1,25-Dihydroxy vitamin D3 inhibits LPS-mediated inflammatory responses in endometriosis

Endometriosis, an oestrogen-dependent, chronic inflammatory disease, is characterized by the presence of endometrial glands and stroma outside the uterine cavity. This disease affects approximately 5–10% of women of reproductive age, posing a significant public health challenge with substantial impacts on women’s quality of life and imposing a considerable economic burden [ 1 , 2 ]. The main symptoms of endometriosis are infertility [ 3 ] and pelvic pain [ 4 ]. At present, the pathogenesis of endometriosis remains unclear, and the widely accepted theory is the retrograde menstruation hypothesis proposed by Sampson in 1940, in addition to immune imbalance, coelomic metaplasia, and other theories [ 2 , 5 ]. A large number of studies have emphasized that chronic inflammation and abnormal immune responses are crucial mechanisms in the development of endometriosis [ 6 ]. Recently, a novel ‘bacterial contamination hypothesis’ has emerged in the endometriosis research landscape. This hypothesis suggests that lipopolysaccharide (LPS), a bacterial product, disrupts the immune-inflammatory balance in the pelvic microenvironment, thereby facilitating the implantation and proliferation of ectopic endometrium [ 7 , 8 ]. LPS can promote the proliferation and invasion of endometrial cells in endometriosis patients by activating the NF-κB transcription factor in the Toll-like receptor (TLR) pathways, thus modulating inflammatory responses [ 9 ]. Intraperitoneal injection of LPS can significantly increase the size of ectopic peritoneal lesions in a murine endometriosis model, further propelling the growth of ectopic endometrium through the induction of a pelvic inflammatory cascade mediated by the NF-κB pathway [ 7 ]. Therefore, the LPS-induced inflammatory response is a crucial factor in the development of endometriosis. Vitamin D (VD), a crucial steroid derivative, plays a pivotal role in regulating the immune response. 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the active form of VD, is involved in the pathogenesis of endometriosis through the VD-VD receptor (VDR) system and the regulation of immunity, the inflammatory response, cell proliferation and apoptosis, angiogenesis, cell adhesion and invasion [ 10 , 11 ]. Notably, endometriosis patients exhibit lower level of 25-hydroxyvitamin D (25(OH)D) compared to healthy women, and an increase in plasma 25(OH)D reserves can significantly reduce the risk of endometriosis by 24% [ 12 ]. In a murine endometriosis model, administration of 1,25(OH) 2 D 3 led to a remarkable 48.8% reduction in the volume of ectopic lesions within the abdominal cavity. Presumably because 1,25(OH) 2 D 3 inhibited the release of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and other proinflammatory cytokines [ 13 , 14 ]. VDR is expressed in various immune cells such as T lymphocytes, B lymphocytes, monocytes, and macrophages. VD can inhibit the maturation and differentiation of monocytes/macrophages, thus affecting the expression of TLR, which leads to immunosuppression [ 15 ]. Additionally, VD enhances the antimicrobial effect of monocytes and also delays the release of IL-6 and TNF-α of macrophages in the inflammatory response. This modulation is particularly evident in the early formation of immune cells. As an inducer of immune cell activation, active VD can upregulate the gene expression of antimicrobial and autophagy pathways in monocytes while downregulating the expression of proinflammatory genes treated with LPS [ 16 ]. To investigate the therapeutic potential of VD in endometriosis, this study examined the effects of active VD on the biological behaviour and cytokine expression of ectopic endometrial stromal cells (EESCs) and Ishikawa cells under LPS exposure. Furthermore, we evaluated the in vivo efficacy of active VD in a rat model of endometriosis, laying a solid experimental foundation for further exploration of active VD as a potential treatment for endometriosis.

