lesions compared to the control group (Fig. 6A). Moreover, compared with the
NC + LPS group, the ERβ + LPS group could promote
the migration and invasion of hESCs (Fig. 6B, C). And
overexpressed NLRC5 can inhibit the positive role ofERβ in promoting migration and invasion of hESCs(Fig. 6B, C). Lastly, we detect whether NLRC5-mediated anti-inflammation contributes to inhibiting thePage 11 of 15

ERβ-mediated development of endometriosis. In both
eutopic and ectopic endometrial tissue, the immunohistochemical results showed that the expressionof pro-inflammatory factors TNF-α and IL-6 in bothepithelium and stroma was increased in the shNLRC5 + ERB-041 group compared to the NC + ERB041 group (Fig. 6D–G). Additionally, the expressionof anti-inflammatory factor IL-10 in both epitheliumand stroma was decreased in the shNLRC5 + ERB-041
group compared to the NC + ERB-041 group (Fig. 6H,I). Further in vitro experiments revealed that NLRC5
could inhibit the role of ERβ in promoting the secretion of pro-inflammatory factors TNF-α and IL-6 andinhibiting the secretion of anti-inflammatory factor
IL-10 in an inflammatory environment (Fig. 6J). The
above results indicate that NLRC5 can inhibit the ERβmediated development of endometriosis by attenuating
the inflammatory response (Fig. 7).

Fig. 6 NLRC5 inhibits ERβ-mediated development of endometriosis by attenuating inflammatory response. A Comparison of the size of ectopic
lesions in the different groups (n = 3). B–C Migration and invasive capability of hESCs in different groups assessed by wound healing assay
and transwell assay. Scale bar = 100 μm. D–I Immunohistochemical and quantitative analyses of TNF-α, IL-6, and IL-10 levels in the eutopic
and ectopic endometrial tissue of C57BL/6 J mice with endometriosis in the different groups. Scale bar = 20 μm. J ELISA analysis of TNF-α, IL-6,
and IL-10 levels in the conditioned media harvested 48 h after plating hESCs. Statistical analysis was performed using a one-way ANOVA followed
by Tukey’s post hoc analysis. **P ≤ 0.01, *P < 0.05

Guo et al. BMC Medicine(2024) 22:351

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Fig. 7 Schematic diagram of NLRC5 exerts anti-endometriosis effects through inhibiting ERβ-mediated inflammatory response

Discussion
Endometriosis is an estrogen-dependent inflammatory
disease that has led to extensive research on the pathophysiology of inflammatory mediators as potential therapeutic targets [25]. Previous studies had demonstrated
that ERβ was recognized to be more highly expressedin ectopic endometriotic tissues than ERα and wasnoted to drive the progression of endometriosis [26].
It has been reported that ERβ can activate the NF-κB
inflammatory pathway and increase the expression of
C–C motif chemokine ligand 2 (CCL2), leading to therecruitment of macrophages (M2). This, in turn, promotes the proliferation, invasion, adhesion, and metastasis of ectopic lesions in endometriosis [27]. Thus, thesignificant presence of ERβ in abnormal growths ofindividuals with endometriosis is likely to contributeto the progression of this condition through its ability to enhance the inflammatory microenvironment
within the endometrium. This subsequently resultsin the increased potential for proliferation, migration,and invasion of the abnormal endometrial tissue. The
role of ERβ in individuals suffering from endometriosis
remains intricate and necessitates additional investigation to fully comprehend its mechanism of action. Conducting a thorough investigation into the mechanism ofERβ-mediated inflammatory regulation in the development of endometriosis patients and exploring potential
therapeutic targets will hold great clinical significanceand offer valuable treatment possibilities. In this study,

