Targeting SGK1 Mitigates Synovial Fibrosis via Suppressing M1 Polarization to Alleviate Osteoarthritis

Targeting SGK1 Mitigates Synovial Fibrosis via Suppressing M1 Polarization to Alleviate Osteoarthritis | 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 Research Article Targeting SGK1 Mitigates Synovial Fibrosis via Suppressing M1 Polarization to Alleviate Osteoarthritis Zhichao Yang, Ming Wei, Yang Liu, Wenwei Li, Zhaoyu Li, Liang Yan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6880630/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Synovial inflammation and fibrosis constitute significant pathological characteristics of osteoarthritis, with their progression being intricately linked to the M1 polarization of synovial macrophages and subsequent synovial fibrosis. Despite this understanding, the precise molecular mechanisms remain elusive. In the present study, we employed both a lipopolysaccharide (LPS)-induced inflammation model and an anterior cruciate ligament transection (ACLT)-induced osteoarthritis rat model to elucidate the role of serum and glucocorticoid-regulated kinase 1 (SGK1) in this context. RNA sequencing analysis revealed that the knockdown of SGK1 markedly suppressed the activity of the JAK-STAT signaling pathway in macrophages. Furthermore, in vitro experiments demonstrated that the silencing of SGK1 led to a reduction in the release of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, and diminished the migratory and invasive capabilities of fibroblast-like synoviocytes (FLS). Mechanistically, the silencing of SGK1 was found to inhibit the expression of M1 polarization markers, specifically iNOS and CD86, by suppressing JAK1-STAT3 phosphorylation. In an ACLT-induced osteoarthritis (OA) rat model, intra-articular administration of an SGK1 inhibitor significantly attenuated synovitis and fibrosis. Histological analyses revealed an up-regulation of collagen II expression and a down-regulation of MMP13, indicating a chondroprotective effect. Collectively, these findings suggest that SGK1 modulates macrophage M1 polarization and synovial fibrosis via the JAK1-STAT3 signaling pathway, and that targeted inhibition of SGK1 may represent a novel therapeutic strategy for OA management. This study thus provides a theoretical foundation for the development of anti-OA pharmacological interventions targeting SGK1. ​​Osteoarthritis M1 polarization Synovial fibrosis SGK1 JAK-STAT signaling Targeted therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteoarthritis (OA) stands as one of the most prevalent degenerative joint disorders worldwide, affecting approximately 7% of the global population, with incidence rates escalating dramatically with advancing age, particularly in weight-bearing joints such as the knees and hips [ 1 , 2 ]. Recognized as a leading cause of disability among the elderly, OA imposes a substantial socioeconomic burden, compounded by the limitations of current therapeutic strategies, which primarily focus on symptom alleviation rather than halting or reversing disease progression [ 3 ]. The pathogenesis of OA is characterized by a complex interplay of biomechanical and biochemical factors that collectively drive the progressive deterioration of articular cartilage, synovial membrane, and periarticular tissues [ 4 ]. Among these mechanisms, emerging evidence underscores synovial inflammation and fibrosis as pivotal contributors to OA progression [ 5 ]. However, the precise molecular pathways governing these processes remain incompletely elucidated, necessitating further investigation to identify novel therapeutic targets. The synovial membrane, a critical structure responsible for joint lubrication and nutrient supply, undergoes profound pathological remodeling during OA. These changes include aberrant immune cell infiltration—particularly of macrophages—dysregulated cytokine production, and excessive collagen deposition, all of which disrupt synovial homeostasis [ 6 ]. Clinically, these alterations manifest as joint effusion, tenderness, and progressive impairment of mobility [ 7 ]. Central to this pathogenic cascade is the polarization of synovial macrophages into functionally distinct subsets: the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype [ 8 ].M1 macrophages, activated by Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide, LPS) or Th1 cytokines (e.g., interferon-gamma, IFN-γ), are characterized by the expression of surface markers such as CD86 and inducible nitric oxide synthase (iNOS). These cells secrete a repertoire of catabolic mediators, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), which exacerbate cartilage degradation and promote synovial fibrosis [ 9 ]. In contrast, M2 macrophages, identified by markers like CD163, foster tissue repair through the secretion of anti-inflammatory cytokines such as transforming growth factor-beta (TGF-β) [ 10 ]. In the inflammatory milieu of OA, the balance between these subsets is skewed toward M1 dominance, creating a self-perpetuating cycle of stromal activation and extracellular matrix (ECM) remodeling [ 11 ]. While macrophage polarization has emerged as a promising therapeutic target, the molecular mechanisms regulating this process in the synovium remain poorly defined. Serum- and glucocorticoid-regulated kinase 1 (SGK1), a serine/threonine kinase from the AGC family, has recently emerged as a critical regulator of inflammation and fibrosis [ 12 ]. Initially identified for its role in renal ion transport and cellular stress responses [ 13 ], SGK1 is now known to influence immune modulation, affecting T-cell differentiation [ 12 ], neutrophil clearance, and macrophage activation in cardiovascular and pulmonary diseases [ 14 ]. Mechanistically, SGK1 interacts with key inflammatory pathways—including NF-κB and JAK-STAT signaling—to enhance cytokine production and fibrotic responses [ 15 ]. In fibrotic conditions such as pulmonary fibrosis and cirrhosis, SGK1 overexpression correlates with ECM deposition via TGF-β/SMAD activation [ 16 ]. Additionally, SGK1 mediates mechanical stress-induced inflammation in cardiac fibroblasts [ 17 ] and facilitates tumor metastasis by promoting epithelial-mesenchymal transition (EMT) [ 18 ]. However, its role in synovial pathology, particularly in macrophage polarization during OA, remains unexplored. Preliminary studies suggest that SGK1 may contribute to joint degeneration. In chondrocytes, SGK1 inhibition reduces inflammatory responses [ 19 ], while murine OA models indicate its involvement in cartilage degradation [ 20 ]. Paradoxically, SGK1 also suppresses innate immune responses to bacterial infections [ 21 ], demonstrating context-dependent functionality. These conflicting findings highlight the need for a systematic investigation into SGK1’s synovial-specific actions. Importantly, no prior studies have examined whether SGK1 regulates synovial fibrosis through macrophage reprogramming or identified its downstream effectors in joint tissues. To elucidate the role of SGK1 in synovial inflammation and fibrosis, we employed a combination of in vitro and in vivo models. LPS stimulated RAW264.7 macrophages were used to investigate SGK1's influence on macrophage polarization, while an ACLT rat model was utilized to recapitulate the pathological features of OA in vivo. Our findings demonstrate that SGK1 knockdown suppresses M1 polarization by inhibiting JAK1/STAT3 signaling, subsequently attenuating FLS proliferation and collagen deposition. These results not only advance our understanding of OA pathogenesis but also position SGK1 as a promising therapeutic target for OA treatment. Materials and methods Clinical Samples and Ethical Approval Knee synovial tissues were obtained from osteoarthritis patients (Kellgren-Lawrence grade III-IV) undergoing total knee arthroplasty. Normal control tissues were collected from traumatic amputation patients with no history of arthritis. This study was approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (Approval No. 2024-ky546), and written informed consent was obtained from all participants in accordance with the ethical principles of the Declaration of Helsinki. Fresh tissues were flash-frozen in liquid nitrogen immediately after collection and stored at -80°C for long-term preservation. Cell Culture RAW264.7 cells (Shanghai Cell Bank, Chinese Academy of Sciences, Cat.SCSP-5036) were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Primary human fibroblast-like synoviocytes (FLSs) were isolated from surgically resected synovial tissues. Briefly, tissues were rinsed with PBS and digested with 0.1% collagenase II (Sigma) at 37°C for 2 hours. The digested suspension was filtered through a 100 μm cell strainer to remove undigested debris, centrifuged at 1000g for 5 minutes, and resuspended in culture medium. The medium was replaced every 48 hours. Cells from passages 4–6 were used for experiments, with purity (>95%) confirmed by flow cytometry (CD90+/CD68−) and vimentin immunofluorescence staining. Cell Viability Assay FLSs were co-cultured with macrophage-conditioned medium for 24 hours. Cell viability was assessed using the CCK-8 kit (Dojindo), following the manufacturer’s protocol. Absorbance was measured at 450 nm using a microplate reader (BioTek). Cell Transfection Based on preliminary screening (Figure 2A), siRNA#2 was selected for subsequent experiments. RAW264.7 cells were seeded in 6-well plates at 1×10⁵ cells/well. After 24 hours, siRNA (20 nM) and riboFECT™ CP transfection reagent (5 μl, RiboBio) were mixed to form complexes. The transfection mixture was added to cells, and the medium was replaced with fresh complete medium 6 hours post-transfection. Knockdown efficiency was validated by qPCR (ΔΔCt method) and Western blotting after 24 hours. siRNA sequences are listed in Table 1. RNA Sequencing Analysis Total RNA was extracted using TRIzol and assessed for integrity (RIN >8.0) via an Agilent 2100 Bioanalyzer. Libraries were prepared with the NEBNext® Ultra II RNA Library Prep Kit and sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads, ~30 million reads/sample). Raw data were quality-checked with Fast QC, aligned to the mm10 genome using STAR (v2.7.9a), and analyzed for differential expression (|log2FC| >1, FDR <0.05) with DESeq2. Functional enrichment (GO and KEGG) was performed using clusterProfiler (v4.0). Quantitative Real-Time PCR Assay Total RNA was isolated from treated RAW264.7 cells using TRIzol (Beyotime, China). RNA purity and concentration were quantified via NanoDrop 2000. cDNA was synthesized by