Therapeutic Effects of a Synthetic Glabridin Derivative on Th17/ B cell Immune Regulation and Salivary gland Regeneration in Experimental Sjögren’s Syndrome

Therapeutic Effects of a Synthetic Glabridin Derivative on Th17/ B cell Immune Regulation and Salivary gland Regeneration in Experimental Sjögren’s Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Therapeutic Effects of a Synthetic Glabridin Derivative on Th17/ B cell Immune Regulation and Salivary gland Regeneration in Experimental Sjögren’s Syndrome Mi-La Cho, Jin-Sil Park, Hye Yeon Kang, Ha Yeon Jeong, JeongWon Choi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7540230/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Sjögren’s syndrome (SS) is a chronic autoimmune disease in which inflammatory cells infiltrate the exocrine glands, reducing glandular secretory function and ultimately resulting in keratoconjunctivitis sicca (dry eyes) and xerostomia (oral dryness). Cardiovascular risk factors are more prevalent in patients with SS than in healthy controls; patients with SS and metabolic syndrome also have higher leptin and inflammatory cytokine levels. In this study, we investigated the effects of HGR4113, a structural analog of glabridin that promotes mitochondrial function and is in clinical trials for obesity treatment, on the development of Sjögren’s syndrome in non-obese diabetic NOD/ShiLtJ (NOD) mice. HGR4113 inhibited IL-17 production by regulating STAT3 activity and the metabolic profile of splenic CD4 + T cells; it also increased the frequency of IL-10-producing regulatory B cells and decreased immunoglobulin production. Oral administration of HGR4113 (100 mg/kg) improved salivary flow rate, reduced lymphocyte infiltration, and lowered inflammatory cytokine levels in the salivary glands of NOD mice. HGR4113 also decreased the frequencies of splenic IL-17-producing T and B cells, germinal center B cells, and plasma cells ex vivo in NOD mice. Additionally, organoid formation from salivary gland stem cells of HGR4113-treated NOD mice increased, as did the levels of E-cadherin-14, aquaporin-5, α-SMA, and cytokeratin-14. Finally, treatment with HGR4113 promoted salivary gland organoid formation in vitro . HGR4113 improves salivary gland hypofunction by inhibiting lymphocyte infiltration and inflammation in the salivary glands and restoring damaged salivary tissue in SS-like NOD mice. Biological sciences/Immunology/Autoimmunity Health sciences/Diseases/Immunological disorders/Autoimmune diseases Biological sciences/Immunology/Inflammation/Chronic inflammation Biological sciences/Immunology/Gene regulation in immune cells Sjögren’s syndrome metabolic abnormality T cell B cell salivary gland organoid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sjögren’s syndrome (SS) is a chronic autoimmune disease in which inflammatory cells infiltrate the exocrine glands, reducing glandular secretory function and ultimately resulting in keratoconjunctivitis sicca (dry eyes) and xerostomia (oral dryness). SS is often accompanied by extraglandular manifestations involving the skin, muscles, kidneys, joints, peripheral nervous system, and blood vessels. 1 , 2 In SS, mortality is increased in patients with extraglandular manifestations and non-Hodgkin lymphoma. 2 The pathogenesis of SS is still under investigation, but lymphocyte infiltration into target organs, including exocrine glands such as the salivary and lacrimal glands, is considered a key mechanism of disease development. 3 Aggregates of B and T cells are observed in ectopic lymphoid structures in the salivary and lacrimal glands, and B cells that receive signals from T cells become hyperactivated, resulting in hypergammaglobulinemia, germinal center (GC) formation, and production of anti-SSA/Ro and anti-SSB/La autoantibodies. 4 , 5 Metabolic imbalance can alter immune cell phenotypes and immune responses, potentially leading to autoimmunity or malignant transformation. 6 , 7 Recently, metabolic abnormalities have been reported to play a role in the pathogenesis of SS. Ultrastructural alterations of mitochondria have been observed in the salivary epithelial cells of patients with SS, 8 and oxidative stress levels are increased in their saliva. 9 Glabridin, a polyphenolic isoflavan isolated from the root of Glycyrrhiza glabra L. (Fabaceae), commonly known as licorice, is a widely used herb-derived natural compound with anti-inflammatory, antioxidant, anticancer, and antiatherogenic effects, as well as the ability to regulate energy metabolism. 10 , 11 However, glabridin has poor stability in formulations and low bioavailability, limiting its application as a commercial clinical therapeutic agent. 12 , 13 A recent structure–activity relationship study identified a novel structural analog of glabridin, HGR4113 ((R)-2-(8,8-dimethyl-2,3,4,8,9,10-hexahydropyrano[2,3-f]chromen-3-yl)-5-propoxyphenol), with improved chemical stability and metabolic characteristics relative to glabridin. 14 In the present study, we evaluated the ability of HGR4113 to modulate inflammatory responses in murine splenic T and B cells and its therapeutic efficacy in NOD/ShiLtJ mice, a preclinical model of SS. We also investigated the role of HGR4113 in salivary gland organoid generation in NOD/ShiLtJ mice ex vivo and in vitro . Materials and methods Animals Seven-week-old female NOD/ShiLtJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and cage of animals maintained under specific pathogen-free conditions at the Institute of Medical Science, Catholic University of Korea. The mice were fed standard mouse chow and water. All procedures were approved by the Animal Research Ethics Committee of the Catholic University of Korea and conformed to the United States National Institutes of Health guidelines (permit number: CUMS-2024-0267-02). All animals were treated and euthanized in accordance with the Guidelines on the Use and Care of Animals of the Catholic University of Korea. Surgery was performed under isoflurane anesthesia, and every effort was made to minimize suffering. At the end of the study, the mice were euthanized in a CO 2 chamber for sample collection. Treatment with HGR4113 and measurement of saliva secretion in NOD/ShiLtJ mice Saliva secretion was stimulated in anesthetized mice by intraperitoneal injection of pilocarpine (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 5 mg/kg body weight. Saliva was collected from the oral cavity for 7 min, beginning 90 s after the injection, using a micropipette. The volume of saliva was determined gravimetrically (µL/g/min). Before drug injection, salivary flow rates were measured in mice, and groups were randomized based on the average salivary flow rate of all mice. HGR4113 was dissolved in dimethyl sulfoxide. Seven-week-old female NOD/ShiLtJ mice were administered 100 mg/kg of HGR4113 or vehicle orally once daily for 12 weeks (n = 10/group). Sample size was determined for statistical significance and experimental feasibility. To minimize potential confounders such as the order of treatments and measurements, we individually marked each mouse with an ear punch. Blood glucose levels were measured using an Accu-Check™ Compact Glucometer (Roche Diagnostics, Indianapolis, IN, USA). Individuals with blood glucose levels of 600 mg/dL were excluded from evaluation due to the development of diabetes. Samples were stored at − 70°C until analysis. Histopathological examination and immunohistochemistry Salivary glands were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer-thick sections were stained with H&E, and the degree of inflammation was quantified as previously described. 15 Sections were incubated with primary antibodies against CD4 (1:50, Abcam, #ab25475), CD19 (1:200, Abcam, #ab203615), IL-6 (1:300, Novus, #NB600-1131), IL-17 (1:200, Abcam, #ab79056), type I collagen (Col-I) (1:100, Invitrogen, #PA5-89281), fibronectin (1:200, Abcam, #ab2413), TGF-β (1:100, Bioss, #BS-0086R), and aquaporin-5 (AQP5) (1:100, Novus, #NBP2-92926) for 2 h at room temperature. Sections were then incubated with a horseradish peroxidase–conjugated secondary antibody for 30 min. The final products were visualized using the chromogen diaminobenzidine. Immunostained sections were examined by photomicroscopy (Olympus). Positive cells were counted in high-power digital images (400× magnification) using Adobe Photoshop (Adobe, San Jose, CA, USA). Counts were performed visually by three individuals, and mean values were calculated. Isolation and stimulation of splenocytes Murine splenocytes were prepared as previously described. 16 Spleens were dissociated using sterilized glass slides with frosted ends, and red blood cells were lysed in hypotonic ACK buffer (0.15 mM NH 4 Cl, 1 mM KCO 3 , and 0.1 mM ethylenediaminetetraacetic acid, pH 7.4). The remaining splenocytes were filtered through a 40-µm cell strainer (Falcon, Durham, NC, USA) and maintained in RPMI 1640 medium containing 5% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). Splenic CD4 + T or CD19 + B cells were purified using a positive cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec). The cells were pretreated with HGR4113 for 2 h. Splenocytes or isolated T cells were cultured with 0.5 µg/mL anti-CD3 (BD Pharmingen, #553057) and 1 µg/mL anti-CD28 (BD Pharmingen, #553294) antibodies, or under Th17-polarizing conditions with anti-CD3, anti-CD28, 20 ng/mL IL-6 (R&D Systems, #406-ML), 2 ng/mL transforming growth factor-β (TGF-β) (PeproTech, #100-21C), 5 µg/mL anti–IFN-γ antibody (R&D Systems, #MAB485), and 5 µg/mL anti–IL-4 antibody (R&D Systems, #MAB404) for 72 h. Splenocytes or isolated B cells were cultured with 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich, #L4391) or 10 µg/mL IgM antibody (Jackson ImmunoResearch Labs), 20 ng/mL recombinant mouse IL-4 protein (R&D Systems), or 1 µg/mL recombinant murine sCD40 ligand (PeproTech) for 72 h. Seahorse metabolic assay The oxygen consumption rate (OCR) was measured using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s instructions. Briefly, 5 × 10 5 purified splenic CD4 + T cells were cultured in RPMI 1640 medium with 5% FBS in the presence or absence of HGR4113 under anti-CD3 and anti-CD28 stimulation for 12 h. The washed cells, resuspended in XF medium containing 1× ITSA (Gibco), were seeded on Cell-Tak–coated (BD Biosciences) Seahorse cell culture plates and incubated in a 37°C incubator without CO 2 for 30 min. Real-time changes in OCR (pmol O 2 /min) were measured after treatment with oligomycin (complex V inhibitor, 7 µM), FCCP (3 µM), and rotenone plus antimycin A (electron transport chain inhibitors, 10 µM). Real-time polymerase chain reaction (PCR) Total RNA was extracted using TRI Reagent (Molecular Research Center), and cDNA was synthesized with the Dyne First-Strand cDNA Synthesis Kit (Dyne Bio) according to the manufacturer’s protocol. Gene expression was measured using the StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Premix (Bioline Inc.). The following primers were used: Ifng , 5′-AGG AAC TGG CAA AAG GAT GGT-3′ and 5′-ATG TTG TTG CTG ATG GCC TG-3′; IL-17 , 5′-CCT CAA AGC TCA GCG TGT CC-3′ (sense) and 5′-GAG CTC ACT TTT GCG CCA AG-3′ (antisense); Foxp3 , 5′-CAC TGC CCC TAG TCA TGG T-3′ (sense) and 5′-GGA GGA GTG CCT GTA AGT GG-3′ (antisense); IL-10 , 5′-GGC CCA GAA ATC AAG GAG CA-3′ (sense) and 5′-AGA AAT CGA TGA CAG CGC CT-3′ (antisense); BAFFR , 5′-GGT GGG TCT AGT GAG TCT GGT-3′ (sense) and 5′-TTC TGA GGA GGG TAC AAA GAC AT-3′ (antisense); activation-induced cytidine deaminase ( AID ), 5′-GCC ACC TTC GCA ACA AGT CT-3′ (sense) and 5′-CCG GGC ACA GTC ATA GCA C-3′ (antisense); Col1a1 , 5′-GCC AAG AAG ACA TCC CTG AAG-3′ (sense) and 5′-TGT GGC AGA TAC AGA TCA AGC-3′ (antisense); and β-actin , 5′-GTA CGA CCA GAG GCA TAC AGG-3′ (sense) and 5′-GAT GAC GAT ATC GCT GCG CTG-3′ (antisense). mRNA levels were normalized to those of β-actin . Immunoblot analysis Immunoblot analysis Cells were lysed in Halt protein lysis buffer containing Halt phosphatase inhibitor (Thermo Pierce). Lysates were centrifuged at 14,000 × g for 15 min at 4°C. Protein concentration was determined using the Bradford protein assay (Bio-Rad). Protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Membranes were incubated with antibodies against phosphorylated STAT3 (Y705, Abcam, #ab76315), STAT3 (Abcam, #ab68153), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam, #ab181602). Hybridized bands were detected using an ECL detection kit (Pierce) and Hyperfilm (Agfa). Western blot analysis was performed using the SNAP i.d. Protein Detection System (Millipore). Protein levels were normalized to β-actin or GAPDH as loading controls, and the relative level of the protein of interest under the nil condition was set to 1. Enzyme-linked immunosorbent assay (ELISA) Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-17, and IL-6 levels in culture supernatants were determined by sandwich ELISA (DuoSet; R&D Systems, Lille, France). Blood was collected from the orbital sinuses of mice, and serum samples were stored at − 20°C until use. Serum IgG concentration was measured using the ELISA Quantitation Set (Bethyl Laboratories) according to the manufacturer’s protocol. Streptavidin-conjugated horseradish peroxidase was used for color development. Absorbance at 450 nm was measured using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). Intracellular staining and flow cytometry To stain surface markers, single-cell suspensions were washed with fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS] with 2% FBS) and incubated with fluorochrome-labeled antibodies for 30 min at 4°C. For intracellular staining, single-cell suspensions were cultured with 25 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) with added GolgiStop (BD Biosciences) for 4 h. After surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm according to the manufacturer’s instructions (BD Biosciences). For intracellular Foxp3 and Bcl-6 staining, a Foxp3/Transcription Factor Staining Buffer Kit (eBioscience) was used following surface staining. After washing with Perm/Wash buffer, antibodies for intracellular staining were added for 30 min at 4°C. The following anti-mouse antibodies were used for FACS: Alexa Fluor® 700 anti-CD4 (RM4-5), PE-Cy7 anti-CD19 (1D3), PerCP anti-CD45R/B220 (RA3-6B2), PE anti-CD138 (281-2), and FITC anti-GL-7 (GL7) from BD Biosciences; PerCP-Cy5.5 anti-CD4 (RM4-5), eFluor 450 anti-CD4 (RM4-5), FITC anti-CD5 (53 − 7.3), PE anti-CD1d (1B1), PE anti-Fas (15A7), PerCP-eFluor 710 anti-CXCR5 (SPRCL5), PE-Cyanine7 anti-ICOS (7E.17G9), APC anti-Bcl-6 (BCL-DWN), PE anti-Foxp3 (FJK-16s), PE anti–IFN-γ (XMG1.2), PE anti–IL-17 (eBio17B7), and APC anti–IL-10 (JES5-16E3) from Invitrogen; and APC anti-CD25 (PC61) from BioLegend. Stained cells were analyzed on a FACSCalibur (BD Biosciences), CytoFLEX (Beckman Coulter), or LSRII (BD Biosciences). Events were collected and analyzed with FlowJo software (Tree Star). Mouse salivary gland organoid primary culture Spheroid-forming mouse salivary gland stem cells were isolated from the submandibular gland. The thin fascia covering the submandibular gland was carefully removed to obtain the tissue. The tissue was washed three times with PBS (Gibco) containing 3% penicillin–streptomycin and minced into small pieces, which were placed in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) containing 1 mg/mL collagenase IV (Gibco) and incubated for 30 min at 37°C in a 5% CO 2 atmosphere. The tissue was then centrifuged, and the cell pellet was resuspended in DMEM and filtered through a 40-µm cell strainer (BD) to obtain single cells. After centrifugation, the cells were cultured in DMEM/F12 (1:1, v/v; Gibco) supplemented with 20 ng/mL epidermal growth factor (PeproTech), 20 ng/mL fibroblast growth factor-2 (PeproTech), 1% N2 supplement (Gibco), 1% insulin–transferrin–selenium (Gibco), 1 µM dexamethasone (Sigma), and 1% antibiotic–antimycotic (GenDEPOT). Salivary gland stem cells were seeded on Matrigel™ (Corning) and cultured for 10 days. Immunofluorescence Salivary gland organoids were snap-frozen in liquid nitrogen and stored at − 70°C. Six-micrometer-thick cryosections were fixed in acetone for 10 min and dried at room temperature. The slides were hydrated in Tris buffer and blocked with 10% goat serum for 30 min at room temperature. They were then incubated overnight at 4°C with the appropriate antibodies diluted in Tris buffer. Sections were mounted with fluorescence mounting medium (Dako). The following anti-mouse antibodies were used for staining: keratin (KRT) 5 (1:100, Abcam, #ab52635), KRT7 (1:200, Invitrogen, #MA1-06315), α-SMA (1:800, Abcam, #ab7817), KRT14 (1:600, Abcam, #ab181595), E-cadherin (1:1000, Abcam, #ab231303), and AQP5 (1:100, Novus, #NBP2-92926). Nuclei were stained with DAPI (D35271; Invitrogen) in PBS. All stained sections were imaged using a confocal laser scanning microscope (LSM700). Statistical analysis Statistical analyses were performed using GraphPad Prism (version 5 for Windows; GraphPad Software, San Diego, CA, USA). Normally distributed continuous data were analyzed using the parametric Student’s t -test. Differences in mean values among groups were analyzed by analysis of variance. Data are presented as mean ± standard deviations (SD). P values < 0.05 (two-tailed) were considered statistically significant. Results HGR4113 reduces the production of inflammatory cytokines in splenocytes from SS-like NOD/ShiLtJ mice To determine the effects of HGR4113 on cell proliferation in vitro , splenocytes from C57BL/6 mice were pretreated with HGR4113 for 2 h and then cultured with or without anti-CD3 antibody (Ab)/anti-CD28 Ab or LPS for 3 days. HGR4113 treatment did not affect the viability of splenocytes that were not stimulated with anti-CD3 Ab/anti-CD28 Ab or LPS. However, HGR4113 treatment dose-dependently decreased the viability of splenocytes stimulated with anti-CD3 Ab/anti-CD28 Ab or LPS (Fig. 1 a). To assess whether HGR4113 exerts a regulatory effect on SS-derived pathogenic immune cells, splenocytes from NOD mice exhibiting SS-like symptoms were stimulated with HGR4113 in the presence of anti-CD3 Ab/anti-CD28 Ab or LPS for 3 days. HGR4113 treatment effectively reduced the amount of IFN-γ that was increased by anti-CD3 Ab and anti-CD28 Ab stimulation. Compared with normal C57BL/6 mice, production of IL-17 after anti-CD3 Ab and anti-CD28 Ab stimulation in splenocytes of NOD mice was significantly increased, and HGR4113 significantly reduced IL-17 levels (Fig. 1 b). Additionally, treatment with HGR4113 effectively reduced the amount of TNF-α that was increased by LPS stimulation. Compared with normal C57BL/6 mice, LPS stimulation significantly increased the production of IL-6 in splenocytes of NOD mice, and HGR4113 treatment significantly reduced IL-6 production (Fig. 1 c). These results demonstrate that HGR4113 can modulate the production of inflammatory factors in immune cells in a preclinical model of SS. HGR4113 inhibits IL-17 production by regulating STAT3 activity and the metabolic profile in splenic CD4 + T cells To determine whether HGR4113 affects cytokine secretion in T cells, splenic CD4 + T cells isolated from C57BL/6 mice were treated with HGR4113 and cultured under anti-CD3 Ab and anti-CD28 Ab stimulation or under Th17-polarizing conditions for 3 days. HGR4113 dose-dependently reduced the frequency of Th17 cells under Th17-polarizing conditions (Fig. 2 a). Treatment with HGR4113 also dose-dependently suppressed the amounts of IFN-γ and IL-17 in the culture supernatant of CD4 + T cells compared with untreated cells (Fig. 2 b). In contrast, under anti-CD3/anti-CD28 Ab stimulation, HGR4113 dose-dependently promoted differentiation into regulatory T cells (Tregs) (Fig. 2 c). Treatment with HGR4113 downregulated IFN-γ and IL-17 mRNA expression under anti-CD3/anti-CD28 Ab stimulation and upregulated the levels of Foxp3 and IL-10 mRNA under anti-CD3/anti-CD28 Ab or Th17-polarizing conditions, respectively (Fig. 2 d). Given that STAT3 is a key transcription factor in Th17 cells, we investigated whether HGR4113 regulates STAT3 activity. Splenic CD4 + T cells from C57BL/6 mice were pretreated with HGR4113 for 2 h and then cultured for 12.5 h under anti-CD3/anti-CD28 Ab or IL-6 stimulation; levels of phosphorylated STAT3 (Tyr705) were measured. HGR4113 inhibited phosphorylation of STAT3 induced by each stimulus (Fig. 2 e). To assess the effects of HGR4113 on mitochondrial respiration, splenic CD4 + T cells were treated with HGR4113 and the OCR was measured. HGR4113 significantly reduced the increased oxidative phosphorylation (OXPHOS) activity induced by anti-CD3/anti-CD28 Ab stimulation (Fig. 2 f). These results indicate that HGR4113 suppresses Th17 cells by regulating STAT3 activity and OXPHOS in CD4 + T cells. HGR4113 increases the frequency of IL-10-producing regulatory B cells and decreases immunoglobulin production To determine whether HGR4113 affects B cell function, splenic CD19 + B cells isolated from C57BL/6 mice were treated with HGR4113 and cultured under LPS stimulation for 4 days. HGR4113 dose-dependently reduced the frequency of IL-17-producing B cells induced by LPS stimulation, while increasing the frequency of IL-10-producing regulatory B cells (B10 cells) reduced by LPS stimulation (Fig. 3 a). HGR4113 treatment also dose-dependently reduced immunoglobulin production under LPS stimulation or stimulation that activates B cell receptors (Fig. 3 b). Furthermore, HGR4113 downregulated the expression of BAFFR mRNA increased by LPS stimulation and attenuated the mRNA levels of AID and collagen 1 increased by BCR activation (Fig. 3 c). These results demonstrate that HGR4113 promotes the differentiation of murine splenic CD19 + B cells into regulatory B cells. HGR4113 