The administration of LPS (100 ng/mL) significantly augmented the proliferation rates of both Ishikawa cells and EESCs ( Figure 1A,B ). Conversely, 1,25(OH) 2 D 3 (10 −9 M) inhibited the proliferation of Ishikawa cells and EESCs, with the suppressive influence on cell viability intensifying as the duration of exposure and the concentration increased ( Figure 1C,D ). When EESCs were exposed to LPS with different concentrations of 1,25(OH) 2 D 3 (10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M) for 24 h, the proliferation rates of EESCs were 90.86%, 97.58%, 101.82%, 105.9%, and 107.66%, respectively, which were all lower than those treated only by LPS (109.69%) ( p  = 0.012, p  < 0.001, p  < 0.001, Figure 1E ). Similarly, when Ishikawa cells were cultured under similar conditions with LPS and various concentrations of 1,25(OH) 2 D 3 (10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M) for 24 h, the proliferation rates of Ishikawa cells were 87.49%, 95.14%, 100.87%, 105.36%, and 109.91%, respectively, all of which were lower than the cell proliferation rate of the LPS-treated only group (112.96%) ( p  < 0.001, Figure 1F ). 1,25(OH) 2 D 3 reduces LPS-induced proliferation of Ishikawa cells and EESCs. Viability of EESCs and Ishikawa cells treated with only LPS (A, B) or 1,25(OH) 2 D 3 for 12-48 h (C, D). Viability of EESCs (E) and Ishikawa cells (F) treated with LPS and different concentrations of 1,25(OH) 2 D 3 for 24 h. Data indicate mean ± SD of n  = 5 replicate experiments. p Values are from LSD multiple comparison test after one-way ANOVA. *, p  < 0.05, **, p  < 0.01, ***, p  < 0.001 (compared with control group); #, p  < 0.05, ##, p  < 0.01, ###, p  < 0.001 (compared with LPS group). Compared with the control group, the migration ability of Ishikawa cells ( p  = 0.009) and EESCs ( p  < 0.001) was significantly enhanced following treatment with LPS. The addition of 1,25(OH) 2 D 3 alone had no significant effect on the migration ability of Ishikawa cells and EESCs. However, compared with the LPS group, 1,25(OH) 2 D 3 could reverse the LPS-induced enhancement of Ishikawa cells and EESCs migration, and there was a dose-time effect on the effect of 1,25(OH) 2 D 3 . 10 −6 M 1,25(OH) 2 D 3 for 48 h exhibited the most profound inhibitory effect on the migration ability of LPS-treated Ishikawa cells ( p  = 0.004) and EESCs ( p  = 0.004) ( Figure 2A–C ). 1,25(OH) 2 D 3 downregulates LPS-induced cell migration of Ishikawa cells and EESCs. The typical images (A) and quantitative analyses (B and C) of Ishikawa cells (scale bars, 600 μm) and EESCs’ (scale bars, 1000 μm) migration with various treatment for 0-48 h by wound healing assay. Data indicate mean ± SD of n  = 3 replicate experiments. p Values are from LSD multiple comparison test after one-way ANOVA. *, p  < 0.05, **, p  < 0.01, ***, p  < 0.001 (compared with LPS group). We next explored the effect of 1,25(OH) 2 D 3 and LPS on the apoptosis of Ishikawa cells and EESCs. Flow cytometry results showed that LPS had no significant effect on the apoptosis rate of Ishikawa cells and EESCs. In contrast, the addition of different concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −7 M, 10 −8 M) promoted the apoptosis of EESCs cells in a dose-dependent manner ( p  < 0.001), and the same phenomenon also occurs in Ishikawa cells ( p  < 0.001, Figure 3A–C ). 1,25(OH) 2 D 3 promotes cell apoptosis of Ishikawa cells and EESCs. Flow cytometry (A) and quantitative analyses (B and C) of cell apoptosis of Ishikawa cells and EESCs receiving different treatments. Data indicate mean ± SD of n  = 4 replicate experiments. p Values are from LSD multiple comparison test after one-way ANOVA. *, p  < 0.05, **, p  < 0.01, ***, p  < 0.001. To evaluate the effect of 1,25(OH) 2 D 3 on inflammatory factors production, we quantify the secretion levels of TNF-α and IL-6 in the culture supernatants by ELISA. Our findings indicate that LPS significantly elevated the production of TNF-α and IL-6 in comparison to non-treated EESCs and Ishikawa cells ( p  < 0.001, Figure 4A–D ). 1,25(OH) 2 D 3 significantly inhibited TNF-α ( p  < 0.001) and IL-6 ( p  < 0.001) production compared to nontreated control EESCs, and also had an inhibitory effect on Ishikawa cells ( p  = 0.003, p  = 0.058). Furthermore, our investigation revealed that 1,25(OH) 2 D 3 significantly reversed the LPS-induced production of TNF-α and IL-6 in both EESCs and Ishikawa cells ( Figure 4A–D ), and it had concentration-dependent characteristics. 