To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004]. Animals were housed under specific pathogen-free conditions at 18–22°C and 40–60% relative humidity with a 12-h light/dark cycle and ad libitum access to standard food and water. Mice were maintained until 20 weeks of age prior to experimental analysis.

The systemic dosage of JSH-23 (MedChemExpress) was set at 3 mg/kg daily based on previously validated pharmacological protocols. This dosage has been reported to effectively inhibit NF-κB activation and its downstream proinflammatory cascades in various murine models. Previous studies have confirmed that this specific dose attenuates inflammatory injury in vital organs, including the lung liver and kidney, without inducing notable systemic toxicity (18,19). Therefore, 3 mg/kg JSH-23 was selected to investigate its mechanistic impact on lacrimal gland injury in the present study. In the NOD/Ltj + JSH-23 treatment group (n=6), mice received the NF-κB inhibitor JSH-23 (3 mg/kg) by oral gavage once daily for 4 weeks. Untreated NOD/Ltj mice and ICR control mice received an equal volume of sterile normal saline by oral gavage at the same frequency to control for handling-related stress. At the end of the treatment period, mice were euthanized by cervical dislocation and lacrimal glands were harvested for subsequent analyses. All procedures were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines (20) and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (21). The experimental protocol was approved by the Animal Care and Welfare Committee of Zhejiang University (approval no. 2022-190; Zhejiang, China).

To evaluate lacrimal gland secretory function, tear production was measured every 5 days during the initial assessment, and every 4 days beginning at treatment initiation (20 weeks of age) using the phenol red-impregnated cotton thread test (Tianjin Jingming New Technological Development Co. Ltd.) (14). Measurements were continued until 24 weeks of age. During each assessment, mice were gently restrained without anesthesia to avoid suppression of basal tear secretion. The lower eyelid was carefully retracted and one end of the phenol red cotton thread was placed into the lateral canthus of the conjunctival fornix. After 1 min, the length of the wetted red portion of the thread was measured in millimeters (mm) using a calibrated ruler with 0.5-mm precision. Each eye was tested three times consecutively at the same time of day to minimize diurnal variation. All measurements were performed independently by two investigators blinded to group allocation. The final tear secretion value for each eye was calculated as the mean of the measurements obtained by the two observers.

To evaluate histopathological alterations and determine in situ expression levels of target proteins and immune cell infiltration in lacrimal glands, hematoxylin and eosin (H&E) staining and IHC were performed. Lacrimal gland tissues were fixed in 4% paraformaldehyde (PFA) for 24 h at room temperature, embedded in paraffin and sectioned (thickness, 4 µm). Paraffin sections were deparaffinized and rehydrated through graded ethanol solutions (100% ethanol, two changes, 3 min each; 95% ethanol, 2 min; 70% ethanol, 2 min), followed by rinsing in distilled water.

For H&E staining, sections were stained with hematoxylin for 5 min and counterstained with eosin for 2 min at room temperature to visualize the overall tissue structure and cell morphology. Stained sections were visualized and images were captured using a light microscope (TS100; Nikon Corporation).

For IHC analysis, lacrimal gland tissues were fixed in 4% PFA for 24 h at room temperature and sectioned at a thickness of 4 µm. Antigen retrieval was performed in citrate buffer (pH, 6.0) at 95–100°C for 15 min, followed by washing with PBS. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at room temperature for 15 min. Sections were then blocked with 5% bovine serum albumin (BSA) containing 0.3% Triton X-100 (for permeabilization) for 30 min at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-CARD14 (cat. no. A16569; 1:50; ABclonal Biotech Co., Ltd.), anti-CCL19 (cat. no. PA5-109488; 1:50; Invitrogen; Thermo Fisher Scientific, Inc.), anti-CD4 (cat. no. MA1-146; 1:50; Invitrogen; Thermo Fisher Scientific, Inc.) and anti-IL-17A (cat. no. sc-374218; 1:50; Santa Cruz Biotechnology, Inc.). After washing with phosphate-buffered saline (PBS), sections were incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. 31460; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Immunoreactivity was visualized using 3,3′-diaminobenzidine substrate, followed by hematoxylin counterstaining at room temperature for 2 min. Sections were dehydrated, cleared and mounted with coverslips. Images were captured using a light microscope (TS100; Nikon Corporation) at ×200 magnification. For quantitative analysis, at least 3 randomly selected non-overlapping fields per section were analyzed using ImageJ software (version 1.48v; National Institutes of Health). The mean integrated optical density or positive staining area per field was calculated for each sample.

