PMC

Therapeutic Mechanisms of MSCs in DM management. Abbreviations ANG-1 Angiopoietin 1, ANG-2 Angiopoietin 2, AT Adipose tissue, CXCR4 C-X-C chemokine receptor type 4, DCs Dendritic cells, DLK1 Delta like non-canonical Notch ligand 1, EGF Epidermal growth factor, ERK Extracellular signal-regulated kinase, FGF Fibroblast growth factor, FoxO1 Forkhead box protein O 1, GLUT4 Glucose transporter 4, GM-CSF Granulocyte macrophage colony stimulating factor, HGF Hepatocyte growth factor, HO-1 Heme oxygenase 1, IDO Indoleamine 2,3-dioxygenase, IL-10 Interleukin 10, IL-4 Interleukin 4, IL-6 Interleukin 6, IGF-1 Insulin-like-growth factor, iNOS Inducible-nitric oxide synthase, IRS-1 Insulin receptor substrate 1, M1 Pro-inflammatory macrophages, M2 Anti-inflammatory macrophages, MCP-1 monocyte-chemotactic protein 1, MC4R melanocortin-4 receptor, NK-cells Natural killer cells, Nrf2 Nuclear factor erythroid 2-related factor, PDGF Platelet-derived growth factor, PGE2 Prostaglandin E2, Reg B cells regulatory B cells, Reg DCs Regulatory dendritic cells, ROS Reactive oxygen species, SDF-1α stromal-derived factor 1 alpha, SOD-1 Superoxide dismutase 1, SOD-2 Superoxide dismutase 2, TIMP-1 Tissue inhibitor of metalloproteinase 1, TIMP-2 Tissue inhibitor of metalloproteinase 2, TGF-β1 Transforming growth factor beta 1, Tregs Regulatory T cells, VEGF Vascular endothelial growth factor, (+): promote/support, (-): inhibit or suppress

Profiles of LH changes induced by estradiol benzoate (EB) alone or in combination with progesterone (P) in ovariectomized WT-ERα (white) or C451A-ERα (gray mice). A, Protocol used to induce a positive feedback: females were ovariectomized (OVX), chronically treated with estradiol (E₂) from day 0 to day 8, injected with estradiol benzoate on day 7, and injected with progesterone or its vehicle (sesame oil) on day 8. B, On day 6, C451A-ERα females (n = 32) showed higher baseline LH levels than WT-ERα females (n = 31; Mann–Whitney test). C, D, Profiles of LH levels measured every 30 min starting 1 h before lights off following treatment on day 8 in WT-ERα and C451A-ERα females, respectively. C, LH profiles obtained in WT-ERα mice (OVX + E₂+ veh + veh n = 12, OVX + E₂+ EB + veh n = 11, OVX + E₂+ EB + P n = 14). D, LH profiles obtained in C451A-ERα mice (OVX + E₂+ veh + Veh n = 10, OVX + E₂+ EB + veh n = 11 OVX + E₂+ EB + P n = 12). E, Regardless of treatment, WT-ERα females showed an increased LH concentration at one time point (peak) between 0 and 2.5 h after lights off compared with prior (day 6, pre) and during 3 h after lights off (post; two-way ANOVA; Tukey’s post hoc test following significant time effect: ⁺⁺p < 0.01 vs “pre”; ^†p < 0.05 vs “post”). F, EB + P induced an increased LH concentration in C451A-ERα females within 0 and 2.5 h after lights off (peak) compared with prior (day 6, pre) and during 3 h after lights off (post; two-way ANOVA; Tukey’s post hoc test, following significant interaction: ⁺⁺⁺p = 0.001 vs “pre” within same treatment; ^†††p = 0.001 vs “post“ within same treatment; ^∇p < 0.05 EB + P “pre” vs “post” within same treatment. Symbols in the statistical boxes: *, **, ***, p < 0.05, 0.01, 0.001; N.S., nonsignificant).