1,25(OH) 2 D 3 downregulates LPS-induced production of proinflammatory cytokines . The levels of IL-6 (A) and TNF-α (B) in the supernatant of EESCs treated with LPS and different concentrations of 1,25(OH) 2 D 3 by ELISA. The levels of IL-6 (C) and TNF-α (D) in the supernatant of Ishikawa cells treated with LPS and different concentrations of 1,25(OH) 2 D 3 by ELISA. The levels of IL-6 (E) and TNF-α (F) in the supernatant of EESCs treated with LPS alone, LPS + 1,25(OH) 2 D 3 , and LPS + 1,25(OH) 2 D 3 co-administered with TEI-9647 by ELISA. The levels of IL-6 (G) and TNF-α (H) in the supernatant of Ishikawa cells treated with LPS alone, LPS + 1,25(OH) 2 D 3 , and LPS + 1,25(OH) 2 D 3 co-administered with TEI-9647 by ELISA. Typical fluorescence microscopy images (I) and corresponding fluorescence quantitative analysis results (J) for COX-2 (green fluorescence), iNOS (green fluorescence) and DAPI (blue fluorescence) in EESCs. Scale bars, 50 μm. Data indicate mean ± SD of n  = 3 replicate experiments. p Values are from LSD multiple comparison test after one-way ANOVA. #, p  < 0.05, ##, p  < 0.01, ###, p  < 0.001 (compared with LPS group); *, p  < 0.05, **, p  < 0.01, ***, p  < 0.001. When EESCs were exposed to LPS with varying concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −7 M, 10 −8 M) for 24 h, the secretion levels of IL-6 were 664.4, 757.23, and 849.73 pg/mL respectively. All of these values were significantly lower compared to those obtained when cells were treated only with LPS (972.07 pg/mL) ( p  < 0.001, Figure 4A ). Similarly, the secretion levels of TNF-α in EESCs were 144.71, 175.58, and 217.18 pg/mL for increasing concentrations of 1,25(OH) 2 D 3 , all significantly lower than LPS-only treated cells (251.22 pg/mL) ( p  < 0.001, Figure 4B ). Analogously, Ishikawa cells cultured with LPS and varying concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −7 M, 10 −8 M) for 24 h exhibited IL-6 secretion levels of 335.98, 419.26, and 583.68 pg/mL, respectively. These levels were all significantly lower than those observed in the LPS-only treatment group (764.64 pg/mL) ( p  < 0.001, Figure 4C ). Likewise, TNF-α secretion in Ishikawa cells were 170.91, 224.29, and 254.36 pg/mL with varying concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −7 M, 10 −8 M), respectively, all significantly lower than LPS-only treated cells (277.69 pg/mL) ( p  = 0.053, p  < 0.001, p  < 0.001, Figure 4D ). As a potent synthetic VDR antagonist, TEI-9647 exerts its inhibitory effect through competitive occupation of the receptor’s ligand-binding domain, which sterically hinders endogenous ligand binding and consequently abolishes VDR-driven transcriptional activation [ 18 ]. To assess the antagonistic activity of TEI-9647 against 1,25(OH) 2 D 3 under inflammatory stimulation, we quantify the secretion levels of TNF-α and IL-6 in the culture supernatants by ELISA and the levels of COX-2 and iNOS in the cells by immunofluorescent staining. TEI-9647 reversed the suppression of LPS-induced TNF-α and IL-6 secretion mediated by 1,25(OH) 2 D 3 ( Figure 4E–H ). Specifically, it restored the cytokine levels to 80.78% for TNF-α and 81.27% for IL-6 in EESCs, and similarly, to 82.63% for TNF-α and 82.31% for IL-6 in Ishikawa cells, as compared to LPS-only controls. NF-κB activation stimulates the production of proinflammatory enzymes like COX-2 and iNOS, which in turn exacerbate inflammatory cascades and tissue damage [ 19 ]. As expected, LPS exposure significantly increased COX-2 (1.6-fold in EESCs and 4.15-fold in Ishikawa cells) and iNOS (2.2-fold in EESCs and 1.37-fold in Ishikawa cells) protein levels compared to controls ( Figure 4I,J ; Supplementary Figure 2A,B ). Notably, 1,25(OH) 2 D 3 significantly attenuated these LPS-induced increases, whereas TEI-9647 co-treatment partially reversed this suppression. TEI-9647 impaired the anti-inflammatory activity of 1,25(OH) 2 D 3 in both cell types, highlighting the importance of VDR engagement in mediating 1,25(OH) 2 D 3 ’s inhibitory effects on LPS-driven inflammation. Previous research underscores that TNF-α serves as a highly effective activator of the NF-κB pathway [ 20 ]. By engaging with the receptor TNFR2, TNF-α modulates the pathway, thereby fostering cancer growth, invasion, and metastasis. In our study, treatment with varying concentrations of TNF-α led to a significant, dose-dependent increase ( p  < 0.001, Supplementary Figure 3A,B ) in the secretion levels of IL-6 in both EESCs and Ishikawa cells. Notably, treatment with 1,25(OH) 2 D 3 was able to counteract this TNF-α-induced enhancement of inflammatory responses ( p  < 0.001, Supplementary Figure 3C,D ), implying that VD may possess antagonistic properties against other factors that promote NF-κB activation. In order to explore the effect of 1,25(OH) 2 D 3 on the NF-κB pathway, the expression of phosphorylated p65 (ser536) was detected by immunofluorescence staining. Compared with the control group, the fluorescence intensity of phosphorylated p65 in LPS-treated Ishikawa cells and EESCs was significantly enhanced, and it was mainly expressed in the nucleus ( Figure 5A,B ). 1,25(OH) 2 D 3 could reverse the LPS-induced increase in fluorescence intensity of phosphorylated p65 ( Figure 5A,B ), confirming that 1,25(OH) 2 D 3 inhibited the LPS-activated NF-κB signaling pathway. 