attenuates the severity of SS in NOD/ShiLtJ mice To evaluate the role of HGR4113 in SS progression, 8-week-old female NOD/ShiLtJ mice were administered HGR4113 (100 mg/kg) or vehicle orally once daily for 12 weeks. With increasing age, the salivary flow rate decreased in the vehicle-treated group; this decrease was alleviated in the HGR4113-treated group (Fig. 4 a). Blood glucose levels were similar between the HGR4113- and vehicle-treated groups (Fig. 4 a). After 12 weeks of HGR4113 administration, serum IgG levels were significantly lower in HGR4113-treated mice compared with the vehicle-treated group (Fig. 4 b). In the HGR4113 group, salivary gland inflammation was reduced compared with controls (Fig. 4 c). To assess the effect of HGR4113 on inflammatory cell infiltration into the salivary glands, salivary gland sections were immunohistochemically stained for CD4, CD19, IL-6, and IL-17. In the HGR4113 group, infiltration of cells expressing CD4 or CD19, as well as cells expressing the inflammatory cytokines IL-6 or IL-17, was reduced in the salivary gland tissue (Fig. 4 c). These results demonstrate that HGR4113 alleviates the development of SS in NOD/ShiLtJ mice. HGR4113 decreases the frequency of ex vivo inflammatory cells and fibrosis in NOD/ShiLtJ mice To determine how HGR4113 treatment affects pathogenic cells in NOD/ShiLtJ mice, we analyzed the populations of T and B cell subsets in ex vivo splenocytes from HGR4113-treated NOD mice. HGR4113 treatment significantly reduced the frequencies of splenic Th17 cells and CD4 + ICOS + IFN-γ + Bcl6 + and CD4 + ICOS + IL-17 + Bcl6 + follicular helper T cells compared with the vehicle-treated group (Fig. 5 a). HGR4113 treatment also significantly reduced the frequencies of splenic IL-17-producing B cells, germinal center B cells, and plasma cells compared with the vehicle-treated group (Fig. 5 b). Increased fibrosis in the minor labial salivary glands of patients with primary SS is correlated with the focus score and leads to impaired function. 17 , 18 To investigate the effect of HGR4113 on fibrosis development, the extent of fibrosis was examined by staining the salivary glands of HGR4113-treated mice with Masson’s trichrome. HGR4113 treatment reduced collagen deposition in the salivary glands (Fig. 5 c). It also reduced the infiltration of cells positive for collagen type I and fibronectin—crucial components of the extracellular matrix—into salivary gland tissue compared with the vehicle-treated group (Fig. 5 c). Furthermore, HGR4113 treatment reduced the infiltration of cells positive for TGF-β, which plays a pivotal role in the pathogenesis of fibrosis, 19 into salivary gland tissue (Fig. 5 c). In contrast, HGR4113 treatment increased the infiltration of cells positive for aquaporin-5, a water channel predominantly located in the apical membrane of salivary gland acinar cells, 20 into salivary gland tissue (Fig. 5 c). These results demonstrate that HGR4113 can alleviate pathogenic cell activation and fibrosis in NOD/ShiLtJ mice. Salivary gland organoid generation in HGR4113-treated NOD/ShiLtJ mice Considering that HGR4113 improved fibrosis in salivary gland tissue and increased aquaporin-5 expression (Fig. 5 c), we isolated salivary epithelial cells from the salivary gland tissue of NOD/ShiLtJ mice administered HGR4113 and cultured them in differentiation medium to determine whether HGR4113 promoted salivary gland organoid generation. After 10 days of culture, we observed substantial acceleration of organoid development from salivary epithelial cells of NOD/ShiLtJ mice given HGR4113 compared with those given vehicle (Fig. 6 a). Salivary gland organoids should contain secretory acinar cells and surrounding myoepithelial cells, which play essential roles in proper saliva secretion and overall gland function. 21 , 22 Compared with controls, organoids developed from salivary epithelial cells of NOD/ShiLtJ mice administered HGR4113 retained KRT5 + basal and KRT7 + luminal duct cells (Fig. 6 b). Moreover, compared with the control, organoids derived from salivary epithelial cells of NOD/ShiLtJ mice administered HGR4113 expressed aquaporin-5 and E-cadherin more strongly, markers of acinar cells and epithelial cell adherens junctions, respectively (Fig. 6 c). Organoids developed from salivary epithelial cells of NOD/ShiLtJ mice administered HGR4113 also contained KRT14 + αSMA + myoepithelial cells (Fig. 6 d). These results demonstrate that HGR4113 promotes the generation of salivary gland organoids in SS-like NOD/ShiLtJ mice. Discussion In this study, we evaluated the effect of the synthetic glabridin derivative HGR4113 on modulating the severity of SS in SS-like NOD/ShiLtJ mice. HGR4113 reduced the production of inflammatory cytokines induced by TCR or LPS stimulation in splenocytes derived from NOD/ShiLtJ mice. In particular, it reduced the development of Th17 cells by downregulating STAT3 phosphorylation in CD4 + T cells and promoted the development of IL-10-producing regulatory B cells in CD19 + B cells. HGR4113 administration effectively alleviated the development of SS in NOD/ShiLtJ mice by reducing the frequency of inflammatory cells in the spleen, alleviating fibrosis, and increasing AQP5 expression in salivary gland tissue. HGR4113 treatment also promoted salivary gland organoid generation in vitro and in vivo . HGR4113 is a synthetic glabridin derivative with a similar skeletal structure; however, it substantially differs from glabridin in that it has a hydroxy-to-propoxy modification at the resorcinol ring at C-4 and double-bond hydrogenation in the pyranobenzene structure. 23 HGR4113 attenuates the LPS-mediated increase in mRNA levels of proinflammatory cytokines, NF-κB activation, and JNK phosphorylation in LPS-stimulated RAW264.7 macrophages. These structural differences result in improved chemical stability and metabolic characteristics compared with glabridin. Moreover, in LPS-stimulated RAW264.7 macrophages, HGR4113 downregulated LPS-mediated increases in mRNA levels of inflammatory cytokines, as well as NF-κB activation and JNK phosphorylation, similarly to or more effectively than glabridin. 23 Although HGR4113 is currently in phase I clinical trials (NCT05642377) for type 2 diabetes mellitus, there are very few reports regarding its mechanism of action and efficacy. Metabolic abnormalities have been reported to be closely related to the pathological development and exacerbation of SS. We have shown that butyric acid or Lactobacillus rhamnosus , which produces butyric acid, can directly increase salivary secretion and suppress autoimmune B cells and autoantibody production. 24 Serum leptin and IL-1β levels were elevated in SS patients with metabolic disorders, and disease severity was aggravated when a glucosamine–nitrosourea compound targeting pancreatic β cells was administered to NOD/ShiLtJ mice, a preclinical model of SS. 25 , 26 Relative to healthy individuals, patients with SS have altered mitochondrial DNA copy numbers and increased levels of factors associated with mitochondrial fission and transcription. 27 Furthermore, mitochondrial swelling and cristae disruption, as well as changes in mitochondrial-related metabolic pathways, have been observed in immune cells infiltrating the salivary glands of SS patients. 28 These findings suggest that mitochondrial dysfunction is a key factor in the pathophysiology of SS and that mitochondria-targeting therapies may represent a novel treatment strategy. In this study, we evaluated the therapeutic potential of HGR4113 in SS associated with metabolic abnormalities. 8 , 9 Phenotypic and functional changes observed during the proliferation and differentiation of B and T cells depend on dynamic mitochondrial metabolism. Resting B cells have low metabolic activity, whereas activation of B cells with BCR or LPS stimulation increases glucose uptake and glycolysis. 29 , 30 When T cells are activated through antigen recognition, they switch to a state of elevated OXPHOS and aerobic glycolysis to meet the high energy and biosynthetic demands required for rapid proliferation and effector activity. 31 , 32 Proinflammatory cytokines such as IL-6 and TNF-α activate glycolysis and anabolic processes to promote differentiation of effector T cells, including Th1, Th2, and Th17 cells, 33 whereas anti-inflammatory cytokines such as TGF-β promote oxidative metabolism and mitochondrial respiration to support the development of Tregs, 34 indicating that each T cell type has unique metabolic characteristics. HGR4113 effectively reduced the production of inflammatory cytokines and OXPHOS activity in CD4 + T cells by inhibiting STAT3 activity. Furthermore, HGR4113 reduced IgG production in CD19 + B cells and promoted their differentiation into regulatory B cells. These results demonstrate that HGR4113 not only alleviates inflammation but also modulates mitochondrial OXPHOS. Research aimed at developing and utilizing salivary gland organoids is gaining increasing attention. A recent study demonstrated that salivary gland organoids derived from patients with SS consistently activated IFN-related genes and that IFN signaling was reduced by treatment with a JAK inhibitor, suggesting that salivary gland organoids derived from SS patients can recapitulate pathological characteristics of the disease. 35 Inhibition of activin receptor-like kinase (Alk) signaling during salivary gland organoid generation has been shown to promote organoid production. 36 Furthermore, in radiation-induced salivary gland damage, YAP translocated to the nucleus of salivary gland cells and activated regeneration-related genes, thereby inducing the proliferation of ductal cells and promoting salivary gland regeneration. 37 We evaluated the efficacy of HGR4113 in salivary gland organoids from NOD mice. Salivary gland organoids derived from NOD mice administered HGR4113 regenerated more actively, with increased development of KRT5 + basal and KRT7 + luminal duct cells, aquaporin-5 + acinar cells, and KRT14 + αSMA + myoepithelial cells. These results suggest that HGR4113 promotes salivary gland cell regeneration. This study revealed that in a preclinical mouse model of SS, HGR4113 suppressed inflammation involving T and B cells, modulated mitochondrial metabolism, and promoted salivary gland organoid regeneration. These findings demonstrate the potential for developing disease-selective therapeutics based on the pathophysiology of SS. Declarations Competing Interests The authors declare that they have no conflict of interests. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00454685) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00208207). Author contributions All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Cho and Park had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. References Vitali C, Bombardieri S, Jonsson R, et al. Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis. 2002; 61:554–8. Beydon M, McCoy S, Nguyen Y, et al. Epidemiology of Sjögren syndrome. Nat Rev Rheumatol. 2024; 20:158–69. Risselada AP, Looije MF, Kruize AA, et al. 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Acta Histochem Cytochem. 2012; 45:251–9. Rocchi C, Barazzuol L, Coppes RP. The evolving definition of salivary gland stem cells. NPJ Regen Med. 2021; 6:4. Yoon YJ, Kim D, Tak KY, et al. Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glands. Nat Commun. 2022; 13:3291. Shin J, Choi LS, Jeon HJ, et al. Synthetic glabridin derivatives inhibit LPS-induced inflammation via MAPKs and NF-κB pathways in RAW264.7 macrophages. Molecules. 2023; 28. Kim DS, Woo JS, Min HK, et al. Short-chain fatty acid butyrate induces IL-10-producing B cells by regulating circadian-clock-related genes to ameliorate Sjögren’s syndrome. J Autoimmun. 2021; 119:102611. Augusto KL, Bonfa E, Pereira RM, et al. Metabolic syndrome in Sjögren’s syndrome patients: a relevant concern for clinical monitoring. Clin Rheumatol. 2016; 35:639–47. Hwang SH, Park JS, Yang S, et al. Metabolic abnormalities exacerbate Sjögren’s syndrome by and is associated with increased the population of interleukin-17-producing cells in NOD/ShiLtJ mice. J Transl Med. 2020; 18:186. De Benedittis G, Latini A, Colafrancesco S, et al. Alteration of mitochondrial DNA copy number and increased expression levels of mitochondrial dynamics-related genes in Sjögren’s syndrome. Biomedicines. 2022; 10. Luo D, Li L, Wu Y, et al. Mitochondria-related genes and metabolic profiles of innate and adaptive immune cells in primary Sjögren’s syndrome. Front Immunol. 2023; 14:1156774. Doughty CA, Bleiman BF, Wagner DJ, et al. Antigen receptor-mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth. Blood. 2006; 107:4458–65. Caro-Maldonado A, Wang R, Nichols AG, et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J Immunol. 2014; 192:3626–36. Chang CH, Curtis JD, Maggi LB, Jr., et al. Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell. 2013; 153:1239–51. Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013; 38:225–36. Saravia J, Chapman NM, Chi H. Helper T cell differentiation. Cell Mol Immunol. 2019; 16:634–43. Liu H, Chen YG. The interplay between TGF-beta signaling and cell metabolism. Front Cell Dev Biol. 2022; 10:846723. Meudec L, Goudarzi N, Silva-Saffar SE, et al. Development of salivary gland organoids derived from patient biopsies: a functional model of Sjögren’s disease. Ann Rheum Dis. 2025; 84:1195–206. Yoshimoto S, Yoshizumi J, Anzai H, et al. Inhibition of Alk signaling promotes the induction of human salivary-gland-derived organoids. Dis Model Mech. 2020; 13. Rocchi C, Cinat D, Serrano Martinez P, et al. The Hippo signaling pathway effector YAP promotes salivary gland regeneration after injury. Sci Signal. 2021; 14:eabk0599. Additional Declarations There is no conflict of interest Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 04 Jan, 2026 Review # 2 received at journal 01 Jan, 2026 Reviewer # 2 agreed at journal 01 Jan, 2026 Review # 1 received at journal 09 Dec, 2025 Reviewer # 1 agreed at journal 01 Dec, 2025 Reviewers invited by journal 27 Nov, 2025 Submission checks completed at journal 07 Sep, 2025 Editor assigned by journal 04 Sep, 2025 First submitted to journal 04 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7540230","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":551526003,"identity":"c9309937-8ada-48d4-b4da-b5f72c58ef23","order_by":0,"name":"Mi-La 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1","display":"","copyAsset":false,"role":"figure","size":151284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGR4113 treatment decreases the production of inflammatory cytokines in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Splenocytes from C57BL/6 mice were stimulated with HGR4113 (1, 5, or 10 μg/mL) or vehicle for 2 h and then cultured with or without anti-CD3 antibody (Ab) (0.5 μg/mL)/anti-CD28 Ab (1 μg/mL) or lipopolysaccharide (LPS; 100 ng/mL) for 3 days. After 3 days, splenocyte viability was determined by the CCK-8 assay. \u003cstrong\u003eb\u003c/strong\u003e Splenocytes from C57BL/6 or NOD/ShiLtJ mice were stimulated with HGR4113 (1, 5, or 10 μg/mL) or vehicle for 2 h and then cultured with anti-CD3 Ab (0.5 μg/mL)/anti-CD28 Ab (1 μg/mL) for 3 days. IFN-γ and IL-17 levels in culture supernatant were determined using ELISA. \u003cstrong\u003ec\u003c/strong\u003e Splenocytes from C57BL/6 or NOD/ShiLtJ mice were stimulated with HGR4113 (1, 5, or 10 μg/mL) or vehicle for 2 h and then cultured with LPS (100 ng/mL) for 3 days. TNF-α and IL-6 levels in culture supernatant were determined using ELISA. Data are presented as the mean ± SD of three independent experiments. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e. vehicle-treated control.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/7702b10ea6edef92fa87902b.png"},{"id":97262624,"identity":"0cff1498-c4c8-4a5e-9927-c1d80351e604","added_by":"auto","created_at":"2025-12-02 14:09:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGR4113 inhibits IL-17 production by suppressing STAT3 phosphorylation and altering the metabolic profile in splenic CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea–d\u003c/strong\u003e Splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells from C57BL/6 mice were stimulated with HGR4113 (1, 5, or 10 μg/mL) or vehicle for 2 h and then cultured with anti-CD3 antibody (Ab) (0.5 μg/mL)/anti-CD28 Ab (1 μg/mL) or under Th17-polarizing conditions (anti-CD3 Ab, anti-CD28 Ab, anti–IFN-γ Ab/anti–IL-4 Ab, each at 5 μg/mL; TGF-β, 2 μg/mL; IL-6, 20 ng/mL) for 3 days. The frequencies of CD4\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e (Th1), CD4\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e (Th17) (\u003cstrong\u003ea\u003c/strong\u003e), and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e (Treg) cells (\u003cstrong\u003ec\u003c/strong\u003e) were assessed by flow cytometry. IFN-γ and IL-17 levels in culture supernatant were determined using ELISA (\u003cstrong\u003eb\u003c/strong\u003e). \u003cem\u003eIFN-γ\u003c/em\u003e, \u003cem\u003eIL-17\u003c/em\u003e, \u003cem\u003eFoxp3\u003c/em\u003e, and \u003cem\u003eIL-10\u003c/em\u003e mRNA expression was determined using real-time PCR (\u003cstrong\u003ed\u003c/strong\u003e). \u003cstrong\u003ee\u003c/strong\u003e Splenic CD4\u003csup\u003e+\u003c/sup\u003e cells from C57BL/6 mice were stimulated with HGR4113 (10 μg/mL) or vehicle for 2 h and then cultured with or without IL-6 (20 ng/mL) for 30 min or anti-CD3 Ab (0.5 μg/mL)/anti-CD28 Ab (1 μg/mL) for 12 h. The levels of p-STAT3 (Tyr705) and STAT3 were measured by immunoblotting, respectively. The graphs are representative of three independent experiments. \u003cstrong\u003ef\u003c/strong\u003e Splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells from C57BL/6 mice (n = 4) were stimulated with HGR4113 (10 μg/mL) or vehicle for 2 h and then cultured with anti-CD3 Ab/anti-CD28 Ab for 12 h. OCR changes during treatment with oligomycin (10 μM), FCCP (3 μM), and rotenone plus antimycin A (10 μM) were assessed by extracellular flux analysis. Data are presented as the mean ± SD of three independent experiments. *\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001 \u003cem\u003evs\u003c/em\u003e. vehicle-treated control.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/3153456f3bf0b8172cafebf3.png"},{"id":97262611,"identity":"7a6bd77b-e57b-449c-bfba-a9f7fd8e4f1c","added_by":"auto","created_at":"2025-12-02 14:09:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGR4113 inhibits immunoglobulin production in splenic CD19\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e B cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea–c\u003c/strong\u003e Splenic CD19\u003csup\u003e+\u003c/sup\u003e B cells from C57BL/6 mice were stimulated with HGR4113 (1, 5, or 10 μg/mL) or vehicle for 2 h and then cultured with LPS (100 ng/mL) or anti-IgM antibody (10 μg/mL), IL-4 (20 ng/mL), and soluble CD40L (1 μg/mL) for 4 days. The frequencies of CD19\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e cells and B10 cells (CD19\u003csup\u003e+\u003c/sup\u003eCD5\u003csup\u003e+\u003c/sup\u003eCD1d\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e) were assessed by flow cytometry (\u003cstrong\u003ea\u003c/strong\u003e). Immunoglobulin levels in culture supernatant were determined using ELISA (\u003cstrong\u003eb\u003c/strong\u003e). \u003cem\u003eBAFF\u003c/em\u003e receptor, \u003cem\u003eAID\u003c/em\u003e, and \u003cem\u003eCol1\u003c/em\u003e mRNA expression was determined using real-time PCR (\u003cstrong\u003ec\u003c/strong\u003e). Data are presented as the mean ± SD of three independent experiments. *\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ****\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001 \u003cem\u003evs\u003c/em\u003e. vehicle-treated control.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/3d24f1efd044d4c00efed073.png"},{"id":97368315,"identity":"2c1d3ffd-e6e6-4282-ae75-16da891cbf80","added_by":"auto","created_at":"2025-12-03 16:22:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1561934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGR4113 attenuates the severity of SS in NOD/ShiLtJ mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old female NOD/ShiLtJ mice were orally administered vehicle (n = 10) or HGR4113 (100 mg/kg; n = 10) daily for 12 weeks. \u003cstrong\u003ea\u003c/strong\u003e Salivary flow rate (left) and blood glucose level (right) were measured at weeks 8, 11, 12, 14, 17, and 20 after HGR4113 administration. \u003cstrong\u003eb\u003c/strong\u003eTotal IgG in serum collected from 20-week-old mice was measured by ELISA. \u003cstrong\u003ec\u003c/strong\u003eSalivary gland sections from 20-week-old mice were stained with hematoxylin and eosin, CD4, CD19, IL-6, and IL-17. Representative histological features are shown. The graph shows the histological grade. Data are presented as the mean ± SD of three independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e. vehicle-treated control.