reverse transcription, and qPCR reactions contained 10 μl Master Mix, 0.5 μl each of forward/reverse primers, 2 μl cDNA, and 7 μl ddH2O. Relative expression levels were calculated using the 2^(-ΔΔCt) method with GAPDH as the reference. Primer sequences are provided in Table 2. Western Blotting Proteins were extracted using RIPA lysis buffer (1% protease inhibitor cocktail) and quantified via BCA assay. Samples (10 μg) were separated by SDS-PAGE and transferred to PVDF membranes (0.45 μm) at 70–120 V under ice cooling. Membranes were blocked with rapid blocking buffer (Beyotime) for 20 minutes, incubated overnight with primary antibodies at 4°C, and then with HRP-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using ECL and analyzed with Image Lab. Antibodies included anti-CD68 (sc-20060), anti-iNOS (ab178945), anti-CD86 (A1199), anti-SGK1 (sc-28338), anti-JAK1 (YT2424), anti-p-JAK1 (YP0154), anti-STAT3 (YT4443), anti-p-STAT3 (YP0250), anti-CTGF (AF7537), anti-α-SMA (AF1032), anti-vimentin (#5741), anti-MMP13 (ab39012), anti-collagen II (28457-1-AP), and anti-β-actin (YM3028). Wound healing experiments FLSs were subjected to diverse treatment conditions and subsequently seeded into 6-well plates. The cells were grown until they achieved 90%-100% confluence. A linear scratch was introduced on the surface of the monolayer using a 200 µL pipette tip. Floating cells were removed with two PBS washes, The cells that stayed attached were cultured in serum-free DMEM, and those at the wound location were viewed with an inverted microscope made by Carl Zeiss in Germany,at both 0 and 24 hours following the scratch. Migration and invasion assays FLSs were subjected to various treatments and then placed into the upper sections of Transwell inserts at a concentration of 2 × 10^4 cells per well in a medium without serum. To clear away floating cells, two PBS washes were executed, and the adherent cells were preserved in serum-free DMEM. An inverted microscope from Carl Zeiss, Germany, was used to view the cells at the wound site. Non-migrating cells were wiped away using a damp cotton swab, and the remaining cells were stained with a 0.1% crystal violet solution. Using an inverted microscope from Carl Zeiss, Germany, images at the microscopic level were obtained, and the migrated cells were tallied. Macrophage conditioned medium (CM) After treating RAW264.7 macrophages with different stimuli for 24 hours, the supernatants were collected and centrifuged at 1000 g for 5 minutes, then stored at -80 °C for future experiments. The macrophage-derived conditioned medium (CM) was diluted in a 1:1 ratio with serum-free medium and subsequently applied to FLSs for further analysis. Animal Model Establishment Twenty-four male SD rats (8 weeks, 250±20 g) were randomized into three groups: sham, ACLT and GSK650394. Under anesthesia (1% pentobarbital sodium, 40 mg/kg), the ACL was transected via medial arthrotomy. Postoperatively, meloxicam (1 mg/kg/day) was administered subcutaneously for 3 days. Procedures followed ARRIVE guidelines and were approved by the Institutional Animal Ethics Committee (Approval No. 2023-N(A)-106). Histological Analysis Knee specimens were fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% EDTA (pH 7.4) for 4 weeks with daily solution changes, then paraffin-embedded and sectioned at 4 μm thickness. For synovitis evaluation, H&E-stained sections were scored using the Krenn scale (0-9) based on synovial hyperplasia, inflammatory infiltration, and stromal density. Cartilage integrity was assessed via Safranin O-fast green staining (0.1% Safranin O for 5 min, 0.02% fast green for 3 min) with OARSI scoring (0-6). Collagen deposition was analyzed through dual approaches: (1) Masson's trichrome staining (Weigert's hematoxylin [5 min], Biebrich scarlet [5 min], phosphomolybdic acid [10 min], aniline blue [5 min]) quantified using ImageJ (v1.53, NIH) thresholding of blue-stained areas; and (2) Picrosirius red staining (0.1% Sirius red in saturated picric acid, 1 h RT) examined under both brightfield and polarized light (Nikon Eclipse Ci-L with polarizing filters), with type I (orange-red birefringence) and type III (green) collagen differentiation. All histological scoring was performed by two blinded investigators (ICC >0.85). Immunohistochemistry and immunofluorescence staining Synovial tissues from humans or rats were preserved in 4% paraformaldehyde for a day, then embedded in paraffin and sliced into sections 4-6 µm thick. The sections underwent deparaffinization with xylene and were rehydrated using a graded series of ethanol prior to retrieving antigens. Before antigen retrieval, the sections were deparaffinized with xylene and rehydrated through a series of graded ethanol. The primary antibodies were subsequently added and left to incubate overnight at 4°C. Sections were incubated with HRP-conjugated secondary antibodies for one hour at room temperature the following day, and images were obtained using a Zeiss light microscope. Statistical analysis All experimental results are presented as the Mean ± Standard Deviation (Mean ± SD), With statistical analyses conducted using GraphPad Prism version 9.0. For assessing differences across multiple groups, the analysis involved a one-way ANOVA, and Tukey's post hoc test was applied for comparing pairs. When comparing two groups, an independent t-test was applied. The criterion for statistical significance was established at P < 0.05. Results SGK1 is highly expressed in synovial macrophages with enhanced M1 polarization and fibrosis in OA. Osteoarthritis (OA) is a chronic joint disorder marked by progressive cartilage degradation, synovial inflammation, and fibrotic changes. Emerging research highlights the pivotal involvement of synovial macrophages in disease pathogenesis. Comparative histopathological examination demonstrated pronounced tissue disorganization and fibrotic alterations in OA synovium relative to healthy controls. Hematoxylin-eosin staining revealed well-preserved cellular architecture in normal synovium, contrasting with the disarranged cellular patterns and prominent inflammatory infiltration observed in OA specimens (Fig. 1 A). The fibrotic transformation of synovial tissue was corroborated through Masson's trichrome and Sirius red staining techniques, which unveiled substantial collagen accumulation in OA samples (Fig. 1 A). Correspondingly, synovitis severity scores were markedly elevated in OA patients compared to healthy individuals (Fig. 1 B). Quantitative assessments further validated these observations, demonstrating a statistically significant rise in collagen deposition within OA synovial tissues (Fig. 1 C), indicative of progressive fibrotic remodeling during OA development. Immunofluorescence analysis revealed a pronounced polarization shift of synovial macrophages toward a proinflammatory M1 phenotype in OA. Comparative evaluation showed substantial upregulation of inducible nitric oxide synthase (iNOS) and CD86—characteristic M1 macrophage markers—in OA synovium relative to normal tissue (Fig. 1 D). Quantitative measurement of fluorescence intensities confirmed the predominant M1 polarization state of macrophages in OA specimens (Fig. 1 F), suggesting their potential contribution to synovial inflammation and structural deterioration. A pivotal observation in this investigation was the heightened expression of serum- and glucocorticoid-regulated kinase 1 (SGK1) within OA synovial macrophages. Immunofluorescence visualization demonstrated robust SGK1 expression (green signal) that colocalized with CD68 + macrophages (red signal) in OA synovium, whereas minimal SGK1 expression was detected in control tissues (Fig. 1 E). Quantitative fluorescence analysis substantiated these findings, revealing significantly intensified SGK1 and CD68 signals in OA samples compared to controls (Fig. 1 G). The correlation between SGK1 expression and macrophage activation implies a potential regulatory role for SGK1 in macrophage polarization and synovial inflammatory responses during OA progression. In vitro experiments employing lipopolysaccharide (LPS)-stimulated macrophages provided additional validation. LPS exposure induced notable upregulation of SGK1/CD68 expression (Fig. 1 H), with fluorescence quantification demonstrating approximately 2.5-fold enhancement compared to unstimulated cells (Fig. 1 I). These results suggest that inflammatory mediators may augment SGK1 expression in macrophages, potentially driving their polarization toward a proinflammatory state. Considering that M1-polarized macrophages exacerbate synovial inflammation and fibrosis through secretion of proinflammatory mediators (e.g., TNF-α, IL-1β, IL-6) and matrix-degrading enzymes, the upregulated SGK1 expression in synovial macrophages may constitute a crucial molecular link between macrophage activation and synovial pathology in OA. Knockdown of SGK1 alleviated synovial inflammation and fibrosis To further elucidate the regulatory role of SGK1 in synovial inflammation and fibrosis, we employed siRNA-mediated knockdown of SGK1 in synovial macrophages. qPCR analysis confirmed efficient silencing of SGK1 mRNA in RAW264.7 cells transfected with si-SGK1 (Fig. 2 A). Western blot analysis (Fig. 2 B) and subsequent quantitative evaluation (Fig. 2 C) demonstrated a significant reduction in SGK1 protein levels compared to the si-NC control group. Notably, LPS stimulation markedly upregulated SGK1 expression, an effect that was substantially attenuated by si-SGK1 transfection, confirming the efficacy of SGK1 knockdown at the protein level. Subsequent investigations revealed that SGK1 knockdown significantly suppressed the LPS-induced upregulation of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 (Fig. 2 D–F), underscoring the pivotal role of SGK1 in mediating inflammatory responses. To assess the downstream effects of SGK1 knockdown on synovial fibroblasts, we cultured these cells with conditioned media from treated RAW264.7 cells. CCK-8 assays (Fig. 2 G) revealed that LPS stimulation enhanced the proliferative capacity of synovial fibroblasts, whereas this effect was markedly inhibited by si-SGK1-conditioned medium. Further functional assays, including scratch, migration, and invasion experiments (Fig. 2 H–J and Fig. S1 A-C), demonstrated that synovial fibroblasts treated with si-SGK1-conditioned medium exhibited significantly impaired migratory and invasive capabilities compared to those exposed to si-NC-conditioned medium. Immunofluorescence and Western blot analyses (Fig. 2 K-L) corroborated these