1,25(OH) 2 D 3 reduces LPS-induced NF-κB pathway activation. (A) Typical fluorescence microscopy images for phosphorylated p65 (ser536) (green fluorescence) and DAPI (blue fluorescence) in EESCs and Ishikawa cells. (B) The average nuclear-cytoplasmic ratio of phosphorylated p65 (ser536) was quantified of each group were measured by ImageJ 1.53e (Wayne Rasband and contributors National Institutes of Health, USA). Typical fluorescence microscopy images for VDR (C, green fluorescence), and NF-κB pathway components (D, green fluorescence), including TLR4, IκBα and NLRP3, counterstained with DAPI (blue fluorescence) in EESCs and corresponding fluorescence quantitative analysis results (E). Scale bars, 50 μm. Data indicate mean ± SD of n  = 3 replicate experiments. p Values are from LSD multiple comparison test after one-way ANOVA. *, p  < 0.05, **, p  < 0.01, ***, p  < 0.001. Building on our observation that 1,25(OH) 2 D 3 inhibits LPS-induced nuclear translocation of p65 (a central NF-κB subunit), we further showed that TEI-9647 (a selective VDR antagonist) substantially reversed this inhibitory effect ( p  = 0.018 and p  = 0.021 vs. LPS + 1,25(OH) 2 D 3 group; Supplementary Figure 4A,B ). Immunofluorescence quantification revealed that LPS stimulation reduced VDR expression intensity by 42.85% in EESCs and 49.78% in Ishikawa cells, whereas 1,25(OH) 2 D 3 partially counteracted this suppression ( Figure 5C ; Supplementary Figure 5A ). Notably, TEI-9647 diminished 1,25(OH) 2 D 3 -mediated VDR upregulation, supporting a role for VDR-dependent mechanisms in regulating these responses. To further explore the broader influence of 1,25(OH) 2 D 3 on NF-κB signaling, we examined additional pathway components ( Figure 5D,E ; Supplementary Figure 5B,C ). LPS stimulation enhanced TLR4 expression by 1.52-fold in EESCs and 3.08-fold in Ishikawa cells, while NLRP3 levels increased by 1.97-fold and 2.24-fold, respectively. These elevations were notably reduced by 1,25(OH) 2 D 3 treatment. Co-treatment with TEI-9647 partially reversed this suppression, restoring TLR4 expression to approximately 100.69% and 89.09% of LPS-only controls in EESCs and Ishikawa cells, respectively, and NLRP3 levels to 84.01% and 78.27% of LPS-stimulated values. Concurrently, 1,25(OH) 2 D 3 significantly elevated IκBα protein levels by 2.27-fold in EESCs ( p  < 0.001 vs. LPS) and 1.17-fold in Ishikawa cells ( p  = 0.003 vs. LPS), which correlated with diminished NF-κB activation. TEI-9647 disrupted IκBα stabilization, implying a role for VDR-mediated transcriptional regulation in 1,25(OH) 2 D 3 -induced IκBα upregulation. Additionally, RT-qPCR findings demonstrated that LPS treatment led to an increased repression of RELA (p65) mRNA expression, along with a reduction in the repression of VDR and NFκBIA (IκBα) mRNA levels in both EESCs and Ishikawa cells. Notably, these effects of LPS were significantly mitigated by 1,25(OH) 2 D 3 ( Supplementary Figure 6A,B ). Together, these observations support a model wherein 1,25(OH) 2 D 3 attenuates inflammatory responses through VDR-dependent stabilization of IκBα and modulation of TLR4/NF-κB signaling, effects that are countered by VDR antagonism via TEI-9647. To further explore the therapeutic effect of 1,25(OH) 2 D 3 on endometriosis, we established an autologous transplantation rat model of the disease. The surgical procedure for model induction and experimental treatment regimens are schematically outlined in Figure 6A . Our findings demonstrated that 1,25(OH) 2 D 3 effectively hindered the growth of endometriotic lesions, with the higher dose of 50 ng/100g exhibiting a more profound effect than the 25 ng/100g dose ( Figure 6B–D ). Furthermore, ELISA assays of inflammatory factors within the lesions indicated that 1,25(OH) 2 D 3 significantly repressed the expression of IL-6 and TNF-α, with a stronger inhibitory effect observed in the higher-concentration group ( Figure 6E,F ). H&E staining showed that following 1,25(OH) 2 D 3 treatment, the glandular epithelium of endometriotic lesions was disordered, the number of glands was reduced, and calcifications appeared ( Figure 6G ). Immunohistochemical analysis further corroborated these findings, showing that 1,25(OH) 2 D 3 suppressed the expression of phosphorylated p65 ( Figure 6H ). Additionally, concentration-dependent 1,25(OH) 2 D 3 treatment significantly upregulated VDR and IκBα protein levels, while downregulating TLR4, NLRP3, COX-2, and iNOS expression in lesions ( Figure 6H ; Supplementary Figure 7 ). These coordinated regulatory mechanisms suggest that 1,25(OH) 2 D 3 may inhibit the NF-κB pathway through both ligand-dependent activation of the VDR and stabilization of IκBα, which inhibits NF-κB signaling. In Vivo therapeutic effect of 1,25(OH) 2 D 3 on endometriosis. (A) The surgical procedure for model induction and experimental treatment regimens. (B) Photographs of endometriotic lesions in a rat model after the process of 1,25(OH) 2 D 3 treatment for 16 d. (C) The average lesion volumes of each group were measured ( n  = 4). (D) All the lesions ( n  = 4) were collected and weighed after the rat were sacrificed. The levels of IL-6 (E) and TNF-α (F) in the lesions by ELISA ( n  = 4). (G) Light micrographs of endometriotic lesion sections stained with H&E. Scale bars, 100 μm. Orange arrows, endometriotic glands. (H) Light micrographs of endometriotic lesion sections with immunohistochemical staining for phosphor-NF-κB p65 (ser536), VDR, TLR4 and IκBα. Scale bars, 100 μm. Data indicate mean ± SD of n  = 3 replicate experiments. p Values are from LSD multiple comparison test after one-way ANOVA. Not significant (ns), *, p  < 0.05, **, p  < 0.01, ***, p  < 0.001.