Effect of mERα absence on the positive feedback of estrogens on LH concentration and the activation of the associated neurocircuits. A, Protocol used to induce positive feedback: following a first round of injections to induce the positive feedback (), the E₂ implant was replaced by a new one on Day 30, and females were treated again with veh + veh, EB + veh, or EB + P on Days 38 and 39. Blood and brains were collected 30–60 min after lights off. B, In WT-ERα females (white), EB + P, but not EB + veh, induced a significant rise in LH (Kruskal–Wallis test: **p < 0.01 vs veh + veh), while in C451A-ERα females (gray), EB + veh induced a significant reduction in LH (Kruskal–Wallis test: *p < 0.05 vs veh + veh; Mann–Whitney tests: ##, ### < 0.01, 0.001 vs WT-ERα within same treatment). C, WT-ERα females displayed more kisspeptin (Kp) neurons in RP3 V (AVPv + PeN) than C451A-ERα females (two-way ANOVA). D, A higher percentage of Kp neurons coexpressed Fos following EB and EB + P than veh + veh in WT-ERα, while only EB + P induced such activation in C451A-ERα (two-way ANOVA; * and ***, p < 0.05 and 0.001 vs veh + veh same genotype; ^$$$p < 0.0001 vs EB + veh same genotype; ^###p < 0.001 vs same treatment in WT-ERα). E, GnRH neurons counted in POA were slightly more abundant in C451A-ERα females than in WT-ERα females (two-way ANOVA). F, A higher percentage of GnRH neurons coexpressed Fos following EB and EB + P than veh + veh in WT-ERα, while only EB + P induced such activation in C451A-ERα (two-way ANOVA; ***, p < 0.001 vs veh + veh same genotype; ^$$$p < 0.0001 vs EB + veh same genotype; ^##p < 0.01 vs same treatment in WT-ERα). Sample size: B, C. WT-ERα: veh + veh, n = 14, veh + EB, n = 14, EB + P, n = 13, C451A-ERα: veh + veh, n = 11, veh + EB, n = 11, EB + P, n = 13. C–F. WT-ERα: veh + veh, n = 13, veh + EB, n = 14, EB + P, n = 13, C451A-ERα: veh + veh, n = 12, veh + EB, n = 11, EB + P, n = 13. Symbols in the statistical boxes: *, **, ***, p < 0.05, 0.01, 0.001; N.S., nonsignificant.

Effect of estetrol on the LH surge induced by estradiol and the neurocircuits underlying this response. A, Protocol used to induce a positive feedback. WT mice were ovariectomized (OVX) on day 0, treated with subcutaneous estradiol (E₂) implant from day 0 to day 9, and injected on day 8 with estradiol benzoate (EB) alone or combined with estetrol (E₄, 200 µg, s.c.) or their vehicle (sesame oil) and on day 9 with progesterone (P) or its vehicle (sesame oil). Blood samples were collected prior to treatment on day 8 and within 1 h of lights off on day 9, when brains were also collected for immunohistological analyses. B, LH levels did not differ between groups (n = 9) on day 8. C, Females treated with EB alone (n = 9) or EB + E₄ (n = 9) did not show a LH surge compared with veh + veh (n = 9) unless they were treated with P (EB + P, n = 9, and EB + E₄+ P, n = 9). D, F, The number of kisspeptin (Kp) neurons in RP3V (AVPv + PeN, D) or GnRH neurons in POA (F) did not differ across treatments (Kp: veh + veh, n = 7, EB, n = 9, EB + P, n = 7, EB + E4, n = 6, EB + E4 + P, n = 7; GnRH: veh + veh, n = 9, EB, n = 8, EB + P, n = 9, EB + E4, n = 8, EB + E4 + P, n = 8). E, G, The percentages of Kp (E) and GnRH (G) neurons coexpressing Fos were higher in females treated with EB, EB + P, and EB + E₄+ P than females treated with veh + P and EB + E₄ (same sample sizes as in D and F). All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test when significant: *, **, and *** p < 0.05, 0.01, and 0.001 versus veh + P; #, p < 0.05 versus EB; ^Δ, ^ΔΔ, and ^ΔΔΔ, p < 0.05, 0.01, and 0.001 versus EB + E₄.