findings, showing that SGK1 knockdown led to a pronounced reduction in the expression of fibrosis-associated proteins, such as Vimentin, α-SMA, and CTGF, in synovial fibroblasts. Quantitative analysis of these proteins (Fig. S1 D–I) further confirmed the significant downregulation of Vimentin, α-SMA, and CTGF in the LPS + si-SGK1 group compared to controls, as evidenced by both fluorescence intensity measurements and protein level ratios normalized to β-actin. Collectively, these results demonstrate that SGK1 silencing not only attenuates pro-inflammatory cytokine production in RAW264.7 cells but also inhibits the fibrotic and proliferative responses of synovial fibroblasts by modulating the paracrine effects of inflammatory mediators. Our findings provide robust evidence supporting the critical role of SGK1 in regulating synovial inflammation and fibrosis, highlighting its potential as a therapeutic target for osteoarthritis. M1 polarization of macrophages leads to a synovial fibrosis phenotype. Experimental results demonstrate that LPS-induced M1 macrophage polarization promotes synovial fibrosis via paracrine mechanisms. Immunofluorescence staining confirmed that LPS-treated macrophages exhibited stronger expression of M1 markers (iNOS in green, CD86 in red) compared to the control group (Ctrl), with merged images (DAPI in blue) clearly indicating enhanced M1 polarization (Fig. 3 A). Quantitative analysis further validated these findings, revealing significantly higher relative fluorescence intensity for both iNOS and CD86 in LPS-stimulated cells (Fig. 3 E). To assess the paracrine effects of M1 macrophages, conditioned medium (CM) from LPS-polarized macrophages was collected and co-cultured with fibroblast-like synoviocytes (FLS), as illustrated in the experimental schematic (Fig. 3 B). Immunofluorescence analysis (Fig. 3 D) demonstrated that CM-treated FLS displayed markedly elevated expression of fibrosis-related markers (Vimentin, α-SMA, and CTGF) compared to control-treated cells, with quantitative data (Fig. 3 H) confirming significantly increased fluorescence intensity for these proteins. Functional assays further supported these observations. Transwell migration and invasion experiments (Fig. 3 C) revealed that CM from M1 macrophages substantially enhanced FLS motility and invasiveness. Quantitative analysis (Fig. 3 F, G) confirmed significantly higher relative migration and invasion rates in CM-treated groups compared to controls. Collectively, these findings demonstrate that M1 macrophage polarization drives synovial fibrosis by activating fibroblasts and augmenting their migratory and invasive capacities through paracrine signaling. The regulation of macrophage polarization by SGK1 contributes to the alleviation of synovial fibrosis To investigate the potential role of SGK1 in regulating synovial fibrosis through macrophage M1 polarization modulation, we employed dexamethasone-mediated SGK1 activation as an experimental approach. Our initial protein analysis demonstrated that LPS challenge substantially elevated the expression of characteristic M1 markers iNOS and CD86 (Fig. 4 A), whereas SGK1 silencing (si-SGK1) effectively counteracted these LPS-triggered changes. Subsequent quantitative evaluation of protein bands verified that si-SGK1 treatment led to significant decreases in both iNOS and CD86 protein abundance relative to control conditions (Fig. 4 B-C). At the transcriptional level, LPS stimulation markedly enhanced the expression of pro-inflammatory cytokine genes (TNF-α, IL-1β, and IL-6) relative to untreated controls (Fig. 4 D-F). Genetic inhibition of SGK1 substantially attenuated this response, decreasing cytokine mRNA levels by 50–60% compared to LPS-treated macrophages. Importantly, dexamethasone treatment produced comparable suppression of cytokine expression, further supporting the critical role of SGK1 in regulating inflammatory signaling pathways. To examine the paracrine influence of macrophage polarization states on synovial fibroblasts, we employed conditioned media from variously treated macrophages. Subsequent functional analyses revealed that SGK1 silencing significantly impaired the LPS-enhanced proliferative (Fig. 4 G), migratory (Fig. 4 H), and invasive (Fig. 4 I) capacities of synovial fibroblasts. Scratch wound healing assays further confirmed that si-SGK1 suppressed fibroblast migration (Fig.S2A-C). These results indicate that SGK1-dependent M1 polarization promotes fibroblast activation. Additional protein-level investigations by WB and immunofluorescence (IF) staining (Fig. 4 J-K) showed that SGK1 knockdown substantially diminished fibrotic markers, including Vimentin, α-SMA, and CTGF, in synovial fibroblasts. Quantitative analysis of WB bands and IF intensity confirmed a significant reduction in these fibrosis-related proteins upon SGK1 inhibition (Fig. S2D-I). For mechanistic validation, we utilized dexamethasone (DEX) to pharmacologically modulate SGK1 activity. As anticipated, DEX administration neutralized the suppressive effects of si-SGK1, reinstating M1 polarization characteristics and re-potentiating fibroblast activation. Together, these results establish that SGK1 inhibition ameliorates synovial fibrosis by interrupting macrophage M1 polarization and consequent inflammatory cytokine secretion, ultimately reducing fibroblast activation and extracellular matrix protein deposition. SGK1 regulates M1 polarization of synovial macrophages through JAK-STAT signaling pathway We performed RNA sequencing (RNA-Seq) to investigate how SGK1 might regulate M1 macrophage polarization by examining gene expression changes in RAW264.7 cells after si-SGK1 treatment (Fig. 5 A). RNA sequencing analysis revealed that SGK1 knockdown (si-SGK1) induced significant transcriptomic alterations in RAW264.7 macrophages, with 688 differentially expressed genes (DEGs) meeting the stringent criteria of padj 2 (Fig.S3). Among these DEGs, 378 genes were upregulated while 310 were downregulated, demonstrating a clear asymmetric distribution pattern. Principal component analysis (Fig. 5 B) showed distinct clustering between siR-SGK1 and control groups (NC), confirming the robustness of the transcriptional changes. Hierarchical clustering of DEGs (Fig. 5 C) revealed consistent expression patterns across biological replicates, with clear separation between experimental conditions. Functional enrichment analysis identified significant involvement of these DEGs in critical immune pathways, particularly the JAK-STAT signaling pathway (Fig. 5 D), which was further supported by GO analysis showing enrichment in inflammatory response and cytokine activity (Fig. 5 E). Detailed pathway analysis demonstrated specific enrichment in JAK-STAT (Fig. 5 F), IL-6 receptor (Fig. 3 G), and TGFβ signaling pathways (Fig. 5 H), suggesting their potential roles in SGK1-mediated macrophage polarization. Western blot validation (Fig. 5 I) confirmed these findings at the protein level, showing significant reduction in phosphorylation of both JAK1 (62.3% decrease) and STAT3 (58.7% decrease) following SGK1 knockdown. Quantitative analysis (Fig. 5 J-K) further demonstrated that this inhibitory effect on JAK-STAT signaling was potentiated by dexamethasone treatment (p < 0.05). These results collectively establish SGK1 as a critical regulator of macrophage polarization through modulation of the JAK-STAT signaling axis, influencing both transcriptional profiles and key protein phosphorylation events in inflammatory responses. SGK1 inhibitors attenuate OA progression and synovitis in vivo To investigate the therapeutic potential of SGK1 inhibition in osteoarthritis (OA), we developed an ACLT-induced rat model that recapitulated key OA features, including cartilage degradation, synovial inflammation, and fibrotic changes. The experimental design involved periodic intra-articular administration of either PBS or GSK650394 (an SGK1 inhibitor) post-surgery (Fig. 6 A). Comprehensive histological evaluation through multiple staining techniques (Fig. 6 B) demonstrated that GSK650394 treatment effectively mitigated ACLT-induced pathological alterations. Specifically, the inhibitor reduced synovial inflammation and fibrosis, preserved cartilage architecture, and maintained extracellular matrix integrity compared to untreated ACLT controls. Immunofluorescence analysis revealed that GSK650394 administration significantly suppressed the upregulation of inflammatory macrophage markers (iNOS and CD86) observed in OA progression (Fig. 6 C, I-J). Histopathological assessment using H&E and Safranin O staining (Fig. 6 D) revealed distinct morphological changes across experimental groups. Sham-operated animals exhibited intact cartilage architecture with uniform chondrocyte distribution and smooth articular surfaces, whereas ACLT-induced OA rats displayed characteristic pathological alterations including joint space narrowing, cartilage erosion, and proteoglycan depletion. Notably, GSK650394 administration substantially ameliorated these degenerative changes, promoting cartilage tissue regeneration. Complementary analysis through Masson and Sirius red staining (Fig. 6 E) demonstrated that ACLT-induced collagen disorganization and diminished birefringence - particularly in superficial cartilage layers - were markedly improved by GSK650394 treatment, indicating enhanced extracellular matrix preservation. Immunohistochemical profiling (Fig. 6 F) further revealed that while ACLT surgery dramatically upregulated MMP-13 expression while suppressing collagen type II production, therapeutic intervention with GSK650394 effectively reversed these metabolic imbalances. Quantitative evaluations (Fig. 6 G-H, K-L) consistently demonstrated that GSK650394 treatment significantly lowered synovitis and OARSI scores while normalizing MMP-13 and collagen type II expression patterns. These collective findings establish that pharmacological inhibition of SGK1 via GSK650394 exerts comprehensive therapeutic effects against OA progression through dual mechanisms of inflammation suppression and cartilage protection, highlighting its potential as a disease-modifying therapeutic agent for osteoarthritis management. Discussion Contemporary research has established a robust association between synovial inflammation, fibrotic transformation, and the pathogenesis of osteoarthritis (OA) [ 22 – 23 ]. Histologically, synovial membranes display a distinct bilayer organization. The synovial intima, which directly interfaces with the articular cavity, contains two principal cell populations: phagocytic