Ectopic endometrial tissues were surgically excised from fifteen patients undergoing laparoscopic ovarian endometrioma cystectomy at our institution between April and September 2024. Inclusion criteria comprised: (i) histologically confirmed endometriosis; (ii) age 20–50 years; (iii) absence of concurrent endometrial hyperplasia or malignancy. Exclusion criteria were strictly defined as: (i) postmenopausal status; (ii) severe dysfunction of major organs; (iii) surgical contraindications; (iv) psychiatric disorders; (v) acute inflammation. The ectopic endometrium collected from ovarian endometrioma of premenopausal patients with laparoscopically and histologically confirmed endometriosis was washed with PBS, and the blood, mucus and fat tissue were removed by ophthalmic scissors. The uterus was fully cut into chyme and placed into a 60 mm culture plate. 2.5 mg/mL type I collagenase (1:5, C8140, Solarbio, Beijing) was added to digest tissue for 90 min in a 5% CO 2 incubator at 37 °C. Then, Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Gibco, USA) containing 1% penicillin/streptomycin (Gibco, USA) and 10% fetal bovine serum (FBS, Gibco, USA) was added to stop digestion, the suspension and undigested tissue were removed through a 70 μm sieve. Epithelial cells and EESCs were separated by using a 40 μm sieve. The filtrate was collected and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The sediment was mainly EESCs. The cells were inoculated in another 60 mm culture plate and cultured at 37 °C in a 5% CO 2 incubator. Immunofluorescence staining results showed that the fluorescent staining of vimentin in EESCs was strong, but there was no obvious fluorescent staining of cytokeratin ( Supplementary Figure 1 ). Based on the proportion of vimentin-positive cells stained ( n  = 6), the purity of the EESC cells was calculated to be approximately 95.18%. The morphological characteristics of the cells were observed by inverted microscopy. The Ishikawa cells were purchased from Zhejiang Meisen Cell Technology Co. LTD (CTCC-003-0095, Zhejiang, China). The Ishikawa cells were cultured with DMEM medium (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator of 5% CO 2 at 37 °C. The cells for all experiments were in logarithmic growth phase. To assess the potential effect of 1,25(OH) 2 D 3 on cell proliferation by EESCs and Ishikawa cells,10 5 cells were cultured in wells of a 96-well culture plate and treated with LPS (100 ng/mL) and/or different concentrations of 1,25(OH) 2 D 3 (10 −5 M, 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M) for 12–48 h. A culture plate subsequently was used to detect cell viability according to the manufacturer’s protocols of cell counting kit-8 (CCK8, GLPBIO, America). 10 μL of CCK8 reagent was added to each well and then incubated for 1.5 h. The absorbance was then analysed at 450 nm using a Synergy 2 modular multi-mode reader (BioTek, America) using wells without cells as blanks. The cell proliferation levels were calculated based on the absorbance. In order to assess the potential effect of 1,25(OH) 2 D 3 on the modulation of LPS-stimulated cell apoptosis,10 5 cells were cultured in 24-well plates and treated with LPS (100 ng/mL) and/or different concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −7 M, 10 −8 M) for 48 h. After that, the cells were digested and centrifuged for subsequent cell apoptosis assay. First, the cells were washed by PBS and then processed according to the manufacturer’s protocols of the Annexin V-FITC/PI Apoptosis Detection Kit (E-CK-A211, Elabscience, Wuhan). Finally, the obtained cells were analyzed using an Attune NxT flow cytometer (Invitrogen, America). Data analysis was performed using NovoExpress 1.4.0 software (Agilent, America). All experiments complied with the ARRIVE guidelines 2.0 (updated guidelines for reporting animal research) [ 17 ]. A total of 12 female Wistar rats aged 5–6 weeks were purchased from the animal centre of Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. and bred under pathogen-free conditions. The room temperature was 25 ± 2 °C, the relative humidity was 50 ± 5%, and alternating 12-h cycles of light or dark were maintained. Before surgery, all rats were intragastrically treated with 0.5 mg/kg estradiol valerate three times, once a day, in order to keep all Wistar rats in the same estrous cycle. About 3% isoflurane was used for anesthesia. After hair removal and iodophor disinfection of the rat’s abdomen, a longitudinal incision of about 2.5 cm was made along the midline of the abdomen about 1 cm above the urethra to find the left uterus. After performing ligation on both sides of the ovarian end and the bifurcation of the uterus, a 2 cm length of the uterus is cut 0.5 to 1 cm away from the ovary, and the endometrial tissue was sutured to the left lower abdominal wall with No. 4-0 absorbable suture. Estradiol valerate 0.5 mg/kg was administered intragastrically from the first day after surgery, once every 4 days, for a total of 3 times. After 4 weeks, the suture site of the heterotopic graft in the abdomen of the rats was obviously bulged when touched, which can be used for treatment. Twelve rats were randomly divided into three groups with 4 rats in each group. The low concentration group was intraperitoneally injected with 25 ng/100g 1,25(OH) 2 D 3 (B2141-1, APExBIO, America) every other day for eight