To identify transcriptomic alterations associated with lacrimal gland dysfunction, mRNA-seq was performed on lacrimal gland tissues obtained from NOD/Ltj mice (n=3) and ICR mice (n=3). Total RNA was extracted using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop^™ spectrophotometer (Thermo Fisher Scientific, Inc.) and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Samples with an RNA integrity number ≥7.0 were used for library preparation. Poly(A) mRNA was enriched using oligo(dT) magnetic beads and fragmented into short segments. Complementary DNA (cDNA) was synthesized using the NEBNext^® Ultra^™ II RNA Library Prep Kit for Illumina^® (cat. no. E7770S; New England BioLabs, Inc.). The concentration of the final libraries was measured using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Inc.), and the final library was loaded at a concentration of 1.5 pM. Libraries were amplified by PCR and validated prior to sequencing. Paired-end sequencing [2×150 bp (PE150)] was performed on an Illumina NovaSeq^™ 6000 platform (Illumina, Inc.) using the NovaSeq 6000 S4 Reagent Kit (cat. no. 20028312; Illumina, Inc.).

Raw sequencing reads were filtered to remove adapter sequences and low-quality reads prior to downstream analysis. Differential gene expression analysis between groups was performed using the ‘DESeq2’ package (version 1.46.0; http://bioconductor.org/packages/DESeq2/) in R software. Genes with |log₂ fold change (FC)|>1 and an adjusted P-value (false discovery rate) <0.05 were considered significantly differentially expressed. To functionally annotate the identified differentially expressed genes (DEGs), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using the ‘clusterProfiler’ package (version 4.14.6; http://bioconductor.org/packages/clusterProfiler/) in R software (R Development Core Team). GO enrichment analysis categorized the DEGs into biological processes, cellular components and molecular functions. KEGG pathway analysis was performed to identify significantly enriched signaling pathways. Pathways containing >2 genes and an adjusted P<0.05 was considered to indicate a statistically significant difference. Transcription factor-associated regulatory networks were constructed using weighted correlation network analysis based on the top 2,000 most variable genes to identify key regulatory modules associated with disease phenotype.

The raw mRNA-seq data generated in the present study have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE306385.

To validate differential gene expression identified by mRNA-seq, total RNA was extracted from lacrimal gland tissues using the RNeasy Mini Kit (Qiagen GmbH) according to the manufacturer's instructions. cDNA was synthesized using the iScript^™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol (25°C for 5 min, 46°C for 20 min and 95°C for 1 min). qPCR was performed using iTaq^™ Universal SYBR^® Green SuperMix (Bio-Rad Laboratories, Inc.). Target-specific primers were designed using NCBI Primer-Basic Local Alignment Search Tool (BLAST) to span at least one exon-exon junction. Primer sequences are listed in Table I. Thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 30 sec. A melting curve analysis (65–95°C) was performed at the end of each run to confirm amplification specificity. Relative gene expression levels were calculated using the 2^−ΔΔCq method (22), with β-actin serving as the internal reference gene. The specific primers for mouse β-actin were designed using the NCBI Primer-BLAST software based on the reference cDNA sequence (GenBank accession no. NM_007393.5).

To evaluate the activation of the NF-κB signaling pathway and the expression levels of downstream inflammatory mediators at the protein level, lacrimal gland tissues were homogenized and lysed in radioimmunoprecipitation assay buffer (cat. no. 87787; Thermo Fisher Scientific, Inc.) supplemented with protease and phosphatase inhibitor cocktails. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (30 µg/lane) were separated in 10% gels using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). Membranes were blocked with 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. After washing with TBST, membranes were incubated with the appropriate infrared dye-conjugated secondary antibodies for 1 h at room temperature in the dark. Protein bands were detected using an Odyssey^® Infrared Imaging System (LI-COR Biosciences). Band intensities were quantified using ImageJ software (Version 1.48v; National Institutes of Health). Relative protein expression levels were normalized to β-actin.