macrophages and matrix-producing synovial fibroblasts [ 24 ]. This superficial layer contrasts with the deeper subintimal region, which consists of dense connective tissue permeated by an extensive microvascular network [ 25 ].The progression of osteoarthritis induces profound architectural reorganization of synovial tissue, manifesting as a triad of interconnected pathological changes: synovial intimal hyperplasia driven by dysregulated cellular proliferation, progressive fibrotic transformation of the superficial synovial compartment, and aberrant neovascularization within the extracellular matrix microenvironment[ 26 ]. These coordinated structural alterations synergistically promote the development of a dysfunctional synovial phenotype that actively contributes to joint degeneration in OA. Emerging evidence highlights the pivotal role of M1-polarized macrophage activation in synovium-mediated joint degeneration [ 27 – 28 ]. Immunohistochemical analyses of OA synovial specimens demonstrate significant upregulation of M1-specific markers (CD86 and iNOS), with expression levels showing strong positive correlation with standardized synovitis scoring systems. Comparative studies utilizing different OA animal models reveal distinct temporal patterns of synovial inflammation. Although the anterior cruciate ligament transection (ACLT) model initially produces less severe synovitis than collagenase-induced models, the inflammatory response exhibits progressive escalation, reaching maximal intensity at 8 weeks post-operation. This chronological progression suggests that synovial microenvironment dysregulation actively contributes to early-stage cartilage degradation, rather than representing a secondary consequence of advanced disease [ 29 ]. Pathological evaluation of ACLT-induced OA models reveals significant synovial immune cell infiltration, marked upregulation of key inflammatory cytokines (TNF-α, IL-1β, and IL-6), and faithful recapitulation of human OA histopathological characteristics. These observations collectively support the critical role of synovial macrophage polarization and the ensuing chronic inflammatory response in driving OA pathogenesis, establishing a mechanistic link between synovial microenvironment dysregulation and progressive joint degeneration. Serum/glucocorticoid-regulated kinase 1 (SGK1) is a pleiotropic enzyme that orchestrates diverse biological processes, including cellular survival, ion homeostasis, metabolic adaptation, and inflammatory regulation [ 30 – 32 ]. As a stress-responsive kinase, its expression exhibits significant tissue-specific variability under the control of multiple physiological and pathological stimuli. In renal physiology, SGK1 serves as a critical modulator of electrolyte balance through its regulatory effects on tubular ion channels and transporters [ 33 ]. Beyond its renal functions, SGK1 has emerged as a key mediator of mechanical stress-induced inflammatory activation in cardiac fibroblasts [ 34 ], while also promoting oncogenic processes through enhancement of tumor cell survival, metastatic potential (via epithelial-mesenchymal transition), and proliferative capacity [ 35 ]. Notably, SGK1 plays a pivotal role in immune cell regulation, particularly in monocyte/macrophage biology. During atherogenesis, it facilitates monocyte migration and matrix metalloproteinase-9 (MMP-9) transcriptional activation [ 36 ]. Pioneering work by Xi et al. demonstrated that SGK1 critically regulates macrophage recruitment and activation in hypoxia-induced pulmonary hypertension, with SGK1-deficient mice exhibiting significantly reduced perivascular and pulmonary macrophage infiltration compared to wild-type counterparts [ 37 ]. Mechanistically, SGK1 orchestrates immunomodulation through two principal molecular cascades: first, by phosphorylating the ubiquitin ligase NEDD4 to promote Th17/Th2 polarization while concurrently inhibiting Treg differentiation, thereby establishing a pro-inflammatory milieu; second, through enhancing SMAD2/3-dependent TGF-β signal transduction, which exacerbates fibrotic pathogenesis [ 38 ]. These dual mechanisms collectively underscore SGK1's pivotal role in bridging inflammatory and fibrotic pathways in disease progression. Furthermore, SGK1 enhances NF-κB transcriptional activity, leading to upregulated expression of various fibro-inflammatory mediators, including connective tissue growth factor. The pathological significance of SGK1 is underscored by its elevated expression across multiple fibrotic disorders, encompassing pulmonary fibrosis, diabetic nephropathy, hepatic cirrhosis, hypertensive cardiac remodeling, peritoneal fibrosis, and Crohn's disease [ 39 ]. Our current findings extend this spectrum to osteoarthritis (OA), suggesting that SGK1 inhibition may confer joint protection through macrophage phenotype modulation. Experimental evidence demonstrates that SGK1 gene silencing significantly attenuates LPS-induced expression of M1 polarization markers (CD86 and iNOS), highlighting its crucial role in innate immune regulation. From a therapeutic standpoint, SGK1 inhibitors demonstrate a novel dual-action mechanism by concurrently suppressing M1 macrophage-mediated synovial inflammation and preventing fibrotic tissue remodeling. This combined pharmacological effect disrupts both the inflammatory cascade and pathological extracellular matrix reorganization, thereby attenuating cartilage degeneration. Such bifunctional properties establish SGK1 as a highly promising therapeutic target for osteoarthritis, offering a comprehensive approach that addresses the intertwined inflammatory and fibrotic pathways driving disease progression. Emerging evidence highlights the pivotal role of the JAK-STAT signaling pathway in modulating inflammatory responses [ 40 ]. Transcriptomic analysis revealed that SGK1 gene silencing markedly reduced JAK and STAT protein phosphorylation in LPS-stimulated RAW 264.7 cells, suggesting SGK1 may serve as an upstream regulator of this critical inflammatory pathway. This study has certain limitations that warrant acknowledgment. Firstly, our investigation was conducted solely using in vitro experiments and in vivo rat models, highlighting the necessity for extensive clinical trials to validate these findings. Additionally, this study did not delve into the specific mechanisms underlying the M1 polarization of synovial macrophages, a critical scientific question that will serve as the focal point of our future research endeavors. Conclusion The current investigation provides novel insights into the functional involvement of SGK1 in osteoarthritis pathogenesis. Our results demonstrate that SGK1 suppression attenuates M1 phenotype polarization in synovial macrophages by modulating the JAK1-STAT3 pathway. This intervention effectively mitigates synovial inflammatory responses and fibrotic changes, while concurrently slowing articular cartilage degradation, collectively contributing to the amelioration of OA progression (Fig. 7 ). These observations significantly broaden the therapeutic prospects of SGK1-targeted interventions for osteoarthritis management. Declarations Acknowledgments The authors thank the Orthopaedic Laboratory of the USTC for providing technical guidance Authors’ Contributions Zhichao Yang: Writing – Original Draft, Review & Editing, Visualization, Verification, Methodology, Formal Analysis, Conceptualization. Ming Wei、 Yang Liu and Wenwei Li: Software, Methodology, Conceptualization. Zhaoyu Li and Liang Yan: Formal Analysis, Data Curation. Yang Lv and Zezhong Guo: Formal Analysis, Data Curation. Wei Zhou and Feng Li: Writing – Review & Editing, Supervision, Resources, Project Administration, Funding Acquisition, Conceptualization. Wei Huang: Writing – Review & Editing, Supervision, Resources, Project Administration, Funding Acquisition, Conceptualization. Funding The study received funding from the National Natural Science Foundation of China (No. 82472503) and the Key Research and Development Program of Anhui Province (No. 2023s07020008). Data Availability The article contains the original contributions discussed in this study; for further questions, please contact the corresponding author. Ethics Approval and Consent to Participate The Ethics Committee of the First Affiliated Hospital of USTC approved all experimental protocols. and the research complied with their animal ethics standards. (Approval No. 2023-N (A) -106) Conflict of interest The authors declare that there are no conflicts of interest concerning this paper. References Prieto-Alhambra, D., Judge, A., Javaid, M. K., Cooper, C., Diez-Perez, A., & Arden, N. K. (2014). Incidence and risk factors for clinically diagnosed knee, hip and hand osteoarthritis: influences of age, gender and osteoarthritis affecting other joints. 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SGK1 enhances Th9 cell differentiation and airway inflammation through NF-κB signaling pathway in asthma. Cell and tissue research , 382 (3), 563–574. https://doi.org/10.1007/s00441-020-03252-3 Artunc, F., & Lang, F. (2014). Mineralocorticoid and SGK1-sensitive inflammation and tissue fibrosis. Nephron. Physiology , 128 (1-2), 35–39. https://doi.org/10.1159/000368267 Harrison D. A. (2012). The Jak/STAT pathway. Cold Spring Harbor perspectives in biology , 4 (3), a011205. https://doi.org/10.1101/cshperspect.a011205 Tables Table 1. Small interfering RNA sequence Gene Sense Antisense si-NC 5'-UUCUCCGAACGU GUCACGUTT-3' 5'-ACGUGACACGUU CGGAGAATT-3' si-SGK1 5'-GUCCAAUCCUCA UGCUAAATT-3' 5'-UUUAGCAUGAGG AUUGGACTT-3' Table 2. Primer sequences of the genes Gene Forward primer Reverse prime GAPDH 5'-GGAAAGCTGTGGCGTGATGG-3' 5'-AGCTCTGGGATGACCTTGCC-3' TNF-a 5'-AGCTCTGGGATGACCTTGCC-3' 5'-CAGAGCAATGACTCCAAAGT-3' 1L-1β 5'-GGCCTTGGGCCTCAAAGGAA-3' 5'-GCTTGGGATCCACACTCTCCA-3' 1L-6 5'-TCTGGGAAATCGTGGAAATGAG-3' 5'-TCTCTGAAGGACTCTGGCTTTGTC-3' Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-6880630","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471722506,"identity":"649b62e0-4a0c-4a80-8353-1b28a1e03862","order_by":0,"name":"Zhichao Yang","email":"","orcid":"","institution":"The First Affiliated Hospital of USTC, University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Yang","suffix":""},{"id":471722507,"identity":"0a16b370-d913-4513-87ff-731afe3ac9a6","order_by":1,"name":"Ming Wei","email":"","orcid":"","institution":"The First 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13:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6880630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6880630/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84824978,"identity":"d1b654e1-e41a-4ef3-bf99-73df62523867","added_by":"auto","created_at":"2025-06-17 16:55:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":816598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe high expression of SGK1 in OA synovial tissue promotes the M1 polarization and fibrotic phenotype of macrophages.