times, while the high concentration group was intraperitoneally injected with 50 ng/100g 1,25(OH) 2 D 3 . The control group was treated with phosphate-buffered saline (PBS, G4202, Servicebio, Wuhan) instead of 1,25(OH) 2 D 3 . After 16 days of treatment, the rats were sacrificed by cervical dislocation and endometriotic lesions were harvested. The volumes of the lesions were calculated according to the following formula: V  = 0.5 × length × width 2 . The removed rat lesions were immersed in 4% paraformaldehyde and fixed at room temperature. The endometrial lesions of the rats were immersed in 4% paraformaldehyde for 72 h, conventionally embedded in paraffin, sectioned at a thickness of 4 μm, dewaxed and hydrated with gradient ethanol, stained with haematoxylin-eosin (H&E), and sealed with neutral gum. The pathological changes in endometrial tissue were observed by light microscopy. 10 5 cells were cultured in wells of a 12-well culture plate with cell slide, and inoculated until 30–50% confluence. The cells were washed in PBS three times and fixed in 4% paraformaldehyde for 15 min. Then, 0.5% Triton X-100 was added to break the membrane for 20 min and normal goat serum was added to block at room temperature for 30 min. After removing the blocking solution, 300ul of diluted primary antibody [vimentin, 1:200; cytokeratin, 1:300; phosphor-NF-κB p65 (ser536), 1:200; VDR, 1:400; TLR4, 1:400; IκBα, 1:400; pyrin domain (PYD)-containing protein 3 (NLRP3), 1:500; cyclooxygenase-2 (COX-2), 1:400; inducible nitric oxide synthase (iNOS), 1:400] was added to each slide and incubated overnight at 4 °C. Then, slides were incubated by the FITC-conjugated goat anti-rabbit IgG ­secondary antibody (1:60) or goat anti-mouse IgG secondary antibody (Alexa Fluor ® 488, 1:600) at room temperature for 1 h. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI, AR1176, Boster, Wuhan) for 5 min. A 10–20 μL anti-fluorescence attenuating tablet (AR1109, Boster, Wuhan) was added to seal the sections. Images were taken with a confocal laser scanning microscopy (CLSM, LSM 710, Carl Zeiss, Germany). To evaluate the effects of 1,25(OH) 2 D 3 on cytokine production by EESCs and Ishikawa cells, 10 5 cells were cultured in wells of a 96-well culture plate and treated with LPS (100 ng/mL) and/or different concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −7 M, 10 −8 M) for 48 h. Furthermore, to assess the antagonistic activity of TEI-9647 (25-dehydro-1α-hydroxyvitamin D 3 -26,23-lactone) against 1,25(OH) 2 D 3 under inflammatory stimulation, three treatment regimens were implemented: (i) LPS (100 ng/mL) control, (ii) LPS + 1,25(OH) 2 D 3 (10 −7 M), and (iii) LPS + 1,25(OH) 2 D 3 co-administered with TEI-9647 (10 −6 M, MC18512, Meilunbio, Guangzhou). Moreover, to investigate the impact of 1,25(OH) 2 D 3 on inflammatory responses triggered by TNF-α, the cells were treated with a range of TNF-α concentrations (0 - 10 ng/mL), either in conjunction with 1,25(OH) 2 D 3 (10 −7 M) or independently, and these treatments were carried out both with and without the inclusion of TEI-9647 (10 −6 M). Conditioned media were collected and stored at −80 °C for enzyme-linked immunosorbent assay (ELISA). The concentrations of TNF-α and IL-6 in cell culture supernatants collected from the EESCs and Ishikawa cells were measured by human IL-6 ( F01310 , Westang, Shanghai) and TNF-α ( F02810 , Westang, Shanghai) ELISA kits. Endometriotic lesions collected from the endometriosis rat model were ground with 9× homogenization medium, and then the grinding solution was centrifuged at 3500 rpm for 10 min. The supernatant was taken to prepare 10% tissue homogenate, which was stored in a 4 °C refrigerator for subsequent inflammatory factor assay. The concentrations of TNF-α and IL-6 in endometrial lesions were measured by mouse IL-6 (GEM0001-1, Servicebio, Wuhan) and TNF-α (GEM0004-1, Servicebio, Wuhan) ELISA kits. The absorbance of each well was measured at 450 nm in sequence. Total RNA was extracted from the samples using a commercially available RNA extraction kit (R711-02, Vazyme, Nanjing), strictly adhering to the manufacturer’s recommended protocol. Subsequently, complementary DNA (cDNA) was synthesized for each sample utilizing a reverse transcription kit (R423-01, Vazyme, Nanjing), following the instructions provided by the manufacturer. For reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the following primer sequences were employed: RELA forward: 5′-TCCCATCTTTGACAATCGTGC-3′, reverse: 5′-AGCCTGGTCCCGTGAAATAC-3′; NFκBIA forward: 5′- CTCCATCCTGAAGGCTACCAACT-3′, reverse: TACAGGGCTCCTGAGCATTGAC-3′; VDR forward: 5′-GTGTGAATGATGGTGGAGGGAG-3′, reverse: 5′- CAGCTCTAGGGTCACAGAAGGGT-3′, and GAPDH was served as a housekeeping gene with following primers: forward: 5′-GGAAGCTTGTCATCAATGGAAATC-3′, reverse: 5′-TGATGACCCTTTTGGCTCCC-3′. The RT-qPCR reactions were performed using the SYBR Green Pro Taq HS Premixed qPCR Kit (AG11741, Accurate Biology, Hunan). Prior to data analysis, the amplification efficiencies of both the target and reference genes were determined. The comparative ΔΔCT method was then applied to analyze the RT-qPCR data. The expression levels of the target genes were compared and subsequently subjected to a detailed comparative analysis. First, lines were drawn across the back of the 6-well board evenly per 0.5-1 cm with a marker. Approximately 1.5 × 10 6 