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/f5ee03246f7a7e73e9408250.png"},{"id":97262609,"identity":"3d1d302a-80ce-47ad-9c98-7e496f5d6e94","added_by":"auto","created_at":"2025-12-02 14:09:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":993942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGR4113 treatment decreases the frequency of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inflammatory cells and fibrosis in NOD/ShiLtJ mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old female NOD/ShiLtJ mice were given vehicle (n = 10) or HGR4113 (100 mg/kg; n = 10) orally daily for 12 weeks. \u003cstrong\u003ea, b\u003c/strong\u003e \u003cem\u003eEx vivo\u003c/em\u003e splenocytes from 20-week-old NOD mice were cultured with PMA, ionomycin, and GolgiStop for 4 h and then immunostained with antibodies against CD4\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003eICOS\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eBcl6\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003eICOS\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003eBcl6\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003ea\u003c/strong\u003e), CD19\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e, B220\u003csup\u003e+\u003c/sup\u003eGL-7\u003csup\u003e+\u003c/sup\u003eFas\u003csup\u003e+\u003c/sup\u003e, and B220⁻CD138\u003csup\u003e+\u003c/sup\u003e cells (\u003cstrong\u003eb\u003c/strong\u003e). The cells were analyzed by flow cytometry. \u003cstrong\u003ec\u003c/strong\u003e Salivary gland sections from 20-week-old mice were stained with Masson’s trichrome, collagen I, fibronectin, TGF-β, and aquaporin-5. Representative histological features are shown, and the graph displays the histological grade. Data are presented as the mean ± SD.\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/5048ec87c99d92a456492765.png"},{"id":97367930,"identity":"a4532001-910c-419a-90de-d00bf13d2201","added_by":"auto","created_at":"2025-12-03 16:21:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":402551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSalivary gland organoid generation in HGR4113-treated NOD/ShiLtJ mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old female NOD/ShiLtJ mice were orally administered vehicle (n = 10) or HGR4113 (100 mg/kg; n = 10) daily for 12 weeks. Salivary epithelial cells isolated from the submandibular glands of 20-week-old NOD mice were cultured for 10 days. \u003cstrong\u003ea\u003c/strong\u003e Bright-field images of salivary gland organoids. \u003cstrong\u003eb\u003c/strong\u003e Representative immunofluorescence images of basal cells (KRT5; green) and luminal cells (KRT7; red) in organoids. \u003cstrong\u003ec\u003c/strong\u003eRepresentative immunofluorescence images of epithelial cell adherens junctions (E-cadherin; green) and acinar cells (AQP5; red) in organoids. \u003cstrong\u003ed\u003c/strong\u003eRepresentative immunofluorescence images of myoepithelial cells (α-SMA; green; cytokeratin-14; red) in organoids.\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/b6ed8184dd3f2ecd5d3f19ca.png"},{"id":97372666,"identity":"734c8eb6-63a1-405b-a3f8-ac1b2682ce3d","added_by":"auto","created_at":"2025-12-03 16:32:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5545851,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540230/v1/88c1e5ac-67f0-4dd6-ba75-838d7da8921e.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Therapeutic Effects of a Synthetic Glabridin Derivative on Th17/ B cell Immune Regulation and Salivary gland Regeneration in Experimental Sjögren’s Syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSj\u0026ouml;gren\u0026rsquo;s syndrome (SS) is a chronic autoimmune disease in which inflammatory cells infiltrate the exocrine glands, reducing glandular secretory function and ultimately resulting in keratoconjunctivitis sicca (dry eyes) and xerostomia (oral dryness). SS is often accompanied by extraglandular manifestations involving the skin, muscles, kidneys, joints, peripheral nervous system, and blood vessels.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e In SS, mortality is increased in patients with extraglandular manifestations and non-Hodgkin lymphoma.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe pathogenesis of SS is still under investigation, but lymphocyte infiltration into target organs, including exocrine glands such as the salivary and lacrimal glands, is considered a key mechanism of disease development.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Aggregates of B and T cells are observed in ectopic lymphoid structures in the salivary and lacrimal glands, and B cells that receive signals from T cells become hyperactivated, resulting in hypergammaglobulinemia, germinal center (GC) formation, and production of anti-SSA/Ro and anti-SSB/La autoantibodies.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Metabolic imbalance can alter immune cell phenotypes and immune responses, potentially leading to autoimmunity or malignant transformation.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Recently, metabolic abnormalities have been reported to play a role in the pathogenesis of SS. Ultrastructural alterations of mitochondria have been observed in the salivary epithelial cells of patients with SS,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and oxidative stress levels are increased in their saliva.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eGlabridin, a polyphenolic isoflavan isolated from the root of \u003cem\u003eGlycyrrhiza glabra\u003c/em\u003e L. (Fabaceae), commonly known as licorice, is a widely used herb-derived natural compound with anti-inflammatory, antioxidant, anticancer, and antiatherogenic effects, as well as the ability to regulate energy metabolism.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e However, glabridin has poor stability in formulations and low bioavailability, limiting its application as a commercial clinical therapeutic agent.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e A recent structure\u0026ndash;activity relationship study identified a novel structural analog of glabridin, HGR4113 ((R)-2-(8,8-dimethyl-2,3,4,8,9,10-hexahydropyrano[2,3-f]chromen-3-yl)-5-propoxyphenol), with improved chemical stability and metabolic characteristics relative to glabridin.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn the present study, we evaluated the ability of HGR4113 to modulate inflammatory responses in murine splenic T and B cells and its therapeutic efficacy in NOD/ShiLtJ mice, a preclinical model of SS. We also investigated the role of HGR4113 in salivary gland organoid generation in NOD/ShiLtJ mice \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eSeven-week-old female NOD/ShiLtJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and cage of animals maintained under specific pathogen-free conditions at the Institute of Medical Science, Catholic University of Korea. The mice were fed standard mouse chow and water. All procedures were approved by the Animal Research Ethics Committee of the Catholic University of Korea and conformed to the United States National Institutes of Health guidelines (permit number: CUMS-2024-0267-02). All animals were treated and euthanized in accordance with the Guidelines on the Use and Care of Animals of the Catholic University of Korea. Surgery was performed under isoflurane anesthesia, and every effort was made to minimize suffering. At the end of the study, the mice were euthanized in a CO\u003csub\u003e2\u003c/sub\u003e chamber for sample collection.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTreatment with HGR4113 and measurement of saliva secretion in NOD/ShiLtJ mice\u003c/h3\u003e\n\u003cp\u003eSaliva secretion was stimulated in anesthetized mice by intraperitoneal injection of pilocarpine (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 5 mg/kg body weight. Saliva was collected from the oral cavity for 7 min, beginning 90 s after the injection, using a micropipette. The volume of saliva was determined gravimetrically (\u0026micro;L/g/min). Before drug injection, salivary flow rates were measured in mice, and groups were randomized based on the average salivary flow rate of all mice. HGR4113 was dissolved in dimethyl sulfoxide. Seven-week-old female NOD/ShiLtJ mice were administered 100 mg/kg of HGR4113 or vehicle orally once daily for 12 weeks (n\u0026thinsp;=\u0026thinsp;10/group). Sample size was determined for statistical significance and experimental feasibility. To minimize potential confounders such as the order of treatments and measurements, we individually marked each mouse with an ear punch. Blood glucose levels were measured using an Accu-Check\u0026trade; Compact Glucometer (Roche Diagnostics, Indianapolis, IN, USA). Individuals with blood glucose levels of 600 mg/dL were excluded from evaluation due to the development of diabetes. Samples were stored at \u0026minus;\u0026thinsp;70\u0026deg;C until analysis.\u003c/p\u003e\n\u003ch3\u003eHistopathological examination and immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eSalivary glands were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer-thick sections were stained with H\u0026amp;E, and the degree of inflammation was quantified as previously described.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Sections were incubated with primary antibodies against CD4 (1:50, Abcam, #ab25475), CD19 (1:200, Abcam, #ab203615), IL-6 (1:300, Novus, #NB600-1131), IL-17 (1:200, Abcam, #ab79056), type I collagen (Col-I) (1:100, Invitrogen, #PA5-89281), fibronectin (1:200, Abcam, #ab2413), TGF-β (1:100, Bioss, #BS-0086R), and aquaporin-5 (AQP5) (1:100, Novus, #NBP2-92926) for 2 h at room temperature. Sections were then incubated with a horseradish peroxidase\u0026ndash;conjugated secondary antibody for 30 min. The final products were visualized using the chromogen diaminobenzidine. Immunostained sections were examined by photomicroscopy (Olympus). Positive cells were counted in high-power digital images (400\u0026times; magnification) using Adobe Photoshop (Adobe, San Jose, CA, USA). Counts were performed visually by three individuals, and mean values were calculated.\u003c/p\u003e\n\u003ch3\u003eIsolation and stimulation of splenocytes\u003c/h3\u003e\n\u003cp\u003eMurine splenocytes were prepared as previously described.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Spleens were dissociated using sterilized glass slides with frosted ends, and red blood cells were lysed in hypotonic ACK buffer (0.15 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 1 mM KCO\u003csub\u003e3\u003c/sub\u003e, and 0.1 mM ethylenediaminetetraacetic acid, pH 7.4). The remaining splenocytes were filtered through a 40-\u0026micro;m cell strainer (Falcon, Durham, NC, USA) and maintained in RPMI 1640 medium containing 5% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). Splenic CD4\u003csup\u003e+\u003c/sup\u003e T or CD19\u003csup\u003e+\u003c/sup\u003e B cells were purified using a positive cell isolation kit according to the manufacturer\u0026rsquo;s instructions (Miltenyi Biotec). The cells were pretreated with HGR4113 for 2 h. Splenocytes or isolated T cells were cultured with 0.5 \u0026micro;g/mL anti-CD3 (BD Pharmingen, #553057) and 1 \u0026micro;g/mL anti-CD28 (BD Pharmingen, #553294) antibodies, or under Th17-polarizing conditions with anti-CD3, anti-CD28, 20 ng/mL IL-6 (R\u0026amp;D Systems, #406-ML), 2 ng/mL transforming growth factor-β (TGF-β) (PeproTech, #100-21C), 5 \u0026micro;g/mL anti\u0026ndash;IFN-γ antibody (R\u0026amp;D Systems, #MAB485), and 5 \u0026micro;g/mL anti\u0026ndash;IL-4 antibody (R\u0026amp;D Systems, #MAB404) for 72 h. Splenocytes or isolated B cells were cultured with 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich, #L4391) or 10 \u0026micro;g/mL IgM antibody (Jackson ImmunoResearch Labs), 20 ng/mL recombinant mouse IL-4 protein (R\u0026amp;D Systems), or 1 \u0026micro;g/mL recombinant murine sCD40 ligand (PeproTech) for 72 h.