(A)​​ \u003c/strong\u003eHistological staining (H\u0026amp;E, Masson, Sirius Red) demonstrating structural disorganization and increased collagen deposition (blue/green in Masson, red in Sirius Red) in OA tissues compared to normal. Scale bars: 100 μm. \u003cstrong\u003e(B)\u003c/strong\u003e​​ Bar graph showing significantly elevated synovitis scores in OA tissues versus normal. \u003cstrong\u003e(C)​​\u003c/strong\u003e Quantification of relative collagen content confirming higher levels in OA tissues. \u003cstrong\u003e(D)\u003c/strong\u003e​​ Immunofluorescence staining of iNOS (pro-inflammatory marker), CD86 (M1 macrophage marker), and CD68 (pan-macrophage marker) with DAPI nuclear counterstain. OA tissues exhibit intensified fluorescence signals. Scale bars: 100 μm.\u003cstrong\u003e(E)​​ \u003c/strong\u003eCo-localization analysis of SGK1 (green) and CD68 (red) in tissues. Scale bars: 100 μm.\u003cstrong\u003e (F)\u003c/strong\u003e Quantitative fluorescence intensity showed that the expression of M1 macrophage markers was up-regulated in osteoarthritis.\u003cstrong\u003e (G)\u003c/strong\u003e​​Quantified fluorescence intensity demonstrating upregulated SGK1 and CD68 in OA.\u003cstrong\u003e (H)​​\u003c/strong\u003e Enhanced SGK1/CD68 co-localization in LPS group versus control. Scale bars: 50 μm. \u003cstrong\u003e(I)\u003c/strong\u003e​​ Quantitative fluorescence intensity showed that the expression of M1 macrophage markers was up-regulated in osteoarthritis. n=3,* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/194ac86d2a4a76663a6e0146.png"},{"id":84826095,"identity":"2d6b620a-0b1f-4e31-87a1-5385c698e399","added_by":"auto","created_at":"2025-06-17 17:11:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":680302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of SGK1 inhibits M1 polarization of synovial macrophages and fibrosis of fibroblasts.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eAnalysis of SGK1 mRNA expression levels using quantitative PCR (qPCR). \u003cstrong\u003e(B-C) \u003c/strong\u003eWestern blotting confirmed the transfection efficiency of SGK1 knockdown. \u003cstrong\u003e(D-F)\u003c/strong\u003eAnalysis of TNF-α, IL-1β, and IL-6 mRNA expression levels using qPCR across various treatment groups. \u003cstrong\u003e(G-I)\u003c/strong\u003e Proliferation, wound healing, migration, and invasion assays of fibroblast-like synoviocytes (FLSs). \u003cstrong\u003e(J-L)\u003c/strong\u003eImmunofluorescence staining images and Western blot analysis of fibrosis marker proteins in various treatment groups. Scale bar: 200 µm. n=3; * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/55782f9e3d6b20a5b07162d0.png"},{"id":84826096,"identity":"31e3cd27-f959-465f-926c-8f7963654cf2","added_by":"auto","created_at":"2025-06-17 17:11:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":638877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPS-induced macrophage polarization promotes fibroblast-like synoviocyte (FLS) activation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)​\u003c/strong\u003e​ Immunofluorescence staining of Mφ cells showing upregulated expression of M1 markers iNOS (green) and CD86 (red) following LPS treatment (1 μg/mL, 24 h). Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. \u003cstrong\u003e(B)​\u003c/strong\u003e​ Schematic of conditioned medium (CM) co-culture system: Mφ cells were polarized to M1 phenotype by LPS, then CM was collected to treat FLS cells for 24 h. \u003cstrong\u003e(C)​\u003c/strong\u003e​ Enhanced migratory and invasive capacities of FLS cells after CM treatment, demonstrated by Transwell assay.\u003cstrong\u003e (D)​\u003c/strong\u003e​ Immunofluorescence analysis showing increased expression of mesenchymal markers Vimentin and α-SMA (red) in CM-treated FLS cells. Nuclei stained with DAPI (blue). Scale bar: 100 μm. \u003cstrong\u003e(E) \u003c/strong\u003eQuantitative analysis of iNOS /CD86 fluorescence intensity in Mφ cells\u003cstrong\u003e (F-G)\u003c/strong\u003eRelative migration/invasion fold changes ​\u003cstrong\u003e​(H)​\u003c/strong\u003e​ Quantitative analysis of Vimentin, α-SMA and CTGF fluorescence intensity. n=3; * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/b48b0ba70e78f6e8c9d6c3b1.png"},{"id":84825395,"identity":"3197a557-ff92-4ca2-94bb-c0a7c8084083","added_by":"auto","created_at":"2025-06-17 17:03:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":600777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of SGK1 inhibits M1 polarization of synovial macrophages and reduces synovial fibrosis. (A-C) \u003c/strong\u003eWestern blot examination of M1 macrophage markers CD86 and iNOS across various treatment groups. \u003cstrong\u003e(D-F)\u003c/strong\u003e Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression levels of TNF-α, IL-1β, and IL-6 across different treatment groups. \u003cstrong\u003e(G-I) \u003c/strong\u003eAssays evaluating proliferation, wound healing, migration, and invasion of fibroblast-like synoviocytes (FLSs). Scale bar: 200 µm \u003cstrong\u003e(J-K)\u003c/strong\u003e Images from immunofluorescence staining and Western blot analysis of proteins related to fibrosis in different treatment groups. Scale bar: 100 µm. n=3; * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/e1adb4bce0b957d197f3d575.png"},{"id":84824980,"identity":"9ab824fd-f910-4f62-bb95-ead8cf7e89ca","added_by":"auto","created_at":"2025-06-17 16:55:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":355457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of SGK1 inhibits M1 polarization of synovial macrophages by inhibiting JAK-STAT signaling pathway. \u003c/strong\u003e(A) Graphical depiction of the RNA sequencing analysis process. (B-C) Identification of differentially expressed genes via a volcano plot and hierarchical clustering heatmap. (D-E) Examination of KEGG pathways along with Gene Ontology (GO) enrichment studies. (F-H) Assessment of relevant pathways through Gene Set Enrichment Analysis (GSEA). (I-K) Western blot analysis to evaluate phosphorylated JAK1 (p-JAK1), total JAK1, phosphorylated STAT3 (p-STAT3), total STAT3, and β-actin levels in RAW264.7 cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/eb23e3654438e1a3c67b4c27.png"},{"id":84824988,"identity":"d46bc511-5c69-46a1-9eab-55992edabbc6","added_by":"auto","created_at":"2025-06-17 16:55:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1117204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGSK650394 alleviates osteoarthritis progression in ACLT-induced rat model through modulation of inflammatory markers and extracellular matrix composition​.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic illustration of experimental design: Anterior cruciate ligament transection (ACLT) surgery to induce osteoarthritis (OA) followed by treatment with GSK650394. \u003cstrong\u003e(B)\u003c/strong\u003e Representative histological staining of synovial tissues (Sham, ACLT and ACLT+GSK650394 groups) : hematoxylin-eosin (H\u0026amp;E), Masson trichrome, and Sirius red staining. Scale bar: 100 μm\u003cstrong\u003e（C）\u003c/strong\u003eImmunofluorescence staining of pro-inflammatory macrophage markers (iNOS and CD86) was performed, and DAPI nuclear counterstaining was performed. Scale bar: 100 μm \u003cstrong\u003e(D-E)\u003c/strong\u003e Representative histological staining of rat knee joints (Sham, ACLT and ACLT+GSK650394 groups): hematoxylin-eosin (H\u0026amp;E) staining, Safranin O staining, Masson trichrome staining, and Sirius red staining. Cartilage degradation, collagen deposition, and proteoglycan loss were shown. Scale bar: 200 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Immunohistochemistry of extracellular matrix components (MMP13, COL-II).\u003cstrong\u003e（G-H）\u003c/strong\u003eQuantitative analysis of synovitis score and cartilage damage. \u003cstrong\u003e(I-J)\u003c/strong\u003e Quantitative analysis of iNOS and CD86 fluorescence intensity,\u003cstrong\u003e（K-L）\u003c/strong\u003eMMP13 and COLL-Ⅱ positive cells were quantified. Scale bar: 100 μm\u003cstrong\u003e. \u003c/strong\u003en = 3; * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/fb54662327d451ffa1b26df3.png"},{"id":84824995,"identity":"80c33e85-3603-4306-9eab-1448fb6cf29b","added_by":"auto","created_at":"2025-06-17 16:55:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":525904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic Framework of SGK1 Inhibitors in the Treatment of Osteoarthritis.\u003c/strong\u003e By targeting the JAK1-STAT3 pathway, the SGK1 inhibitor (GSK650394) prevents synovial M1 macrophage polarization and synovial fibrosis, which helps in relieving osteoarthritis.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/127d879db9218dcfc1cbd3a4.png"},{"id":87307033,"identity":"0799a4d3-df71-49e1-91cf-04e0552462ac","added_by":"auto","created_at":"2025-07-22 14:17:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5846795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/ed97e262-8bbf-4ff8-87b7-ae837e50296f.pdf"},{"id":84825396,"identity":"f3f91c4a-f8b9-4f07-ad4e-9f56b5150a9a","added_by":"auto","created_at":"2025-06-17 17:03:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":912318,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6880630/v1/8661806e4a1ae555947fe31b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting SGK1 Mitigates Synovial Fibrosis via Suppressing M1 Polarization to Alleviate Osteoarthritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) stands as one of the most prevalent degenerative joint disorders worldwide, affecting approximately 7% of the global population, with incidence rates escalating dramatically with advancing age, particularly in weight-bearing joints such as the knees and hips [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recognized as a leading cause of disability among the elderly, OA imposes a substantial socioeconomic burden, compounded by the limitations of current therapeutic strategies, which primarily focus on symptom alleviation rather than halting or reversing disease progression [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The pathogenesis of OA is characterized by a complex interplay of biomechanical and biochemical factors that collectively drive the progressive deterioration of articular cartilage, synovial membrane, and periarticular tissues [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these mechanisms, emerging evidence underscores synovial inflammation and fibrosis as pivotal contributors to OA progression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the precise molecular pathways governing these processes remain incompletely elucidated, necessitating further investigation to identify novel therapeutic targets.