we have confirmed that the anti-endometriosis effectmediated by NLRC5 is achieved through the inhibitionof the ERβ-mediated inflammatory response. ERβ and
NLRC5 have limited expression in normal endometrium, whereas their expression is specifically observedin endometriotic tissue. Therefore, we believe that ERβ
and NLRC5 may be the next generation of promisingtherapeutic targets compared with the current treatment of endometriosis.
ERβ has emerged as an important player in the pathogenesis of endometriosis [28]. Studies have revealed thathuman endometriotic lesions, either ovarian or deeply
infiltrating endometriosis, display higher ERβ expression when compared to that of normal human tissues[29]. The ERβ to ERα ratio was over 1 in both human andanimal models of endometriosis lesions, while ERα was
predominately expressed in normal endometrial cells[30, 31]. We proved that the level of ERβ expression in
the epithelium and stroma of endometriosis patientswas considerably greater compared to the level in theepithelium and stroma of normal endometrium. Studies
have demonstrated that ERβ plays an important role in
the progression of endometriosis by modulating inflammation [32]. However, little is known about the targets
that can be involved in the ERβ-mediated inflammatory
response of endometriosis. In this study, we uncoveredthat NLRC5 inhibits the ERβ-mediated development ofendometriosis by attenuating the inflammatory responseboth in vitro and in vivo.

Guo et al. BMC Medicine(2024) 22:351

NLRs are pattern recognition receptor families in thecytoplasm that are implicated in inflammatory diseases
and promote rapid removal of invasive pathogens [33].
NLRC5 is the largest protein of the NLR family, contains
a nucleotide-binding domain and leucine-rich repeats,
and regulates the inflammatory response [34]. Evidence
has been steadily increasing, indicating that NLRC5 hinders the activation of NF-κB signaling caused by LPS,
TNF-α, or IL-1β [35]. By extracting secretory ectopic
endometrial stromal cells, we noted that NLRC5 inhibited inflammation by promoting autophagy [21]. Generally, the identified protein expression profile is helpful forstudying its function. NLRC5 expression patterns haverevealed that NLRC5 is expressed in various tissues andcells [36]. Compared to the normal group, our previous
study showed a significant increase in NLRC5 expression
in both the eutopic and ectopic endometrium of patientswith endometriosis [24]. It has also been reported that
NLRC5 expression levels in gastric cancer, lung cancer,
and hepatocellular carcinoma are higher than in normal
tissue [37–39]. Another study revealed that, comparedto healthy colorectal tissue, there is increased expression of NLRC5 in colorectal cancer (CRC), and the special high inflammatory state of this cancer type probably
explains this phenomenon [40]. Consistently, our previous research noted that NLRC5 was upregulated inectopic endometrial stromal cells (EESCs) of patientswith ovarian endometriosis in a highly inflammatory
state [21]. Since ERβ is a specific molecule of endometriosis, we observed the effect of ERβ on NLRC5. Our
results confirmed that ERB-041 significantly upregulated
the expression of NLRC5 in both eutopic and ectopic
endometrial tissues of an endometriosis mouse model,
as compared to the control group. And compared withthe control group, PHTPP inhibited the expression of
NLRC5. In vitro, we noted that the expression levels of
NLRC5 in the hESCs were significantly increased due toERβ overexpression. Further in vitro and in vivo experiments revealed that ERβ promotes the expression of
pro-inflammatory factors TNF-α and IL-6 and inhibits
the expression of anti-inflammatory factor IL-10 in the
inflammatory environment. And NLRC5 can inhibit theERβ-mediated inflammatory response in endometriosis. Therefore, we put forward our view that it may be acompensatory response in which NLRC5 is activateddependent on abundant ERβ in endometriosis in order toalleviate ERβ-mediated inflammatory response.
One limitation of this study is that in the tissue analysisof population samples, the sample size of the organization was relatively insufficient. In this study, due to theexcessive length of the NLRC5 overexpressed transcriptfrom the mouse source, it is difficult to construct an
NLRC5 overexpression AAV. Therefore, we constructed