Diagram representing the molecular orchestration of decidualisation and its dysregulation by SOCS3 and PTPN2 in obese mothers. Decidualisation is largely regulated by the steroid hormones progesterone (P4) and oestradiol (E2), which in association with the signal transducer and activator of the transcription 3 (STAT3) activate genes molecularly regulating decidualisation. In obese mothers, excessive levels of leptin bind to the leptin receptor b (ObRb), phosphorylating the Janus kinase 2 (JAK2) and STAT3 (pSTAT3), leading to the transcription of leptin signalling regulators like suppressor of cytokine signalling 3 (SOCS3), and protein tyrosine phosphatase non-receptor type 2 (PTPN2), known to inhibit pSTAT3. Decreased pSTAT3 levels disrupts the pSTAT3-P4 hub, which affects the expression of genes involved in cell proliferation and differentiation like prolactin family 8, subfamily a, member 2 (Dtprp), bone morphogenetic protein 2 (Bmp2), heart and neural crest derivatives expressed 2 (Hand2), and homeobox A10 (Hoxa10); extracellular matrix remodelling (ECM) as matrix metallopeptidase 13 (Mmp13), ADAM metallopeptidase with thrombospondin type 1 motif 18, (Adamts18); angiogenesis like calcitonin related polypeptide alpha (Calca), fibroblast growth factor 12 (Fgf12), Fms related receptor tyrosine kinase 1 (Flt1); and cell metabolism including CD36 molecule (Cd36), or insulin receptor substrate 1 (Irs1)

Mechanism of tamoxifen resistance. The activation of the E2/ERα signaling pathway contributes to tamoxifen sensitivity. By contrast, the activation of the PI3K/AKT/mTOR signaling pathway leads to tamoxifen resistance. E2, oestrogen; ERα, estrogen receptor α; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; IRS, insulin receptor substrate; PTEN, phosphatase and tensin homolog; PDK1, 3-phosphoinositide-dependent protein kinase 1; TSC, tuberous sclerosis complex; mTORC1, target of rapamycin complex 1; 4E-BP1, eukaryotic translation initiation factor 4E binding protein; S6K, p70/85 S6 kinase; PPARγ, peroxisome proliferator-activated receptor gamma; Elf4e, eukaryotic initiation factor 4E.

Potential mechanism for effects of PS-NPs on zebrafish neurobehavior and reproductive toxicity. (A) NPs carry other contaminants and bacteria and viruses []; (B) three main mechanisms; (C) interaction of behavioral changes and reproductive toxicity in zebrafish []; (D) potential effects on human reproduction. NPs, Nanoplastics; PS-NPs, Polystyrene nanoplastics; BPA, Bisphenol A; CAT, Catalase; MDA, Malondialdehyde; TNF-α, Tumor necrosis fac-tor-alpha; AChE, Acetylcholinesterase; E2, Estradiol; VTG, Vitellogenin; T, Testosterone; vtg2, Vitellogenin 2; cyp19a, Cytochrome P450 19A; lhb, Luteinizing hormone beta; cyp17, Cytochrome P450 17; erα, Estrogen receptor alpha; vtg1, Vitellogenin 1; cyp19b, Cytochrome P450 19B; fshr, Follicle-stimulating hormone receptor; ar, Androgen receptor.

(A) The effect of 25 μM curcumin (CUR) for 48 h on cell viability in the MCF-7 human breast cancer cell line, an estrogen receptor-positive cell line. (B) 17ß-estradiol (E2) at 1 × 10⁻⁷ for 48 h. The viability was measured using (C) an MTT assay and (D) crystal violet staining, and DMSO was used as a control (Ct). (E) The effect of CUR, E2, and a combination of both on anchorage-independent growth in the MCF-7 cell line, measured using colony formation assays in semisolid agar for 31 days. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (A–D). The data are expressed as the mean ± average with standard deviations (**: p < 0.01; ***: p < 0.001).

Effect of CUR, E2, and combination of both on proliferating nuclear antigen (PCNA) protein expression in MCF-7 cell line using (A) Western blot (β-actin served as control for loading) and (B) graph that represents PCNA protein expression according to relative peroxidase intensity in MCF-7 cell line, and its corresponding representative peroxidase images of immunocytochemistry analysis of PCNA (sc-56, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in MCF-7 cells treated with E2, CUR, or both combined for 48 h. Pictures were captured at 40× magnification using an Olympus CX31 optical microscope. Data are expressed as mean ± average with standard deviations (*: p < 0.05).