\u003c/p\u003e \u003cp\u003eThe synovial membrane, a critical structure responsible for joint lubrication and nutrient supply, undergoes profound pathological remodeling during OA. These changes include aberrant immune cell infiltration\u0026mdash;particularly of macrophages\u0026mdash;dysregulated cytokine production, and excessive collagen deposition, all of which disrupt synovial homeostasis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Clinically, these alterations manifest as joint effusion, tenderness, and progressive impairment of mobility [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Central to this pathogenic cascade is the polarization of synovial macrophages into functionally distinct subsets: the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].M1 macrophages, activated by Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide, LPS) or Th1 cytokines (e.g., interferon-gamma, IFN-γ), are characterized by the expression of surface markers such as CD86 and inducible nitric oxide synthase (iNOS). These cells secrete a repertoire of catabolic mediators, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), which exacerbate cartilage degradation and promote synovial fibrosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, M2 macrophages, identified by markers like CD163, foster tissue repair through the secretion of anti-inflammatory cytokines such as transforming growth factor-beta (TGF-β) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the inflammatory milieu of OA, the balance between these subsets is skewed toward M1 dominance, creating a self-perpetuating cycle of stromal activation and extracellular matrix (ECM) remodeling [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While macrophage polarization has emerged as a promising therapeutic target, the molecular mechanisms regulating this process in the synovium remain poorly defined.\u003c/p\u003e \u003cp\u003eSerum- and glucocorticoid-regulated kinase 1 (SGK1), a serine/threonine kinase from the AGC family, has recently emerged as a critical regulator of inflammation and fibrosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Initially identified for its role in renal ion transport and cellular stress responses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], SGK1 is now known to influence immune modulation, affecting T-cell differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], neutrophil clearance, and macrophage activation in cardiovascular and pulmonary diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mechanistically, SGK1 interacts with key inflammatory pathways\u0026mdash;including NF-κB and JAK-STAT signaling\u0026mdash;to enhance cytokine production and fibrotic responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In fibrotic conditions such as pulmonary fibrosis and cirrhosis, SGK1 overexpression correlates with ECM deposition via TGF-β/SMAD activation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, SGK1 mediates mechanical stress-induced inflammation in cardiac fibroblasts [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and facilitates tumor metastasis by promoting epithelial-mesenchymal transition (EMT) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, its role in synovial pathology, particularly in macrophage polarization during OA, remains unexplored.\u003c/p\u003e \u003cp\u003ePreliminary studies suggest that SGK1 may contribute to joint degeneration. In chondrocytes, SGK1 inhibition reduces inflammatory responses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], while murine OA models indicate its involvement in cartilage degradation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Paradoxically, SGK1 also suppresses innate immune responses to bacterial infections [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], demonstrating context-dependent functionality. These conflicting findings highlight the need for a systematic investigation into SGK1\u0026rsquo;s synovial-specific actions. Importantly, no prior studies have examined whether SGK1 regulates synovial fibrosis through macrophage reprogramming or identified its downstream effectors in joint tissues.\u003c/p\u003e \u003cp\u003eTo elucidate the role of SGK1 in synovial inflammation and fibrosis, we employed a combination of in vitro and in vivo models. LPS stimulated RAW264.7 macrophages were used to investigate SGK1's influence on macrophage polarization, while an ACLT rat model was utilized to recapitulate the pathological features of OA in vivo. Our findings demonstrate that SGK1 knockdown suppresses M1 polarization by inhibiting JAK1/STAT3 signaling, subsequently attenuating FLS proliferation and collagen deposition. These results not only advance our understanding of OA pathogenesis but also position SGK1 as a promising therapeutic target for OA treatment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eClinical Samples and Ethical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKnee synovial tissues were obtained from osteoarthritis patients (Kellgren-Lawrence grade III-IV) undergoing total knee arthroplasty. Normal control tissues were collected from traumatic amputation patients with no history of arthritis. This study was approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (Approval No. 2024-ky546), and written informed consent was obtained from all participants in accordance with the ethical principles of the Declaration of Helsinki. Fresh tissues were flash-frozen in liquid nitrogen immediately after collection and stored at -80\u0026deg;C for long-term preservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 cells (Shanghai Cell Bank, Chinese Academy of Sciences, Cat.SCSP-5036) were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Primary human fibroblast-like synoviocytes (FLSs) were isolated from surgically resected synovial tissues. Briefly, tissues were rinsed with PBS and digested with 0.1% collagenase II (Sigma) at 37\u0026deg;C for 2 hours. The digested suspension was filtered through a 100 \u0026mu;m cell strainer to remove undigested debris, centrifuged at 1000g for 5 minutes, and resuspended in culture medium. \u0026nbsp;The medium was replaced every 48 hours. Cells from passages 4\u0026ndash;6 were used for experiments, with purity (\u0026gt;95%) confirmed by flow cytometry (CD90+/CD68\u0026minus;) and vimentin immunofluorescence staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFLSs were co-cultured with macrophage-conditioned medium for 24 hours. Cell viability was assessed using the CCK-8 kit (Dojindo), following the manufacturer\u0026rsquo;s protocol. Absorbance was measured at 450 nm using a microplate reader (BioTek).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on preliminary screening (Figure 2A), siRNA#2 was selected for subsequent experiments. \u0026nbsp;RAW264.7 cells were seeded in 6-well plates at 1\u0026times;10⁵ cells/well. After 24 hours, siRNA (20 nM) and riboFECT\u0026trade; CP transfection reagent (5 \u0026mu;l, RiboBio) were mixed to form complexes. \u0026nbsp; The transfection mixture was added to cells, and the medium was replaced with fresh complete medium 6 hours post-transfection. \u0026nbsp;Knockdown efficiency was validated by qPCR (\u0026Delta;\u0026Delta;Ct method) and Western blotting after 24 hours. siRNA sequences are listed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Sequencing Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol and assessed for integrity (RIN \u0026gt;8.0) via an Agilent 2100 Bioanalyzer. Libraries were prepared with the NEBNext\u0026reg; Ultra II RNA Library Prep Kit and sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads, ~30 million reads/sample). Raw data were quality-checked with Fast QC, aligned to the mm10 genome using STAR (v2.7.9a), and analyzed for differential expression (|log2FC| \u0026gt;1, FDR \u0026lt;0.05) with DESeq2. Functional enrichment (GO and KEGG) was performed using clusterProfiler (v4.0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Real-Time PCR Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from treated RAW264.7 cells using TRIzol (Beyotime, China). RNA purity and concentration were quantified via NanoDrop 2000. cDNA was synthesized by reverse transcription, and qPCR reactions contained 10 \u0026mu;l Master Mix, 0.5 \u0026mu;l each of forward/reverse primers, 2 \u0026mu;l cDNA, and 7 \u0026mu;l ddH2O. Relative expression levels were calculated using the 2^(-\u0026Delta;\u0026Delta;Ct) method with GAPDH as the reference. Primer sequences are provided in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Proteins were extracted using RIPA lysis buffer (1% protease inhibitor cocktail) and quantified via BCA assay. Samples (10 \u0026mu;g) were separated by SDS-PAGE and transferred to PVDF membranes (0.45 \u0026mu;m) at 70\u0026ndash;120 V under ice cooling. Membranes were blocked with rapid blocking buffer (Beyotime) for 20 minutes, incubated overnight with primary antibodies at 4\u0026deg;C, and then with HRP-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using ECL and analyzed with Image Lab. Antibodies included anti-CD68 (sc-20060), anti-iNOS (ab178945), anti-CD86 (A1199), anti-SGK1 (sc-28338), anti-JAK1 (YT2424), anti-p-JAK1 (YP0154), anti-STAT3 (YT4443), anti-p-STAT3 (YP0250), anti-CTGF (AF7537), anti-\u0026alpha;-SMA (AF1032), anti-vimentin (#5741), anti-MMP13 (ab39012), anti-collagen II (28457-1-AP), and anti-\u0026beta;-actin (YM3028).