\u003c/p\u003e\n\u003ch3\u003eSeahorse metabolic assay\u003c/h3\u003e\n\u003cp\u003eThe oxygen consumption rate (OCR) was measured using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer\u0026rsquo;s instructions. Briefly, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e purified splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells were cultured in RPMI 1640 medium with 5% FBS in the presence or absence of HGR4113 under anti-CD3 and anti-CD28 stimulation for 12 h. The washed cells, resuspended in XF medium containing 1\u0026times; ITSA (Gibco), were seeded on Cell-Tak\u0026ndash;coated (BD Biosciences) Seahorse cell culture plates and incubated in a 37\u0026deg;C incubator without CO\u003csub\u003e2\u003c/sub\u003e for 30 min. Real-time changes in OCR (pmol O\u003csub\u003e2\u003c/sub\u003e/min) were measured after treatment with oligomycin (complex V inhibitor, 7 \u0026micro;M), FCCP (3 \u0026micro;M), and rotenone plus antimycin A (electron transport chain inhibitors, 10 \u0026micro;M).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eReal-time polymerase chain reaction (PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using TRI Reagent (Molecular Research Center), and cDNA was synthesized with the Dyne First-Strand cDNA Synthesis Kit (Dyne Bio) according to the manufacturer\u0026rsquo;s protocol. Gene expression was measured using the StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Premix (Bioline Inc.). The following primers were used: \u003cem\u003eIfng\u003c/em\u003e, 5\u0026prime;-AGG AAC TGG CAA AAG GAT GGT-3\u0026prime; and 5\u0026prime;-ATG TTG TTG CTG ATG GCC TG-3\u0026prime;; \u003cem\u003eIL-17\u003c/em\u003e, 5\u0026prime;-CCT CAA AGC TCA GCG TGT CC-3\u0026prime; (sense) and 5\u0026prime;-GAG CTC ACT TTT GCG CCA AG-3\u0026prime; (antisense); \u003cem\u003eFoxp3\u003c/em\u003e, 5\u0026prime;-CAC TGC CCC TAG TCA TGG T-3\u0026prime; (sense) and 5\u0026prime;-GGA GGA GTG CCT GTA AGT GG-3\u0026prime; (antisense); \u003cem\u003eIL-10\u003c/em\u003e, 5\u0026prime;-GGC CCA GAA ATC AAG GAG CA-3\u0026prime; (sense) and 5\u0026prime;-AGA AAT CGA TGA CAG CGC CT-3\u0026prime; (antisense); \u003cem\u003eBAFFR\u003c/em\u003e, 5\u0026prime;-GGT GGG TCT AGT GAG TCT GGT-3\u0026prime; (sense) and 5\u0026prime;-TTC TGA GGA GGG TAC AAA GAC AT-3\u0026prime; (antisense); activation-induced cytidine deaminase (\u003cem\u003eAID\u003c/em\u003e), 5\u0026prime;-GCC ACC TTC GCA ACA AGT CT-3\u0026prime; (sense) and 5\u0026prime;-CCG GGC ACA GTC ATA GCA C-3\u0026prime; (antisense); \u003cem\u003eCol1a1\u003c/em\u003e, 5\u0026prime;-GCC AAG AAG ACA TCC CTG AAG-3\u0026prime; (sense) and 5\u0026prime;-TGT GGC AGA TAC AGA TCA AGC-3\u0026prime; (antisense); and \u003cem\u003eβ-actin\u003c/em\u003e, 5\u0026prime;-GTA CGA CCA GAG GCA TAC AGG-3\u0026prime; (sense) and 5\u0026prime;-GAT GAC GAT ATC GCT GCG CTG-3\u0026prime; (antisense). mRNA levels were normalized to those of \u003cem\u003eβ-actin\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunoblot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eImmunoblot analysis\u003c/div\u003e\u003cp\u003eCells were lysed in Halt protein lysis buffer containing Halt phosphatase inhibitor (Thermo Pierce). Lysates were centrifuged at 14,000 \u0026times; g for 15 min at 4\u0026deg;C. Protein concentration was determined using the Bradford protein assay (Bio-Rad). Protein samples were separated by 10% sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis and transferred to Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Membranes were incubated with antibodies against phosphorylated STAT3 (Y705, Abcam, #ab76315), STAT3 (Abcam, #ab68153), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam, #ab181602). Hybridized bands were detected using an ECL detection kit (Pierce) and Hyperfilm (Agfa). Western blot analysis was performed using the SNAP i.d. Protein Detection System (Millipore). Protein levels were normalized to β-actin or GAPDH as loading controls, and the relative level of the protein of interest under the nil condition was set to 1.\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eTumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-17, and IL-6 levels in culture supernatants were determined by sandwich ELISA (DuoSet; R\u0026amp;D Systems, Lille, France). Blood was collected from the orbital sinuses of mice, and serum samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. Serum IgG concentration was measured using the ELISA Quantitation Set (Bethyl Laboratories) according to the manufacturer\u0026rsquo;s protocol. Streptavidin-conjugated horseradish peroxidase was used for color development. Absorbance at 450 nm was measured using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIntracellular staining and flow cytometry\u003c/h2\u003e\u003cp\u003eTo stain surface markers, single-cell suspensions were washed with fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS] with 2% FBS) and incubated with fluorochrome-labeled antibodies for 30 min at 4\u0026deg;C. For intracellular staining, single-cell suspensions were cultured with 25 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) with added GolgiStop (BD Biosciences) for 4 h. After surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm according to the manufacturer\u0026rsquo;s instructions (BD Biosciences). For intracellular Foxp3 and Bcl-6 staining, a Foxp3/Transcription Factor Staining Buffer Kit (eBioscience) was used following surface staining. After washing with Perm/Wash buffer, antibodies for intracellular staining were added for 30 min at 4\u0026deg;C. The following anti-mouse antibodies were used for FACS: Alexa Fluor\u0026reg; 700 anti-CD4 (RM4-5), PE-Cy7 anti-CD19 (1D3), PerCP anti-CD45R/B220 (RA3-6B2), PE anti-CD138 (281-2), and FITC anti-GL-7 (GL7) from BD Biosciences; PerCP-Cy5.5 anti-CD4 (RM4-5), eFluor 450 anti-CD4 (RM4-5), FITC anti-CD5 (53\u0026thinsp;\u0026minus;\u0026thinsp;7.3), PE anti-CD1d (1B1), PE anti-Fas (15A7), PerCP-eFluor 710 anti-CXCR5 (SPRCL5), PE-Cyanine7 anti-ICOS (7E.17G9), APC anti-Bcl-6 (BCL-DWN), PE anti-Foxp3 (FJK-16s), PE anti\u0026ndash;IFN-γ (XMG1.2), PE anti\u0026ndash;IL-17 (eBio17B7), and APC anti\u0026ndash;IL-10 (JES5-16E3) from Invitrogen; and APC anti-CD25 (PC61) from BioLegend. Stained cells were analyzed on a FACSCalibur (BD Biosciences), CytoFLEX (Beckman Coulter), or LSRII (BD Biosciences). Events were collected and analyzed with FlowJo software (Tree Star).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMouse salivary gland organoid primary culture\u003c/h2\u003e\u003cp\u003eSpheroid-forming mouse salivary gland stem cells were isolated from the submandibular gland. The thin fascia covering the submandibular gland was carefully removed to obtain the tissue. The tissue was washed three times with PBS (Gibco) containing 3% penicillin\u0026ndash;streptomycin and minced into small pieces, which were placed in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Gibco) containing 1 mg/mL collagenase IV (Gibco) and incubated for 30 min at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The tissue was then centrifuged, and the cell pellet was resuspended in DMEM and filtered through a 40-\u0026micro;m cell strainer (BD) to obtain single cells. After centrifugation, the cells were cultured in DMEM/F12 (1:1, v/v; Gibco) supplemented with 20 ng/mL epidermal growth factor (PeproTech), 20 ng/mL fibroblast growth factor-2 (PeproTech), 1% N2 supplement (Gibco), 1% insulin\u0026ndash;transferrin\u0026ndash;selenium (Gibco), 1 \u0026micro;M dexamethasone (Sigma), and 1% antibiotic\u0026ndash;antimycotic (GenDEPOT). Salivary gland stem cells were seeded on Matrigel\u0026trade; (Corning) and cultured for 10 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eSalivary gland organoids were snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;70\u0026deg;C. Six-micrometer-thick cryosections were fixed in acetone for 10 min and dried at room temperature. The slides were hydrated in Tris buffer and blocked with 10% goat serum for 30 min at room temperature. They were then incubated overnight at 4\u0026deg;C with the appropriate antibodies diluted in Tris buffer. Sections were mounted with fluorescence mounting medium (Dako). The following anti-mouse antibodies were used for staining: keratin (KRT) 5 (1:100, Abcam, #ab52635), KRT7 (1:200, Invitrogen, #MA1-06315), α-SMA (1:800, Abcam, #ab7817), KRT14 (1:600, Abcam, #ab181595), E-cadherin (1:1000, Abcam, #ab231303), and AQP5 (1:100, Novus, #NBP2-92926). Nuclei were stained with DAPI (D35271; Invitrogen) in PBS. All stained sections were imaged using a confocal laser scanning microscope (LSM700).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 5 for Windows; GraphPad Software, San Diego, CA, USA). Normally distributed continuous data were analyzed using the parametric Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Differences in mean values among groups were analyzed by analysis of variance. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD). \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (two-tailed) were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eHGR4113 reduces the production of inflammatory cytokines in splenocytes from SS-like NOD/ShiLtJ mice\u003c/h2\u003e\u003cp\u003eTo determine the effects of HGR4113 on cell proliferation \u003cem\u003ein vitro\u003c/em\u003e, splenocytes from C57BL/6 mice were pretreated with HGR4113 for 2 h and then cultured with or without anti-CD3 antibody (Ab)/anti-CD28 Ab or LPS for 3 days. HGR4113 treatment did not affect the viability of splenocytes that were not stimulated with anti-CD3 Ab/anti-CD28 Ab or LPS. However, HGR4113 treatment dose-dependently decreased the viability of splenocytes stimulated with anti-CD3 Ab/anti-CD28 Ab or LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To assess whether HGR4113 exerts a regulatory effect on SS-derived pathogenic immune cells, splenocytes from NOD mice exhibiting SS-like symptoms were stimulated with HGR4113 in the presence of anti-CD3 Ab/anti-CD28 Ab or LPS for 3 days. HGR4113 treatment effectively reduced the amount of IFN-γ that was increased by anti-CD3 Ab and anti-CD28 Ab stimulation. Compared with normal C57BL/6 mice, production of IL-17 after anti-CD3 Ab and anti-CD28 Ab stimulation in splenocytes of NOD mice was significantly increased, and HGR4113 significantly reduced IL-17 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Additionally, treatment with HGR4113 effectively reduced the amount of TNF-α that was increased by LPS stimulation. Compared with normal C57BL/6 mice, LPS stimulation significantly increased the production of IL-6 in splenocytes of NOD mice, and HGR4113 treatment significantly reduced IL-6 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results demonstrate that HGR4113 can modulate the production of inflammatory factors in immune cells in a preclinical model of SS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHGR4113 inhibits IL-17 production by regulating STAT3 activity and the metabolic profile in splenic CD4\u0026thinsp;+\u0026thinsp;T cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine whether HGR4113 affects cytokine secretion in T cells, splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from C57BL/6 mice were treated with HGR4113 and cultured under anti-CD3 Ab and anti-CD28 Ab stimulation or under Th17-polarizing conditions for 3 days. HGR4113 dose-dependently reduced the frequency of Th17 cells under Th17-polarizing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Treatment with HGR4113 also dose-dependently suppressed the amounts of IFN-γ and IL-17 in the culture supernatant of CD4\u003csup\u003e+\u003c/sup\u003e T cells compared with untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, under anti-CD3/anti-CD28 Ab stimulation, HGR4113 dose-dependently promoted differentiation into regulatory T cells (Tregs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Treatment with HGR4113 downregulated \u003cem\u003eIFN-γ\u003c/em\u003e and \u003cem\u003eIL-17\u003c/em\u003e mRNA expression under anti-CD3/anti-CD28 Ab stimulation and upregulated the levels of \u003cem\u003eFoxp3\u003c/em\u003e and \u003cem\u003eIL-10\u003c/em\u003e mRNA under anti-CD3/anti-CD28 Ab or Th17-polarizing conditions, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Given that STAT3 is a key transcription factor in Th17 cells, we investigated whether HGR4113 regulates STAT3 activity. Splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells from C57BL/6 mice were pretreated with HGR4113 for 2 h and then cultured for 12.5 h under anti-CD3/anti-CD28 Ab or IL-6 stimulation; levels of phosphorylated STAT3 (Tyr705) were measured. HGR4113 inhibited phosphorylation of STAT3 induced by each stimulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). To assess the effects of HGR4113 on mitochondrial respiration, splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells were treated with HGR4113 and the OCR was measured. HGR4113 significantly reduced the increased oxidative phosphorylation (OXPHOS) activity induced by anti-CD3/anti-CD28 Ab stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). These results indicate that HGR4113 suppresses Th17 cells by regulating STAT3 activity and OXPHOS in CD4\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eHGR4113 increases the frequency of IL-10-producing regulatory B cells and decreases immunoglobulin production\u003c/h2\u003e\u003cp\u003eTo determine whether HGR4113 affects B cell function, splenic CD19\u003csup\u003e+\u003c/sup\u003e B cells isolated from C57BL/6 mice were treated with HGR4113 and cultured under LPS stimulation for 4 days. HGR4113 dose-dependently reduced the frequency of IL-17-producing B cells induced by LPS stimulation, while increasing the frequency of IL-10-producing regulatory B cells (B10 cells) reduced by LPS stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). HGR4113 treatment also dose-dependently reduced immunoglobulin production under LPS stimulation or stimulation that activates B cell receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Furthermore, HGR4113 downregulated the expression of \u003cem\u003eBAFFR\u003c/em\u003e mRNA increased by LPS stimulation and attenuated the mRNA levels of \u003cem\u003eAID\u003c/em\u003e and \u003cem\u003ecollagen 1\u003c/em\u003e increased by BCR activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These results demonstrate that HGR4113 promotes the differentiation of murine splenic CD19\u003csup\u003e+\u003c/sup\u003e B cells into regulatory B cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eHGR4113 attenuates the severity of SS in NOD/ShiLtJ mice\u003c/h2\u003e\u003cp\u003eTo evaluate the role of HGR4113 in SS progression, 8-week-old female NOD/ShiLtJ mice were administered HGR4113 (100 mg/kg) or vehicle orally once daily for 12 weeks. With increasing age, the salivary flow rate decreased in the vehicle-treated group; this decrease was alleviated in the HGR4113-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Blood glucose levels were similar between the HGR4113- and vehicle-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After 12 weeks of HGR4113 administration, serum IgG levels were significantly lower in HGR4113-treated mice compared with the vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In the HGR4113 group, salivary gland inflammation was reduced compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). To assess the effect of HGR4113 on inflammatory cell infiltration into the salivary glands, salivary gland sections were immunohistochemically stained for CD4, CD19, IL-6, and IL-17. In the HGR4113 group, infiltration of cells expressing CD4 or CD19, as well as cells expressing the inflammatory cytokines IL-6 or IL-17, was reduced in the salivary gland tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results demonstrate that HGR4113 alleviates the development of SS in NOD/ShiLtJ mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHGR4113 decreases the frequency of\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003einflammatory cells and fibrosis in NOD/ShiLtJ mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine how HGR4113 treatment affects pathogenic cells in NOD/ShiLtJ mice, we analyzed the populations of T and B cell subsets in \u003cem\u003eex vivo\u003c/em\u003e splenocytes from HGR4113-treated NOD mice. HGR4113 treatment significantly reduced the frequencies of splenic Th17 cells and CD4\u003csup\u003e+\u003c/sup\u003eICOS\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eBcl6\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003eICOS\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003eBcl6\u003csup\u003e+\u003c/sup\u003e follicular helper T cells compared with the vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). HGR4113 treatment also significantly reduced the frequencies of splenic IL-17-producing B cells, germinal center B cells, and plasma cells compared with the vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Increased fibrosis in the minor labial salivary glands of patients with primary SS is correlated with the focus score and leads to impaired function.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e To investigate the effect of HGR4113 on fibrosis development, the extent of fibrosis was examined by staining the salivary glands of HGR4113-treated mice with Masson\u0026rsquo;s trichrome. HGR4113 treatment reduced collagen deposition in the salivary glands (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). It also reduced the infiltration of cells positive for collagen type I and fibronectin\u0026mdash;crucial components of the extracellular matrix\u0026mdash;into salivary gland tissue compared with the vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Furthermore, HGR4113 treatment reduced the infiltration of cells positive for TGF-β, which plays a pivotal role in the pathogenesis of fibrosis,\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e into salivary gland tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In contrast, HGR4113 treatment increased the infiltration of cells positive for aquaporin-5, a water channel predominantly located in the apical membrane of salivary gland acinar cells,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e into salivary gland tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These results demonstrate that HGR4113 can alleviate pathogenic cell activation and fibrosis in NOD/ShiLtJ mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSalivary gland organoid generation in HGR4113-treated NOD/ShiLtJ mice\u003c/h2\u003e\u003cp\u003eConsidering that HGR4113 improved fibrosis in salivary gland tissue and increased aquaporin-5 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), we isolated salivary epithelial cells from the salivary gland tissue of NOD/ShiLtJ mice administered HGR4113 and cultured them in differentiation medium to determine whether HGR4113 promoted salivary gland organoid generation. After 10 days of culture, we observed substantial acceleration of organoid development from salivary epithelial cells of NOD/ShiLtJ mice given HGR4113 compared with those given vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Salivary gland organoids should contain secretory acinar cells and surrounding myoepithelial cells, which play essential roles in proper saliva secretion and overall gland function.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Compared with controls, organoids developed from salivary epithelial cells of NOD/ShiLtJ mice administered HGR4113 retained KRT5\u003csup\u003e+\u003c/sup\u003e basal and KRT7\u003csup\u003e+\u003c/sup\u003e luminal duct cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Moreover, compared with the control, organoids derived from salivary epithelial cells of NOD/ShiLtJ mice administered HGR4113 expressed aquaporin-5 and E-cadherin more strongly, markers of acinar cells and epithelial cell adherens junctions, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Organoids developed from salivary epithelial cells of NOD/ShiLtJ mice administered HGR4113 also contained KRT14\u003csup\u003e+\u003c/sup\u003eαSMA\u003csup\u003e+\u003c/sup\u003e myoepithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). These results demonstrate that HGR4113 promotes the generation of salivary gland organoids in SS-like NOD/ShiLtJ mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the effect of the synthetic glabridin derivative HGR4113 on modulating the severity of SS in SS-like NOD/ShiLtJ mice. HGR4113 reduced the production of inflammatory cytokines induced by TCR or LPS stimulation in splenocytes derived from NOD/ShiLtJ mice. In particular, it reduced the development of Th17 cells by downregulating STAT3 phosphorylation in CD4\u003csup\u003e+\u003c/sup\u003e T cells and promoted the development of IL-10-producing regulatory B cells in CD19\u003csup\u003e+\u003c/sup\u003e B cells. HGR4113 administration effectively alleviated the development of SS in NOD/ShiLtJ mice by reducing the frequency of inflammatory cells in the spleen, alleviating fibrosis, and increasing AQP5 expression in salivary gland tissue. HGR4113 treatment also promoted salivary gland organoid generation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eHGR4113 is a synthetic glabridin derivative with a similar skeletal structure; however, it substantially differs from glabridin in that it has a hydroxy-to-propoxy modification at the resorcinol ring at C-4 and double-bond hydrogenation in the pyranobenzene structure.