(A) The effect of curcumin (CUR) alone and in the presence of 17β-estradiol (E2) on ERα gene (ESR1) expression levels in the MCF-7 cell line. The cells were treated with DMSO as the control (Ct), E2, CUR, and CUR+E2 combined for 48 h. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls. (B) The effect of E2 and CUR, both alone and combined, on the ERα protein expression, with a Western blot and representative graphs, in MCF-7 cells treated for 96 h. β-actin was the loading control. (C) This graph represents the ERα protein expression according to the relative peroxidase intensity in the MCF-7 cell line and the corresponding representative peroxidase images for the immunocytochemistry of ERα (D6R2W, sourced from Cell Signaling, CA, USA) in MCF-7 cells. The images were taken with 40× magnification using an Olympus CX31 optical microscope. The data are expressed as the average with the standard deviation (*: p < 0.05; **: p < 0.01).

(A) The impact of curcumin (CUR) by itself and when combined with 17β-estradiol (E2) on the protein expression of Bcl-2 and Bax in the MCF-7 cell line, using a Western blot analysis and graphs for 48 h. DMSO was used as the control (Ct) and β-actin as the loading control. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (*: p < 0.05). (B) The graphs represent Bcl-2 and Bax protein expressions according to the relative peroxidase intensity in the MCF-7 cell line and the corresponding representative peroxidase images of the immunocytochemistry of Bcl-2 and Bax (sc-492 and sc-526, respectively, both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in the same cell line. The pictures were captured at 40× magnification using an Olympus CX31 light microscope.

The effect of curcumin (CUR) alone and in the presence of 17β-estradiol (E2) for 48 h on the levels of the cathepsin D gene (CTSD), the epidermal growth factor receptor gene (EGFR), the cyclin D1 gene (CCND1), and the BCL2 apoptosis regulator gene in MCF-7 cells, with DMSO as the control (Ct). The data are expressed as the average with the standard deviation. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (*: p < 0.05; **: p < 0.01).

The effect of curcumin (CUR) alone and in the presence of 17β-estradiol (E2) on (A) the β-catenin, Vimentin, and E-cadherin protein expression in MCF-7 cells treated for 48 h. Western blot analysis: DMSO was used as a control (Ct) and β-actin was used as the loading control. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (*: p < 0.05). (B) Graphs that represent ß-catenin, Vimentin, and E-cadherin protein expressions according to the relative peroxidase intensity in the MCF-7 cell line and the corresponding representative peroxidase images of the immunocytochemistry of β-catenin, Vimentin, and E-cadherin (sc-1496, sc-7557, and sc-8426, respectively; provided by Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in the same cell line. The images were taken with 40× magnification in an Olympus CX31 optical microscope.

This figure illustrates the complexity of cellular interactions among TME cells in PDAC. These interactions form a network that helps the tumor grow, evade from the immune system, and spread somewhere else in the body. The main players of this landscape are represented by CAFs, TAMs (M2 type), TANs (N2 type), Tregs, effector T cells, MDSCs, and PSCs. All the abovementioned cells communicate with PDAC cells through the production of several chemokines and cytokines which regulate tumor growth, progression as well as metastasis through angiogenesis and lymphangiogenesis. This complex interconnected network determines the development and behavior of PDAC. TME, tumor microenvironment; PDAC, pancreatic ductal adenocarcinoma; CAFs, cancer-associated fibroblasts; TAMs, tumor-associated macrophages; TANs, tumor-associated neutrophils; Tregs, regulatory T cells; MDSCs, myeloid-derived suppressor cells; PSCs, pancreatic stellate cells; IL-1/4/6/10/11/13/17/35, interleukin-1/4/6/10/11/13/17/35; TGFβ, transforming growth factor β, VEGF, vascular endothelial growth factor; MMPs; matrix metalloproteinases; PGE2, prostaglandin E2; GM-CSF, granulocyte–macrophage colony-stimulating factor; αSMA, α smooth muscle actin; CCL-2/4/5/17/20, C-C motif chemokine ligand -2/4/5/17/20; NE, neutrophil elastase; ROS, reactive oxygen species; RNS, reactive nitrogen species; NETs, neutrophil extracellular traps; CCR5, C-C chemokine receptor type 5, CXCL-1/10/12/18; C-X-C motif chemokine ligand-1/10/12/18; ECM, extracellular matrix; Cox2, cyclooxygenase-2; CSF-3, colony-stimulating factor 3; FAP, fibroblast activation protein; SDF-1, stromal cell-derived factor 1; HGF, hepatocyte growth factor; Arg1, arginase 1; PD-L1, programmed death-ligand 1.