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing experiments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFLSs were subjected to diverse treatment conditions and subsequently seeded into 6-well plates. The cells were grown until they achieved 90%-100% confluence. A linear scratch was introduced on the surface of the monolayer using a 200 \u0026micro;L pipette tip. Floating cells were removed with two PBS washes, The cells that stayed attached were cultured in serum-free DMEM, and those at the wound location were viewed with an inverted microscope made by Carl Zeiss in Germany,at both 0 and 24 hours following the scratch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration and invasion assays\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFLSs were subjected to various treatments and then placed into the upper sections of Transwell inserts at a concentration of 2 \u0026times; 10^4 cells per well in a medium without serum. To clear away floating cells, two PBS washes were executed, and the adherent cells were preserved in serum-free DMEM. An inverted microscope from Carl Zeiss, Germany, was used to view the cells at the wound site. Non-migrating cells were wiped away using a damp cotton swab, and the remaining cells were stained with a 0.1% crystal violet solution. Using an inverted microscope from Carl Zeiss, Germany, images at the microscopic level were obtained, and the migrated cells were tallied.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMacrophage conditioned medium (CM)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter treating RAW264.7 macrophages with different stimuli for 24 hours, the supernatants were collected and centrifuged at 1000 g for 5 minutes, then stored at -80 \u0026deg;C for future experiments. The macrophage-derived conditioned medium (CM) was diluted in a 1:1 ratio with serum-free medium and subsequently applied to FLSs for further analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Model Establishment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four male SD rats (8 weeks, 250\u0026plusmn;20 g) were randomized into three groups: sham, ACLT and GSK650394. Under anesthesia (1% pentobarbital sodium, 40 mg/kg), the ACL was transected via medial arthrotomy. Postoperatively, meloxicam (1 mg/kg/day) was administered subcutaneously for 3 days. Procedures followed ARRIVE guidelines and were approved by the Institutional Animal Ethics Committee (Approval No. 2023-N(A)-106).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKnee specimens were fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% EDTA (pH 7.4) for 4 weeks with daily solution changes, then paraffin-embedded and sectioned at 4 \u0026mu;m thickness. For synovitis evaluation, H\u0026amp;E-stained sections were scored using the Krenn scale (0-9) based on synovial hyperplasia, inflammatory infiltration, and stromal density. Cartilage integrity was assessed via Safranin O-fast green staining (0.1% Safranin O for 5 min, 0.02% fast green for 3 min) with OARSI scoring (0-6). Collagen deposition was analyzed through dual approaches: (1) Masson\u0026apos;s trichrome staining (Weigert\u0026apos;s hematoxylin [5 min], Biebrich scarlet [5 min], phosphomolybdic acid [10 min], aniline blue [5 min]) quantified using ImageJ (v1.53, NIH) thresholding of blue-stained areas; and (2) Picrosirius red staining (0.1% Sirius red in saturated picric acid, 1 h RT) examined under both brightfield and polarized light (Nikon Eclipse Ci-L with polarizing filters), with type I (orange-red birefringence) and type III (green) collagen differentiation. All histological scoring was performed by two blinded investigators (ICC \u0026gt;0.85).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry and immunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynovial tissues from humans or rats were preserved in 4% paraformaldehyde for a day, then embedded in paraffin and sliced into sections 4-6 \u0026micro;m thick. The sections underwent deparaffinization with xylene and were rehydrated using a graded series of ethanol prior to retrieving antigens. Before antigen retrieval, the sections were deparaffinized with xylene and rehydrated through a series of graded ethanol. The primary antibodies were subsequently added and left to incubate overnight at 4\u0026deg;C. Sections were incubated with HRP-conjugated secondary antibodies for one hour at room temperature the following day, and images were obtained using a Zeiss light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll experimental results are presented as the Mean \u0026plusmn; Standard Deviation (Mean \u0026plusmn; SD), With statistical analyses conducted using GraphPad Prism version 9.0. For assessing differences across multiple groups, the analysis involved a one-way ANOVA, and Tukey\u0026apos;s post hoc test was applied for comparing pairs. When comparing two groups, an independent t-test was applied. The criterion for statistical significance was established at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSGK1 is highly expressed in synovial macrophages with enhanced M1 polarization and fibrosis in OA.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOsteoarthritis (OA) is a chronic joint disorder marked by progressive cartilage degradation, synovial inflammation, and fibrotic changes. Emerging research highlights the pivotal involvement of synovial macrophages in disease pathogenesis. Comparative histopathological examination demonstrated pronounced tissue disorganization and fibrotic alterations in OA synovium relative to healthy controls. Hematoxylin-eosin staining revealed well-preserved cellular architecture in normal synovium, contrasting with the disarranged cellular patterns and prominent inflammatory infiltration observed in OA specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The fibrotic transformation of synovial tissue was corroborated through Masson's trichrome and Sirius red staining techniques, which unveiled substantial collagen accumulation in OA samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Correspondingly, synovitis severity scores were markedly elevated in OA patients compared to healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Quantitative assessments further validated these observations, demonstrating a statistically significant rise in collagen deposition within OA synovial tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicative of progressive fibrotic remodeling during OA development. Immunofluorescence analysis revealed a pronounced polarization shift of synovial macrophages toward a proinflammatory M1 phenotype in OA. Comparative evaluation showed substantial upregulation of inducible nitric oxide synthase (iNOS) and CD86\u0026mdash;characteristic M1 macrophage markers\u0026mdash;in OA synovium relative to normal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Quantitative measurement of fluorescence intensities confirmed the predominant M1 polarization state of macrophages in OA specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting their potential contribution to synovial inflammation and structural deterioration. A pivotal observation in this investigation was the heightened expression of serum- and glucocorticoid-regulated kinase 1 (SGK1) within OA synovial macrophages. Immunofluorescence visualization demonstrated robust SGK1 expression (green signal) that colocalized with CD68\u0026thinsp;+\u0026thinsp;macrophages (red signal) in OA synovium, whereas minimal SGK1 expression was detected in control tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Quantitative fluorescence analysis substantiated these findings, revealing significantly intensified SGK1 and CD68 signals in OA samples compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The correlation between SGK1 expression and macrophage activation implies a potential regulatory role for SGK1 in macrophage polarization and synovial inflammatory responses during OA progression. In vitro experiments employing lipopolysaccharide (LPS)-stimulated macrophages provided additional validation. LPS exposure induced notable upregulation of SGK1/CD68 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), with fluorescence quantification demonstrating approximately 2.5-fold enhancement compared to unstimulated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These results suggest that inflammatory mediators may augment SGK1 expression in macrophages, potentially driving their polarization toward a proinflammatory state. Considering that M1-polarized macrophages exacerbate synovial inflammation and fibrosis through secretion of proinflammatory mediators (e.g., TNF-α, IL-1β, IL-6) and matrix-degrading enzymes, the upregulated SGK1 expression in synovial macrophages may constitute a crucial molecular link between macrophage activation and synovial pathology in OA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eKnockdown of SGK1 alleviated synovial inflammation and fibrosis\u003c/h3\u003e\n\u003cp\u003eTo further elucidate the regulatory role of SGK1 in synovial inflammation and fibrosis, we employed siRNA-mediated knockdown of SGK1 in synovial macrophages. qPCR analysis confirmed efficient silencing of SGK1 mRNA in RAW264.7 cells transfected with si-SGK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and subsequent quantitative evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) demonstrated a significant reduction in SGK1 protein levels compared to the si-NC control group. Notably, LPS stimulation markedly upregulated SGK1 expression, an effect that was substantially attenuated by si-SGK1 transfection, confirming the efficacy of SGK1 knockdown at the protein level. Subsequent investigations revealed that SGK1 knockdown significantly suppressed the LPS-induced upregulation of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F), underscoring the pivotal role of SGK1 in mediating inflammatory responses. To assess the downstream effects of SGK1 knockdown on synovial fibroblasts, we cultured these cells with conditioned media from treated RAW264.7 cells. CCK-8 assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) revealed that LPS stimulation enhanced the proliferative capacity of synovial fibroblasts, whereas this effect was markedly inhibited by si-SGK1-conditioned medium. Further functional assays, including scratch, migration, and invasion experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u0026ndash;J and Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C), demonstrated that synovial fibroblasts treated with si-SGK1-conditioned medium exhibited significantly impaired migratory and invasive capabilities compared to those exposed to si-NC-conditioned medium. Immunofluorescence and Western blot analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L) corroborated these findings, showing that SGK1 knockdown led to a pronounced reduction in the expression of fibrosis-associated proteins, such as Vimentin, α-SMA, and CTGF, in synovial fibroblasts. Quantitative analysis of these proteins (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u0026ndash;I) further confirmed the significant downregulation of Vimentin, α-SMA, and CTGF in the LPS\u0026thinsp;+\u0026thinsp;si-SGK1 group compared to controls, as evidenced by both fluorescence intensity measurements and protein level ratios normalized to β-actin. Collectively, these results demonstrate that SGK1 silencing not only attenuates pro-inflammatory cytokine production in RAW264.7 cells but also inhibits the fibrotic and proliferative responses of synovial fibroblasts by modulating the paracrine effects of inflammatory mediators. Our findings provide robust evidence supporting the critical role of SGK1 in regulating synovial inflammation and fibrosis, highlighting its potential as a therapeutic target for osteoarthritis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eM1 polarization of macrophages leads to a synovial fibrosis phenotype.