Primary antibodies against NF-κB p65 (cat. no. 8242T), phosphorylated (p-) NF-κB p65 (cat. no. 3033T), inhibitor of κB kinase (IKK)α (cat. no. 11930T), IKKβ (cat. no. 8943T), p-IKKα/β (cat. no. 2697T), inhibitor of κBα (IκBα; cat. no. 4814T) and p-IκBα (cat. no. 2859T) were purchased from Cell Signaling Technology, Inc., and used at a dilution of 1:1,000. Antibodies against tumor necrosis factor-α (TNF-α; cat. no. 60291-1-1G) was obtained from Proteintech Group, Inc. and used at a dilution of 1:1,000. The β-actin antibody (cat. no. 20536-1-AP; Proteintech Group, Inc.) was used at a dilution of 1:5,000. The secondary antibodies used were IRDye 800CW Goat anti-Rabbit IgG (cat. no. 926-32211; 1:10,000; LI-COR Biosciences) and IRDye 680RD Goat anti-Mouse IgG (cat. no. 926-68070; 1:10,000; LI-COR Biosciences).

At the experimental endpoint, peripheral whole blood samples (~0.8 ml per mouse) were collected from the mice in each experimental group (ICR controls, NOD/Ltj and NOD/Ltj + JSH-23) via retro-orbital venous plexus bleeding. No other clinical procedures or experimental manipulations were performed on the mice on the day of sacrifice prior to blood collection. All samples were obtained as the primary procedure under deep anesthesia. Specifically, anesthesia was induced using 3–4% inhaled isoflurane and maintained at 1.5–2% isoflurane. Deep anesthesia was confirmed by the complete loss of the pedal withdrawal reflex and corneal reflex before initiating the blood collection. The blood was immediately collected into tubes containing EDTA. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll-Paque^™ PLUS (cat. no. 17-1440-02; GE Healthcare) according to standard protocols. Briefly, the collected whole blood samples were diluted 1:1 with PBS and carefully layered onto lymphocyte separation medium. Samples were centrifuged at 800 × g for 30 min at room temperature. After centrifugation, the mononuclear cell layer was collected, transferred to a 15 ml tubes and washed twice with PBS at 250 × g for 10 min at room temperature. For intracellular cytokine staining, PBMCs were stimulated with Cell Stimulation Cocktail plus protein transport inhibitors (cat. no. 00-4975-93; 1:500; eBioscience; Thermo Fisher Scientific, Inc.) for 6 h at 37°C in a 5% CO₂ incubator. Cells were filtered through a 30 µm CELLTrics^® filter (Sysmex Corporation) and resuspended in staining buffer. Surface staining was performed using CD4 antibody (cat. no. MA1-146; 1:100; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at 4°C in the dark. After washing, the cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (cat. no. 88-8824-00; eBioscience; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Subsequently, the cells were incubated with IL-17 antibodies (cat. no. 45-7177-80; 1:100; Invitrogen; Thermo Fisher Scientific, Inc.) were added to the cell suspension, mixed gently by pulse vortex and incubated for 30 min in the dark at 4°C. After washing, PBMCs were centrifuged at 300 × g for 5 min at 4°C and resuspended in PBS for analysis and cell sorting.

Flow cytometric analysis and cell sorting were performed using a BD FACSAria^™ III cell sorter (BD Biosciences) equipped with a 100 µm nozzle at 20 psi. Compensation was performed using single-stained controls. Dead cells were excluded using 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Inc.) staining at room temperature for 5 min. Fluorescence signals were detected using the 488 nm laser with a 530/30 nm filter for Alexa Fluor^™ 488 (CD4), a 695/40 nm filter for PerCP-Cy5.5 (IL-17A), and the 355 nm laser with a 450/50 nm filter for DAPI. Data were analyzed using FlowJo software (version 10.8.1; FlowJo, LLC; BD Biosciences). Gating was established using isotype controls and fluorescence minus one controls. CD4+IL-17A+ cells were gated within the lymphocyte population based on forward and side scatter characteristics.