The schema of dual roles of MSCs in TME and their pro- and anti-tumorigenic activity. (A) Dual effect of tumour-associated mesenchymal stromal cells (MSCs) in head and neck cancer (HNC). MSCs can affect both tumour progression or tumorigenesis inhibition, with a tendency to promote the former. MSCs in the TME show pro-tumorigenic activity on neoplastic cells directly via cell-to-cell contact or extracellular vesicles (MSC-EVs). MSC-EVs include key immune-modulating agents, proangiogenic factors, pro-survival biological agents or soluble factors stimulating cell mobility and extracellular matrix modulators, which favour higher tumour aggressiveness. MSCs isolated from squamous cell carcinomas can mediate cancer progression by secreting pro-inflammatory and pro-angiogenic cytokines such as IL-6 and IL8, which also promotes the recruitment of TAMs, CAFs, T cells, neutrophils and other MSCs. In addition, TGF-β1,VEGF, EGF, PDGF and cytokines IL-6 and IL-8, as well as B2M, CCN2, SCGF, SDF-1 and chemokines such as CXCL 1/5/7/8 are all primarily responsible for tissue remodelling and growth. The formation of new vessels in the tumour niche, enabling the initial growth of neoplastic lesions and then tumour metastasis, is associated with the action of VEGF, IGF-1, TGF-β1, FGF, angiopoetin-1, endothelin-1, and cytokines IL-6 and IL-8, which facilitate further tumour progression and growth by enhancing angiogenesis, increasing vascularisation and the local/regional and general spread of tumour cells. Most of these MSC-derived factors directly regulate the epithelial–mesenchymal plasticity, proliferation, invasion and migration and even drug resistance in HNSCC cells. MSCs can also activate other key signalling pathways, soluble agents and modulators, having an opposite effect on tumour progression and aggressiveness. The anti-tumour MSC-related paracrine soluble secreted factors and MSC-derived molecules released from exosomes that inhibit tumorigenesis include anti-proliferative factors such as Dkk-1, oncostatin-M, a soluble Wnt antagonist, PTEN, BMP, cytotoxic agents such as TRAIL, IFN-α, IGFBP and TNF-α, anti-angiogenic factors or immunomodulatory agents. MSCs may also actively inhibit further tumour development by blocking cancer-related signalling, such as the PI3K/AKT, Wnt/β-catenin, and JAK/STAT pathways. (B) The immunomodulation mechanisms of MSCs and their role in immune cell activity. Adaptive immune system: the active MSCs in the tumour milieu inhibit the adaptive immune response through the secretion of mediators contained in exosomes (MSCs-EV) and soluble factors, such as IDO, TGFβ1, TNF-α, IFN-γ, PGE2, NO, HLA-G, HGF, IL-1β, IL-1α, IL-4 and IL-6; they also interact with various immune cell types, including T cells, B cells, DC cells, NK cells, monocytes and TAMs. This MSC activity constrains dendritic cell maturation, reduces T cell proliferation, enhances macrophage activation and polarises them from M1 towards M2; it also facilitates neutrophil mobility and affects the regulation of NK cells and invariant natural killer T (iNKT) cells. In addition, it can shift the balance of T cell differentiation from the Th1 to an anti-inflammatory Th2 phenotype and enhance the maturation of T helper cells into the CD4⁺CD25⁺Foxp3⁺Treg pathways, which can inhibit effector T cell responses and thus reduce anti-tumour immunity. The innate immune system: in tumorigenic tissues, local factors, such as the cytokine milieu TNF-α and endotoxin LPS, hypoxia and Toll-like receptor (TLRs) ligands, stimulate MSCs, promoting the large-scale secretion of growth factors such as VEGF, FGF2, IGF-1, or HGF by an NFκB-dependent mechanism, with the effect of driving tissue regeneration, angiogenesis, reducing anti-tumour immunity and allowing the effective escape of the tumour from immune surveillance. Abbreviations: MSCs: mesenchymal stromal cells; CSCs: cancer stem cells; BM-MSCs: bone-marrow-derived MSCs; A-MSCs: adipose tissue-derived MSCs; N-MSC: naïve MSCs; CAFs: cancer-associated fibroblasts; ECM: extracellular matrix; CIS: carcinoma in situ; MMPs (MT-MMPs): matrix metalloproteinases, also known as matrix metallopeptidases; TGF-β1: transforming growth factor beta 1; CXCL1/2/12: C-X-C motif chemokine ligand 1/2/12; CCL5: C-C motif chemokine ligand 5; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; PDGF: platelet-derived growth factor; FGF: fibroblast growth factors; TRAIL: TNF-related apoptosis-inducing ligand; INF-α: type-I interferon alpha; IGFBP: insulin-like growth factor-binding protein; Dkk-1: Dickkopf-related protein 1; HGF: hepatocyte growth factor; PTEN: phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase; BMP: bone morphogenetic protein; TRAIL: TNF-related apoptosis-inducing ligand; PGE2: prostaglandin E2; B2M: beta 2 microglobulin; CCN2: cellular communication network factor 2; SCGF: stromal cell growth factor-beta; SDF-1: stromal cell-derived factor 1; IDO: indoleamine 2,3-dioxygenase; iNOS: nitric oxide synthases; HO1: heme oxygenase 1; CTLs: cytotoxic T cells; CD4⁺CD25⁺Foxp3⁺T_reg: regulatory T cells, known as suppressor T cells; TAMs M1/2: tumour-associated macrophages M1/2, CD56^dim/CD16^bright NK cells: activated natural killer cells, TLRs: Toll-like receptors; → activation mechanisms; ¦ inhibitory mechanisms; ↑: increase in expression and activity, ↓: decrease in expression and activity.