cells were added to each well. Approximately 20 h later, when the cells grew to approximately 80%, the tip of the spear was used to scratch the horizontal line perpendicular to the back of the ruler. The cells were washed twice with serum-free medium and treated with LPS (100 ng/mL) and/or different concentrations of 1,25(OH) 2 D 3 (10 −6 M, 10 −8 M) for 48 h. Three different visual fields were selected to measure the wound healing of the scratch at 0 and 48 h and were photographed. Cell migration capacity was expressed by mobility, which was calculated by the following formula: cell mobility = (average edge spacing of 0 h – average edge spacing of 48 h)/average edge spacing of 0 h × 100%. Sections were incubated with 3% BSA to reduce nonspecific binding, followed by primary antibody [p65 (ser536), 1:100, AF2006; VDR, 1:100, AF6159; TLR4, 1:100, AF7017; IκBα, 1:100, AF5005; COX-2, 1:100, AF7003; iNOS, 1:100, AF0199; NLRP3, 1:200, 60102-1-lg] at room temperature overnight. Slides were then incubated with either horseradish peroxidase (HRP) -labeled goat anti-rabbit secondary antibody (1:200, GB23303) or HRP-labeled goat anti-mouse secondary antibody (1:200, GB23301) for 50 min. The DBA reaction was used to develop color for 5 min, and then stained with hematoxylin for 3 min. After drying, the slides were sealed with neutral gum, and observed under a microscope. In this study, ectopic endometrial tissue from humans was employed for the extraction of EESCs. All specimens from patients with ovarian endometrioma were collected with written informed consent, and this work was approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University (Ethics Code: 2024ZSLYEC-238, Date: 30 April 2024). All procedures involving human participants in our study were conducted in accordance with the ethical standards of the Declaration of Helsinki and relevant ethical guidelines. The animal experimentation was approved by the Ethics Department of the Sixth Affiliated Hospital of Sun Yat-sen University (No. IACUC-2022053101). SPSS 21.0 software was used for statistical analysis, and GraphPad Prism 9.3.1 was used for plotting. Measurement data were expressed as the mean ± standard deviation (x ± s), and Student’s t test was used to compare the means between two groups, while univariate analysis of variance (ANOVA) was used for comparisons among three or more groups. LSD test was used for the multirange test. A p value < 0.05 indicated that the difference was statistically significant.

Endometriosis, a debilitating disorder characterized by pelvic pain and infertility, exhibits complex pathogenesis involving immune dysregulation, aberrant steroidogenesis, and enhanced cellular aggressiveness [ 21 ]. Central to its progression is chronic inflammation, as evidenced by elevated pro-inflammatory cytokines and dysregulated immune responses [ 22 ]. Our study confirmed that LPS amplifies TNF-α and IL-6 secretion while promoting proliferation of Ishikawa cells and EESCs, aligning with its role as a key mediator of immune-inflammatory cascades in endometriosis. Notably, 1,25(OH) 2 D 3 , a potent immunomodulator, antagonized LPS-induced effects by suppressing cytokine production and cell proliferation. This aligns with its established functions in dampening inflammation and antigen presentation [ 23 , 24 ]. Ectopic endometrial cells inherently display heightened proliferative, anti-apoptotic, and migratory capacities compared to eutopic counterparts [ 25 ], further exacerbated by LPS exposure in wound healing assays. Elevated levels of pro-inflammatory cytokines, including monocyte chemoattractant protein 1, IL-6, IFN-γ, TNF-α, and others, amplify the propensity of endometriotic cells to aggregate, colonize, migrate, adhere, and invade [ 26 , 27 ]. Critically, 1,25(OH) 2 D 3 attenuated LPS-enhanced cell migration, underscoring its therapeutic potential in disrupting endometriosis-associated aggressive phenotypes. These findings elucidate the intricate crosstalk between LPS-mediated inflammatory signaling and VD-dependent immunomodulatory pathways in endometriosis pathogenesis, thereby establishing VDR activation as a compelling therapeutic target for mitigating disease progression and associated cellular dysregulation. Apoptosis, a tightly regulated self-destruction mechanism maintaining endometrial homeostasis, is frequently dysregulated in endometriosis [ 28 ]. EESCs exhibit resistance to apoptosis, characterized by downregulated pro-apoptotic Bax and upregulated anti-apoptotic Bcl-2, which disrupts apoptotic signaling cascades [ 29 , 30 ]. This anti-apoptotic phenotype contributes to lesion persistence and disease progression. 