Page 13 of 15

an shNLRC5 AAV vector. We believe that this is also oneof the limitations of our present study. We noticed thatthe excessive length of the NLRC5 caused application
constrained also drew great attention by other researchgroups. For example, the NLRC5/CIITA chimeric construct (DD-335-CIITA) generated by Neerincx et al. is
considerably shorter than NLRC5 and shows improved
activity to induce MHC I and MHC II promoters compared with NLRC5 and CIITA [41]. Santharam et al.
examined a smaller NLRC5-CIITA fusion protein,
dubbed NLRC5-superactivator (NLRC5-SA) as it retains
the ability to induce MHC-I, could be used for tumor
growth control [42]. We thought these studies provided
ideas for our future research. The lack of clarity regarding the specific mechanism of ERβ on NLRC5 is anotherlimitation of the current study. It is critical to elucidate
the specific mechanism of ERβ on NLRC5 to develop
therapeutic strategies for patients with endometriosis. In
endometriosis, ERβ acts on a molecular target througha specific mechanism of action, thereby promoting thedevelopment of endometriosis. Our study demonstrated
that ERβ promotes NLRC5 expression in endometriosis
in vitro and in vivo. We believe that further explorationof the specific mechanism of action of ERβ on NLRC5 ishighly significant in understanding the pathogenesis ofendometriosis and improving therapeutic effectiveness.

Conclusions
To summarize, we have observed noteworthy increases
in the expression of ERβ and NLRC5 in both the eutopic
and ectopic endometrium of individuals with endometriosis, as compared to the control group. Importantly,
our results also confirmed that the activation of NLRC5
in endometriosis is specifically dependent on abundant
ERβ. And overexpression of NLRC5 inhibits the ERβmediated development of endometriosis by attenuating
the inflammatory response. These findings indicate that
NLRC5 is a novel therapeutic target for endometriosis.
Abbreviations
ELISA	Enzyme-linked immunosorbent assay
ER	Estrogen receptor
FBS	Fetal bovine serum
hESCs	Human endometrial stromal cells
IHC	ImmunohistochemistryIL	Interleukin
LPS	Lipopolysaccharide
MEM	Minimum Essential Medium
NC	Negative controls
NLRs	NOD-like receptors
NLRC5	NLRs family CARD domain-containing 5
NF-κB	Nuclear factor-kappaB
PS	Penicillin/streptomycin
PVDF	Polyvinylidene fluoride
qRT-PCR	Quantitative real-time PCR
RIPA	Radioimmunoprecipitation assay
shRNA	Short hairpin RNA
TNF-α	Tumor necrosis factor-alphaGuo et al. BMC Medicine(2024) 22:351

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03571-0.
Additional file 1: Table S1. The PCR primers used in the study.
Acknowledgements
We are grateful to all women who consented and donated samples for thisstudy.
Authors’ contributions
BG, HZ, and CX conducted the study and wrote the manuscript. HZ, JZ, and
XL recruited patients and collected samples. BG, HZ, and YF performed theexperiments. BG and HZ performed data analyses. LZ, YC, JZ, and BW designed
and supervised the study. All authors reviewed and agreed to the final versionof the manuscript.
Funding
This work was supported by the National Nature Science Foundation ofChina (No: 81802586); Natural Science Foundation of Anhui Province (No:2308085MH244); Research Fund of Anhui Institute of Translational Medicine(No: 2022zhyx-C41, ZHYX2020A001); and University Research Fund of Anhui
Medical University (No: 2022xkj017).
Availability of data and materials
The datasets generated or analyzed during this study are available from thecorresponding author on reasonable request.

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Declarations
Ethics approval and consent to participate
This study was approved by the Ethics Review Committee of the Second
Affiliated Hospital of Anhui Medical University (No. SL-YX [YS] 2023-SZR010).
Animal experiments were approved by the Ethics Review Committee of the
Department of Laboratory Animal Science of Anhui Medical University (No.
LLSC20231700).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no known competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.
Author details
1
Department of Obstetrics and Gynecology, The First Affiliated Hospitalof Anhui Medical University, No 218 Jixi Road, Hefei 230022, Anhui, China.
2
Department of Obstetrics and Gynecology, The Second Affiliated Hospitalof Anhui Medical University, No 678 Furong Road, Hefei 230601, Anhui, China.
3
Department of Obstetrics and Gynecology, Bengbu Hospital of Shanghai
General Hospital, Bengbu 233040, Anhui, China. 4 Department of Obstetrics
and Gynecology, The Second Affiliated Hospital of Bengbu Medical University,
Bengbu 233040, Anhui, China.

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15.
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17.
18.
19.

20.
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22.
23.

Received: 11 June 2024 Accepted: 19 August 2024
24.
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