Sorted cells were collected into 1.5 ml RNase-free tubes pre-coated with 1% BSA in PBS containing RNasin^® Plus RNase inhibitor (1:25 dilution) for downstream analysis.

To quantitatively assess systemic inflammatory cytokine levels, whole blood samples were collected into sterile tubes without anticoagulant and allowed to clot at room temperature for 20 min. Samples were centrifuged at 1,000 × g for 20 min at 4°C and the serum supernatants were carefully collected and stored at −80°C until analysis. Serum cytokine concentrations were measured using commercial ELISA kits (Jiangsu Meimian Industrial Co., Ltd.), according to the manufacturer's instructions. The following kits were used: IFN-γ (cat. no. MM-0182M1), TNF-α (cat. no. MM-0132M1), IL-17A (cat. no. MM-0759M1) and IL-6 (cat. no. MM-0163M1). Briefly, serum samples and standards were added to antibody-coated 96-well microplates and incubated for 30 min at 37°C. After washing to remove unbound proteins enzyme-conjugated detection antibodies were added and incubated for 30 min at 37°C. Following substrate addition, color development was conducted for 15 min at 37°C and terminated using stop solution and absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated from standard curves generated using recombinant standards provided in the kits. All samples were analyzed in duplicate and mean values were used for statistical analysis.

Apoptosis in lacrimal gland tissues was assessed using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Tissues were fixed in 4% PFA for 24 h at room temperature. Paraffin-embedded tissue sections (4-µm thickness) were deparaffinized, rehydrated and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Sections were blocked with 5% BSA for 30 min at room temperature and washed with PBS. TUNEL staining was performed using a commercial kit (cat. no. RC-01; http://www.recordbio.com; Shanghai Recordbio Biological Technology Co., Ltd.) according to the manufacturer's instructions. Sections were incubated with the TUNEL reaction mixture for 1 h and counterstained with DAPI (1 µg/ml; 5 min at room temperature). Sections were then mounted using antifade mounting medium (cat. no. S2100; Beijing Solarbio Science & Technology Co., Ltd.).

Images were captured using a fluorescence microscope (TS100-F; Nikon Corporation). Apoptosis was quantified by calculating the percentage of TUNEL⁺ cells compared with the total number of DAPI-stained nuclei. At least 3 randomly selected non-overlapping fields per section were analyzed using ImageJ software (Version 1.48v; National Institutes of Health).

To evaluate immune cell infiltration and spatial co-localization in lacrimal gland tissues, paraffin sections (thickness, 4 µm) were fixed in 4% PFA for 10 min at room temperature, permeabilized with 0.1% Triton X-100 and blocked with 5% BSA for 30 min at room temperature.

Sections were incubated overnight at 4°C with primary antibodies against CD4 (cat. no. MA1-146; 1:20; Invitrogen; Thermo Fisher Scientific, Inc.) and IL-17A (cat. no. sc-374218; 1:50; Santa Cruz Biotechnology, Inc.). After washing, sections were incubated for 45 min at room temperature in the dark with Alexa Fluor^™ 488-conjugated secondary antibodies (cat. no. A-21121; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) or Alexa Fluor^™ 594 (cat. no. A-11007; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.). Nuclei were counterstained with DAPI (1 µg/ml) for 5 min at room temperature.

Fluorescent and phase-contrast images were captured using a Zeiss Axiovert 200 inverted microscope equipped with AxioCam MRm digital camera and analyzed using AxioVision software (version 4.9.1; Carl Zeiss AG). Confocal images were acquired using a Leica DM6000 B confocal laser scanning microscope (Leica Microsystems GmbH).

All experiments were performed using at least three independent biological replicates unless stated otherwise. Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism (version 8.0; Dotmatics). For comparisons between two independent groups, an unpaired two-tailed Student's t-test was used. For comparisons among three groups, one-way analysis of variance followed by Tukey's multiple comparisons test was performed. P<0.05 was considered to indicate a statistically significant difference.