E2 and SERM2 effects on DSS colitis activity. Schematic diagram of intact mice colitis experiment, where shaded area represents 7‐day DSS challenge (A). In the intact mice, DAI AUC was increased by DSS (n = 8) compared to vehicle control (n = 4), and the DSS + SERM2 (“S2”, n = 8) was statistically significantly lower compared to DSS control (B). The total colitis score (endpoint DAI combined with the histological scoring) showed a similar pattern (C). Representative HE staining of intact DSS colon with severe erosion and immune infiltration (D) and DSS + SERM2 colon, where architecture is intact and immune infiltration is evident (E). Schematic diagram of OVX mice colitis experiment, where again, shaded area represents 7‐day DSS challenge (F). In the OVX animals DSS (n = 7) induced an increase of the DAI score AUC compared to vehicle control (n = 4). Compared to DSS‐group, DSS + SERM2 (n = 7) mice exhibited slightly decreased disease activity, but in the DSS + E2 (n = 7) mice, AUC was significantly increased (G). Total colitis score of OVX mice showed no difference between DSS control and DSS + SERM2, while in the DSS + E2 mice score was statistically significantly increased (H). E2 serum levels correlated with the total colitis score (I). Representative HE staining of OVX DSS colon with minor signs of inflammation (J). Representative HE staining of OVX DSS + E2 colon with edema, hyperproliferation, extensive immune infiltration and crypt loss (K). Data presented as the mean ± SD, statistical significance of AUC's and scoring data was calculated with ordinary one‐way ANOVA, Brown‐Forsythe and Welch ANOVA or Kruskal‐Wallis nonparametrical test according to distribution and variance. Statistical significance was defined as *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001.

Colon shortening and immune infiltration analysis. Colon length was decreased by DSS (n = 8) in intact mice and SERM2 (n = 5, DSS + SERM2 n = 7) did not significantly alter the length (A). F4/80+ (B) and CD3ε + (C) cells increased with DSS in intact mice. Representative images of anti‐F4/80 (D) and anti‐CD3ε (E) immunostaining in intact DSS mice. In OVX mice, DSS (n = 7) did not affect colon length compared to vehicle (n = 4), but E2 (n = 7) reduced colon length both compared to DSS + SERM2 (n = 7) and DSS (F). F4/80+ (G) and CD3ε + (H) cell counts increased by DSS + E2 (n = 7) and trended toward an increase in DSS + SERM2. Representative images of F4/80 (I) and CD3ε (J) in the colons of OVX DSS mice. In figures B and C, one data point in DSS + SERM2 group is missing due to loss of sample. Data presented as mean ± SD, statistical significance was calculated with ordinary one‐way ANOVA, Brown–Forsythe and Welch ANOVA or Kruskal–Wallis nonparametrical test according to distribution and variance. Statistical significance was defined as *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001.