1,25(OH) 2 D 3 emerges as a critical modulator of this imbalance [ 31 ]. Preclinical studies demonstrate its capacity to sensitize EESCs to apoptosis by suppressing anti-apoptotic proteins (Bcl-2, Bcl-xL) and restoring pro-apoptotic pathways [ 32 , 33 ]. Our findings corroborate these observations, showing that 1,25(OH) 2 D 3 partially reverses apoptosis resistance in EESCs, evidenced by increased apoptotic/necrotic cell populations and reduced viability. These data highlight 1,25(OH) 2 D 3 ’s dual role in resolving inflammatory-proliferative pathologies while reactivating cell death mechanisms, offering a therapeutic avenue to counter endometriosis-associated cellular aggressiveness. TLR4 functions as the central sensor for LPS [ 34 ], orchestrating both innate and adaptive immune responses to bacterial endotoxins at nanomolar concentrations. LPS recognition initiates with its binding to LPS-binding protein, promoting complex formation with MD2 and CD14 co-receptors [ 35 ]. This ternary complex is specifically recognized by the TLR4 ectodomain, triggering receptor dimerization and conformational changes that propagate intracellular signals via cytoplasmic adaptors. The activated TLR4 complex engages myeloid differentiation primary response protein 88 (MyD88), TNF receptor-associated factors (TRAFs), and IL-1 receptor-associated kinases (IRAKs), culminating in activation of the IκB kinase (IKK) complex [ 36 ]. IKK-mediated phosphorylation of IκBα triggers its ubiquitination and subsequent degradation by the 26S proteasome, releasing sequestered NF-κB (p65/p50) dimers. Liberated p65/p50 translocates to the nucleus, where it drives transcription of pro-inflammatory cytokines and anti-apoptotic genes, thereby executing the TLR4-mediated immune response [ 37 ]. Our study demonstrates that 1,25(OH) 2 D 3 suppresses LPS-induced NF-κB inflammatory signaling through VDR-mediated mechanisms ( Figure 7 ). Key findings reveal that 1,25(OH) 2 D 3 inhibits LPS-driven nuclear translocation of p65, a pivotal step in NF-κB activation [ 38 ], an effect reversed by the VDR antagonist TEI-9647 [ 39 ], underscoring VDR’s essential role in mediating VD’s anti-inflammatory actions. Notably, LPS downregulated VDR expression, while 1,25(OH) 2 D 3 restored VDR levels and stabilized IκBα to block NF-κB pathway activation. TEI-9647 antagonism of both VDR expression and IκBα stabilization highlights a dual regulatory mechanism underlying 1,25(OH) 2 D 3 ’s anti-inflammatory effects. Regulation of NF-κB pathway by 1,25(OH) 2 D 3 and LPS . 1,25(OH) 2 D 3 suppresses LPS-induced NF-κB activation by stabilizing IκBα and blocking p65 nuclear translocation to dampen pro-inflammatory cascades, whereas TEI-9647 reverses this inhibitory effect functioning as a VDR antagonist. Arrows indicate activation, blocked arrows indicate inhibition. Furthermore, 1,25(OH) 2 D 3 significantly suppressed LPS-induced upregulation of TLR4 and NLRP3, two critical activators of NF-κB signaling that drive innate immune hyperactivation [ 40 ]. Reversal of these effects by TEI-9647 reinforced VDR’s central role in suppressing pro-inflammatory cascades. These findings extend prior work on VD’s inhibition of TLR4/MyD88 signaling in macrophages [ 41 ] and NLRP3 inflammasome activity in chronic inflammation [ 42 ] by establishing a direct VDR-dependent link in endometrial stromal cells, offering novel insights for endometriosis management. IκBα stabilization by 1,25(OH) 2 D 3 provides an additional regulatory layer, likely delaying proteasomal degradation to retain NF-κB in the cytoplasm. This mechanism synergizes with transcriptional repression of NF-κB targets, creating a multi-pronged anti-inflammatory strategy. TEI-9647 antagonism confirmed VDR dependence of IκBα stabilization, suggesting potential mechanisms involving transcriptional upregulation or post-translational modifications, which warrant further investigation. The ‘bacterial contamination hypothesis’ has recently gained traction in endometriosis research, positing that LPS may perturb immune-inflammatory homeostasis within the pelvic microenvironment, thereby acting as a potential pathogenic contributor to endometriosis development [ 7 , 8 ]. While LPS-induced inflammation in cellular and animal models recapitulates key features of endometriotic lesions, including cytokine dysregulation and aberrant tissue remodeling, emerging evidence highlights the involvement of additional inflammatory mediators that more closely mirror the complex human inflammatory milieu associated with this disease. For instance, elevated levels of prostaglandin E2 (PGE2) and matrix metalloproteinases (MMPs) in peritoneal fluid of endometriosis patients suggest their roles in pain pathogenesis and ectopic tissue invasion, respectively [ 43 , 44 ]. These findings underscore the necessity for expanded investigations to elucidate the interplay between bacterial products, endogenous inflammatory agents, and disease progression, ultimately paving the way for targeted therapeutic strategies that address the multifactorial nature of endometriosis pathophysiology.

This study demonstrates that 1,25(OH) 2 D 3 effectively counteracts LPS-induced proliferation and migration of EESCs and Ishikawa cells in vitro. In vivo validation using an autologous rat endometriosis model reveals significant suppression of endometriotic lesion growth and associated inflammatory responses by 1,25(OH) 2 D 3 . Mechanistically, we identify a VDR-dependent pathway wherein 1,25(OH) 2 D 3 inhibits NF-κB activation through suppressing TLR4 activity and stabilizing IκBα. These findings provide a molecular basis for VD’s anti-inflammatory effects in endometriosis and related pathologies. Given VDR’s ubiquitous expression, further studies are needed to establish optimal therapeutic dosing and systemic physiological impacts of 1,25(OH) 2 D 3 . Critically, as pregnancy remains a primary treatment goal for endometriosis patients, elucidating whether VD modulates endometrial receptivity and enhances fertility in this population represents a critical next step in translational research.