Phenol red thread assay demonstrated significantly reduced tear secretion in NOD/Ltj mice compared with ICR controls at all assessed time points (all P<0.001), with a mean decrease >2 mm (Fig. 1A). Serum cytokine analysis by ELISA revealed significantly elevated levels of IFN-γ, IL-17A and TNF-α in NOD/Ltj mice compared with ICR mice (P<0.05), whereas IL-6 levels did not differ significantly between groups (Fig. 1B). Histopathological examination of lacrimal gland sections stained with H&E demonstrated marked glandular atrophy and prominent lymphocytic infiltration in NOD/Ltj mice compared with ICR controls (Fig. 1C). These findings confirmed the presence of lacrimal gland dysfunction and systemic inflammatory activation in NOD/Ltj mice.

To investigate molecular mechanisms associated with reduced tear production in NOD/Ltj mice, mRNA-seq was performed on lacrimal gland tissues. A total of 1,109 DEGs were identified compared with ICR controls, including 874 upregulated and 235 downregulated genes. Differential expression patterns are presented in the heatmap (Fig. 2A) and volcano plot (Fig. 2C). GO enrichment analysis categorized the DEGs into biological processes, cellular components and molecular functions, with significant enrichment in immune and inflammatory response-related categories, specifically including ‘cellular response to interferon-β’, ‘positive regulation of T cell proliferation’, ‘T cell differentiation’ and ‘adaptive immune response’ (Fig. 2B). KEGG pathway analysis demBonstrated significant enrichment of pathways associated with ‘NF-κB signaling’ and ‘Th17 cell differentiation’ (Fig. 2D). The NF-κB signaling pathway exhibited an adjusted P-value of 6.71×10⁻¹² with a rich factor of 0.29, whereas the Th17 differentiation pathway demonstrated an adjusted P-value of 3.46×10⁻¹² with a rich factor of 0.29.

Based on pathway enrichment and differential expression magnitude, six candidate genes were selected for validation: One downregulated gene (CARD14: log₂FC, −5.02) and five upregulated genes (TNFSF14: log₂FC, 11.64; TNFSF13: log₂FC, 0.14; CCL19: log₂FC, 3.99; CCL20: log₂FC, 4.18; CCL12: log₂FC, 2.09). Consistent with the mRNA-seq data, the RT-qPCR analysis confirmed the significant downregulation of CARD14 (P=0.0412) and upregulation of CCL19 (P=0.001), consistent with the sequencing results. The expression levels of CCL12 (P=0.1464) and CCL20 (P=0.0762), TNFSF13 (P=0.7954), and TNFSF14 (P=0.2265) also showed trends consistent with the sequencing data, although these changes did not reach statistical significance (Fig. 3A).

To further assess NF-κB pathway activation at the protein level, western blotting analysis was performed. Expression levels of NF-κB pathway components, specifically p-NF-κB (relative to total NF-κB; P=0.0048) and p-IκBα (relative to total IκBα; P=0.0003), were significantly increased in lacrimal glands of NOD/Ltj mice compared with that of ICR controls. Furthermore, a significantly elevated level of downstream inflammatory mediator TNF-α was observed (P=0.0112) (Fig. 3B and C). These findings indicated the activation of NF-κB signaling in the lacrimal glands of NOD/Ltj mice.

To evaluate the functional contribution of NF-κB signaling in pSS-associated lacrimal gland pathology, the NF-κB inhibitor JSH-23 was administered to NOD/Ltj mice. Western blotting analysis demonstrated significantly increased p-NF-κB (P<0.001), -IKKα/β (P=0.0004) and -IκBα (P=0.0002), accompanied by significantly reduced total IκBα expression in NOD/Ltj mice compared with ICR controls (Fig. 4A). Treatment with JSH-23 significantly reduced phosphorylation of NF-κB pathway components (p-NF-κB, P=0.0003; p-IKKα/β, P=0.0009; p-IκBα, P=0.0054 vs. NOD group) and significantly restored IκBα expression (P=0.0023 vs. NOD group) toward levels observed in ICR mice (compared to ICR controls: p-NF-κB, P=0.1525; p-IKKα/β, P=0.0480; p-IκBα, P=0.5786; IκBα, P=0.0878) (Fig. 4A and B). Furthermore, the elevated expression level of downstream inflammatory mediator TNF-α (P=0.0002 vs. ICR group) was significantly reduced following JSH-23 treatment (P=0.0022 vs. NOD group), showing no significant difference compared with the ICR controls (P=0.0586) (Fig. 4A and B). These findings indicated that pharmacological inhibition of NF-κB suppresses pathway activation and downstream inflammatory signaling in NOD/Ltj lacrimal glands.