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e HGR4113 attenuates the LPS-mediated increase in mRNA levels of proinflammatory cytokines, NF-κB activation, and JNK phosphorylation in LPS-stimulated RAW264.7 macrophages. These structural differences result in improved chemical stability and metabolic characteristics compared with glabridin. Moreover, in LPS-stimulated RAW264.7 macrophages, HGR4113 downregulated LPS-mediated increases in mRNA levels of inflammatory cytokines, as well as NF-κB activation and JNK phosphorylation, similarly to or more effectively than glabridin.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Although HGR4113 is currently in phase I clinical trials (NCT05642377) for type 2 diabetes mellitus, there are very few reports regarding its mechanism of action and efficacy.\u003c/p\u003e\u003cp\u003eMetabolic abnormalities have been reported to be closely related to the pathological development and exacerbation of SS. We have shown that butyric acid or \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, which produces butyric acid, can directly increase salivary secretion and suppress autoimmune B cells and autoantibody production.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Serum leptin and IL-1β levels were elevated in SS patients with metabolic disorders, and disease severity was aggravated when a glucosamine\u0026ndash;nitrosourea compound targeting pancreatic β cells was administered to NOD/ShiLtJ mice, a preclinical model of SS.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Relative to healthy individuals, patients with SS have altered mitochondrial DNA copy numbers and increased levels of factors associated with mitochondrial fission and transcription.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Furthermore, mitochondrial swelling and cristae disruption, as well as changes in mitochondrial-related metabolic pathways, have been observed in immune cells infiltrating the salivary glands of SS patients.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e These findings suggest that mitochondrial dysfunction is a key factor in the pathophysiology of SS and that mitochondria-targeting therapies may represent a novel treatment strategy.\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the therapeutic potential of HGR4113 in SS associated with metabolic abnormalities.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Phenotypic and functional changes observed during the proliferation and differentiation of B and T cells depend on dynamic mitochondrial metabolism. Resting B cells have low metabolic activity, whereas activation of B cells with BCR or LPS stimulation increases glucose uptake and glycolysis.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e When T cells are activated through antigen recognition, they switch to a state of elevated OXPHOS and aerobic glycolysis to meet the high energy and biosynthetic demands required for rapid proliferation and effector activity.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Proinflammatory cytokines such as IL-6 and TNF-α activate glycolysis and anabolic processes to promote differentiation of effector T cells, including Th1, Th2, and Th17 cells,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e whereas anti-inflammatory cytokines such as TGF-β promote oxidative metabolism and mitochondrial respiration to support the development of Tregs,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e indicating that each T cell type has unique metabolic characteristics. HGR4113 effectively reduced the production of inflammatory cytokines and OXPHOS activity in CD4\u003csup\u003e+\u003c/sup\u003e T cells by inhibiting STAT3 activity. Furthermore, HGR4113 reduced IgG production in CD19\u003csup\u003e+\u003c/sup\u003e B cells and promoted their differentiation into regulatory B cells. These results demonstrate that HGR4113 not only alleviates inflammation but also modulates mitochondrial OXPHOS.\u003c/p\u003e\u003cp\u003eResearch aimed at developing and utilizing salivary gland organoids is gaining increasing attention. A recent study demonstrated that salivary gland organoids derived from patients with SS consistently activated IFN-related genes and that IFN signaling was reduced by treatment with a JAK inhibitor, suggesting that salivary gland organoids derived from SS patients can recapitulate pathological characteristics of the disease.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Inhibition of activin receptor-like kinase (Alk) signaling during salivary gland organoid generation has been shown to promote organoid production.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Furthermore, in radiation-induced salivary gland damage, YAP translocated to the nucleus of salivary gland cells and activated regeneration-related genes, thereby inducing the proliferation of ductal cells and promoting salivary gland regeneration.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e We evaluated the efficacy of HGR4113 in salivary gland organoids from NOD mice. Salivary gland organoids derived from NOD mice administered HGR4113 regenerated more actively, with increased development of KRT5\u003csup\u003e+\u003c/sup\u003e basal and KRT7\u003csup\u003e+\u003c/sup\u003e luminal duct cells, aquaporin-5\u003csup\u003e+\u003c/sup\u003e acinar cells, and KRT14\u003csup\u003e+\u003c/sup\u003eαSMA\u003csup\u003e+\u003c/sup\u003e myoepithelial cells. These results suggest that HGR4113 promotes salivary gland cell regeneration.\u003c/p\u003e\u003cp\u003eThis study revealed that in a preclinical mouse model of SS, HGR4113 suppressed inflammation involving T and B cells, modulated mitochondrial metabolism, and promoted salivary gland organoid regeneration. These findings demonstrate the potential for developing disease-selective therapeutics based on the pathophysiology of SS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflict of interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00454685) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00208207).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eAll authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Cho and Park had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVitali C, Bombardieri S, Jonsson R, et al. Classification criteria for Sj\u0026ouml;gren\u0026rsquo;s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis. 2002; 61:554\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeydon M, McCoy S, Nguyen Y, et al. Epidemiology of Sj\u0026ouml;gren syndrome. Nat Rev Rheumatol. 2024; 20:158\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRisselada AP, Looije MF, Kruize AA, et al. The role of ectopic germinal centers in the immunopathology of primary Sj\u0026ouml;gren\u0026rsquo;s syndrome: a systematic review. 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The Hippo signaling pathway effector YAP promotes salivary gland regeneration after injury. Sci Signal. 2021; 14:eabk0599.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sjögren’s syndrome, metabolic abnormality, T cell, B cell, salivary gland organoid","lastPublishedDoi":"10.21203/rs.3.rs-7540230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7540230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSj\u0026ouml;gren\u0026rsquo;s syndrome (SS) is a chronic autoimmune disease in which inflammatory cells infiltrate the exocrine glands, reducing glandular secretory function and ultimately resulting in keratoconjunctivitis sicca (dry eyes) and xerostomia (oral dryness). Cardiovascular risk factors are more prevalent in patients with SS than in healthy controls; patients with SS and metabolic syndrome also have higher leptin and inflammatory cytokine levels. In this study, we investigated the effects of HGR4113, a structural analog of glabridin that promotes mitochondrial function and is in clinical trials for obesity treatment, on the development of Sj\u0026ouml;gren\u0026rsquo;s syndrome in non-obese diabetic NOD/ShiLtJ (NOD) mice. HGR4113 inhibited IL-17 production by regulating STAT3 activity and the metabolic profile of splenic CD4\u0026thinsp;+\u0026thinsp;T cells; it also increased the frequency of IL-10-producing regulatory B cells and decreased immunoglobulin production. Oral administration of HGR4113 (100 mg/kg) improved salivary flow rate, reduced lymphocyte infiltration, and lowered inflammatory cytokine levels in the salivary glands of NOD mice. HGR4113 also decreased the frequencies of splenic IL-17-producing T and B cells, germinal center B cells, and plasma cells \u003cem\u003eex vivo\u003c/em\u003e in NOD mice. Additionally, organoid formation from salivary gland stem cells of HGR4113-treated NOD mice increased, as did the levels of E-cadherin-14, aquaporin-5, α-SMA, and cytokeratin-14. Finally, treatment with HGR4113 promoted salivary gland organoid formation \u003cem\u003ein vitro\u003c/em\u003e. HGR4113 improves salivary gland hypofunction by inhibiting lymphocyte infiltration and inflammation in the salivary glands and restoring damaged salivary tissue in SS-like NOD mice.\u003c/p\u003e","manuscriptTitle":"Therapeutic Effects of a Synthetic Glabridin Derivative on Th17/ B cell Immune Regulation and Salivary gland Regeneration in Experimental Sjögren’s Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 14:08:50","doi":"10.21203/rs.3.rs-7540230/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-01-04T23:06:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-01T14:01:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-01T13:17:33+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-09T12:39:22+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-12-01T09:37:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-11-27T05:58:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-07T23:21:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T03:06:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-09-05T03:06:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a179fdf6-8939-4f9a-89a9-a936a169ad9e","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58689073,"name":"Biological sciences/Immunology/Autoimmunity"},{"id":58689074,"name":"Health sciences/Diseases/Immunological disorders/Autoimmune diseases"},{"id":58689075,"name":"Biological sciences/Immunology/Inflammation/Chronic inflammation"},{"id":58689076,"name":"Biological sciences/Immunology/Gene regulation in immune cells"}],"tags":[],"updatedAt":"2026-04-21T06:52:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 14:08:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7540230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7540230","identity":"rs-7540230","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}