Immune activity‐associated gene expression in distal colon. Gene expression of Mrc1 was increased by SERM2 (“S2”, n = 5) in intact mice (A). Tgfb expression increased with DSS (n = 8) and decreased in the DSS + SERM2 (n = 8, B). Tnf (D) was not affected by DSS + SERM2, while Il10 (C), Il1b (E), Il6 (F), Ifng (G) expression increased by DSS and demonstrated consistent trends toward downregulation by DSS + SERM2, while only Foxp3 (H) the altered statistically significantly. In OVX mice, the treatment did not affect Mrc1 expression (I). Il10, Tnf, Il1b, Il6, Ifng, and Il17a (K–O) expressions were statistically significantly increased in the DSS + E2 group and trended toward an increase by DSS + SERM2, while FoxP3 increased only by DSS + E2 (P). Please note the two‐part y‐axis in figures (L, M, O). Statistical outliers were identified using ROUT with Q = 1% and one datapoint was removed in the DSS group of figure (F) and DSS + E2 group of figure (K). Data was normalized to DSS group as a part of the ΔΔCt analysis and presented as mean ± SD, statistical significance was calculated with ordinary one‐way ANOVA, Brown‐Forsythe and Welch ANOVA or Kruskal‐Wallis nonparametrical test according to distribution and variance. Statistical significance was defined as *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001.

The serum cytokine response to DSS colitis. In intact animals, serum IL‐5 (A) was unaffected by treatment, while IL‐6, IFNγ, TNFα, and CCL2 increased by DSS (n = 8 vs vehicle n = 4, B–E). In OVX mice circulating IL‐5, IL‐6 and TNFα increased by both DSS + SERM2 (n = 7, “S2”) and DSS + E2 (n = 7), although SERM2 increase was statistically significant only in TNFα (F, G, I), The alterations in IFNγ and CCL2 (H, J) levels were not statistically significant. Statistical outliers were identified using ROUT with Q = 1% and one datapoint has been removed in each figure: A DSS + SERM2, D vehicle, F DSS and DSS + E2, G DSS. Data presented as mean ± SD, statistical significance was calculated with ordinary one‐way ANOVA, Brown–Forsythe and Welch ANOVA or Kruskal–Wallis nonparametrical test according to distribution and variance. Statistical significance was defined as *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001.

COX2 and substance P expression in distal colon. DSS + SERM2 (“S2”, n = 7) trended toward a decrease in Ptsg2 expression compared to DSS (n = 7, A) while COX2 protein levels measured by Western blots showed a similar pattern, though statistically significant (B). Representative image of COX2 immunostaining of the colon in intact and OVX mice (C). The expression of Tacr1 was statistically significantly increased by DSS compared to vehicle (n = 4) and trended toward decrease by DSS + SERM2 (D). In OVX mice the transcription of Ptgs2 was highly elevated in the DSS + E2 group (n = 8, E) and COX2 protein levels were elevated by both DSS + SERM2 and DSS + E2 (F). COX2 gene (G) and protein (H) expression correlated with serum E2 levels. Tacr1 levels were increased by DSS + E2 (I), while neuropeptide substance P was not affected by treatments (J). Representative image of substance P immunostaining in the colon of intact and OVX mice (K). Gene expression data was normalized to DSS group as a part of the ΔΔCt analysis and substance P peak area was normalized to reference peptide. Statistical outliers were identified using ROUT with Q = 1% and one datapoint was removed in DSS and DSS + SERM2 group of figures (A) and DSS in figure (B). Data presented as mean ± SD, statistical significance was calculated with ordinary one‐way ANOVA, Brown‐Forsythe and Welch ANOVA or Kruskal‐Wallis nonparametrical test according to distribution and variance. Statistical significance was defined as *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001. The correlation of E2 with Ptgs2 and COX2 was calculated using Spearman's Rank Correlation Coefficient analysis.