To assess in situ protein expression of selected DEGs, immunohistochemical staining was performed (Fig. 5A). Compared with ICR mice, NOD/Ltj mice exhibited significantly decreased CARD14 protein expression (P=0.0228) and significantly increased CCL19 expression (P=0.0489) in lacrimal gland tissues. Administration of JSH-23 significantly reversed these alterations (CARD14, P=0.0192; CCL19, P=0.0405 vs. NOD group), restoring expression levels similar to those in ICR controls (CARD14, P=0.2349; CCL19, P=0.7356) (Fig. 5B). Therefore, these results demonstrated that NF-κB inhibition modulates CARD14 and CCL19 expression in lacrimal gland tissues of NOD/Ltj mice.

To evaluate the effect of NF-κB inhibition on systemic immune responses, PBMCs were analyzed by flow cytometry to quantify CD4+ T cells and CD4+IL-17A+ Th17 cells (Fig. 6A and B). The proportion of CD4+IL-17A+ cells within the CD4⁺ T cell population was significantly increased in NOD/Ltj mice compared with ICR controls (P=0.0047), indicating the expansion of Th17 cells. Treatment with JSH-23 significantly reduced the CD4+IL-17A+/CD4+ ratio compared with the untreated NOD/Ltj mice (P=0.0011), restoring levels toward those observed in ICR controls.

Consistent with changes in Th17 cell frequency, ELISA demonstrated significantly elevated serum levels of IFN-γ (P=0.0009), IL-17A (P=0.0002) and TNF-α (P<0.0001) in NOD/Ltj mice compared with ICR controls (Fig. 6C). Administration of JSH-23 significantly reduced the cytokine levels compared with untreated NOD/Ltj mice (IFN-γ, P=0.0151; IL-17A, P=0.0099; and TNF-α, P=0.0019), and restored them to levels with no significant differences compared with the ICR controls (P=0.0590, P=0.0599 and P=0.0502, respectively), indicating suppression of systematic inflammatory cytokine production.

To evaluate local immune cell infiltration in lacrimal gland tissues, IHC and IF analyses were performed (Fig. 7). IHC demonstrated significantly increased CD4+ T cell (P=0.0091) and IL-17A expression (P=0.0095) in lacrimal glands of NOD/Ltj mice compared with ICR controls (Fig. 7A and B). Treatment with JSH-23 significantly reduced CD4⁺ and IL-17A staining intensity (P=0.0190 and P=0.0170 vs. NOD group, respectively), restoring levels toward those observed in the ICR mice. Consistently, IF staining confirmed significantly increased infiltration of CD4⁺ (P=0.0033) and IL-17A⁺ (P=0.0434) cells in the lacrimal gland tissues of NOD/Ltj mice, which was significantly attenuated following JSH-23 administration (P=0.0042 and P=0.0302 vs. NOD group, respectively), showing no significant differences compared with the ICR baseline (P=0.9849 and P=0.5101, respectively) (Fig. 7C and D). These findings indicated that inhibition of NF-κB signaling reduces local Th17-associated inflammatory infiltration in lacrimal glands of NOD/Ltj mice.

TUNEL staining demonstrated a significantly higher proportion of apoptotic cells in lacrimal gland tissues of NOD/Ltj mice compared with ICR controls (P=0.0476; Fig. 8A and B). Quantitative analysis demonstrated that JSH-23 treatment reduced the percentage of TUNEL⁺ cells compared with untreated NOD/Ltj mice (P=0.0428), restoring apoptosis levels toward those observed in ICR controls (P=0.8763).

To assess the functional impact of NF-κB inhibition on tear secretion, phenol red thread assay was performed. Tear production in NOD/Ltj mice was significantly reduced compared with ICR mice at the final assessment time point (53 days; P=0.0038) (Fig. 8C). Treatment with JSH-23 significantly increased tear production compared with untreated NOD/Ltj mice (P=0.0320), indicating partial restoration of lacrimal gland secretory function.