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExperimental results demonstrate that LPS-induced M1 macrophage polarization promotes synovial fibrosis via paracrine mechanisms. Immunofluorescence staining confirmed that LPS-treated macrophages exhibited stronger expression of M1 markers (iNOS in green, CD86 in red) compared to the control group (Ctrl), with merged images (DAPI in blue) clearly indicating enhanced M1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Quantitative analysis further validated these findings, revealing significantly higher relative fluorescence intensity for both iNOS and CD86 in LPS-stimulated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). To assess the paracrine effects of M1 macrophages, conditioned medium (CM) from LPS-polarized macrophages was collected and co-cultured with fibroblast-like synoviocytes (FLS), as illustrated in the experimental schematic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Immunofluorescence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) demonstrated that CM-treated FLS displayed markedly elevated expression of fibrosis-related markers (Vimentin, α-SMA, and CTGF) compared to control-treated cells, with quantitative data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) confirming significantly increased fluorescence intensity for these proteins. Functional assays further supported these observations. Transwell migration and invasion experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) revealed that CM from M1 macrophages substantially enhanced FLS motility and invasiveness. Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G) confirmed significantly higher relative migration and invasion rates in CM-treated groups compared to controls. Collectively, these findings demonstrate that M1 macrophage polarization drives synovial fibrosis by activating fibroblasts and augmenting their migratory and invasive capacities through paracrine signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe regulation of macrophage polarization by SGK1 contributes to the alleviation of synovial fibrosis\u003c/h3\u003e\n\u003cp\u003eTo investigate the potential role of SGK1 in regulating synovial fibrosis through macrophage M1 polarization modulation, we employed dexamethasone-mediated SGK1 activation as an experimental approach. Our initial protein analysis demonstrated that LPS challenge substantially elevated the expression of characteristic M1 markers iNOS and CD86 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), whereas SGK1 silencing (si-SGK1) effectively counteracted these LPS-triggered changes. Subsequent quantitative evaluation of protein bands verified that si-SGK1 treatment led to significant decreases in both iNOS and CD86 protein abundance relative to control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). At the transcriptional level, LPS stimulation markedly enhanced the expression of pro-inflammatory cytokine genes (TNF-α, IL-1β, and IL-6) relative to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Genetic inhibition of SGK1 substantially attenuated this response, decreasing cytokine mRNA levels by 50\u0026ndash;60% compared to LPS-treated macrophages. Importantly, dexamethasone treatment produced comparable suppression of cytokine expression, further supporting the critical role of SGK1 in regulating inflammatory signaling pathways. To examine the paracrine influence of macrophage polarization states on synovial fibroblasts, we employed conditioned media from variously treated macrophages. Subsequent functional analyses revealed that SGK1 silencing significantly impaired the LPS-enhanced proliferative (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), migratory (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), and invasive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI) capacities of synovial fibroblasts. Scratch wound healing assays further confirmed that si-SGK1 suppressed fibroblast migration (Fig.S2A-C). These results indicate that SGK1-dependent M1 polarization promotes fibroblast activation. Additional protein-level investigations by WB and immunofluorescence (IF) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K) showed that SGK1 knockdown substantially diminished fibrotic markers, including Vimentin, α-SMA, and CTGF, in synovial fibroblasts. Quantitative analysis of WB bands and IF intensity confirmed a significant reduction in these fibrosis-related proteins upon SGK1 inhibition (Fig. S2D-I). For mechanistic validation, we utilized dexamethasone (DEX) to pharmacologically modulate SGK1 activity. As anticipated, DEX administration neutralized the suppressive effects of si-SGK1, reinstating M1 polarization characteristics and re-potentiating fibroblast activation. Together, these results establish that SGK1 inhibition ameliorates synovial fibrosis by interrupting macrophage M1 polarization and consequent inflammatory cytokine secretion, ultimately reducing fibroblast activation and extracellular matrix protein deposition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSGK1 regulates M1 polarization of synovial macrophages through JAK-STAT signaling pathway\u003c/h2\u003e \u003cp\u003eWe performed RNA sequencing (RNA-Seq) to investigate how SGK1 might regulate M1 macrophage polarization by examining gene expression changes in RAW264.7 cells after si-SGK1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). RNA sequencing analysis revealed that SGK1 knockdown (si-SGK1) induced significant transcriptomic alterations in RAW264.7 macrophages, with 688 differentially expressed genes (DEGs) meeting the stringent criteria of padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |FoldChange| \u0026gt; 2 (Fig.S3). Among these DEGs, 378 genes were upregulated while 310 were downregulated, demonstrating a clear asymmetric distribution pattern. Principal component analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) showed distinct clustering between siR-SGK1 and control groups (NC), confirming the robustness of the transcriptional changes. Hierarchical clustering of DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) revealed consistent expression patterns across biological replicates, with clear separation between experimental conditions. Functional enrichment analysis identified significant involvement of these DEGs in critical immune pathways, particularly the JAK-STAT signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), which was further supported by GO analysis showing enrichment in inflammatory response and cytokine activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Detailed pathway analysis demonstrated specific enrichment in JAK-STAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), IL-6 receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), and TGFβ signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), suggesting their potential roles in SGK1-mediated macrophage polarization. Western blot validation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI) confirmed these findings at the protein level, showing significant reduction in phosphorylation of both JAK1 (62.3% decrease) and STAT3 (58.7% decrease) following SGK1 knockdown. Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-K) further demonstrated that this inhibitory effect on JAK-STAT signaling was potentiated by dexamethasone treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results collectively establish SGK1 as a critical regulator of macrophage polarization through modulation of the JAK-STAT signaling axis, influencing both transcriptional profiles and key protein phosphorylation events in inflammatory responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSGK1 inhibitors attenuate OA progression and synovitis in vivo\u003c/h2\u003e \u003cp\u003eTo investigate the therapeutic potential of SGK1 inhibition in osteoarthritis (OA), we developed an ACLT-induced rat model that recapitulated key OA features, including cartilage degradation, synovial inflammation, and fibrotic changes. The experimental design involved periodic intra-articular administration of either PBS or GSK650394 (an SGK1 inhibitor) post-surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Comprehensive histological evaluation through multiple staining techniques (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) demonstrated that GSK650394 treatment effectively mitigated ACLT-induced pathological alterations. Specifically, the inhibitor reduced synovial inflammation and fibrosis, preserved cartilage architecture, and maintained extracellular matrix integrity compared to untreated ACLT controls. Immunofluorescence analysis revealed that GSK650394 administration significantly suppressed the upregulation of inflammatory macrophage markers (iNOS and CD86) observed in OA progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, I-J). Histopathological assessment using H\u0026amp;E and Safranin O staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) revealed distinct morphological changes across experimental groups. Sham-operated animals exhibited intact cartilage architecture with uniform chondrocyte distribution and smooth articular surfaces, whereas ACLT-induced OA rats displayed characteristic pathological alterations including joint space narrowing, cartilage erosion, and proteoglycan depletion. Notably, GSK650394 administration substantially ameliorated these degenerative changes, promoting cartilage tissue regeneration. Complementary analysis through Masson and Sirius red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) demonstrated that ACLT-induced collagen disorganization and diminished birefringence - particularly in superficial cartilage layers - were markedly improved by GSK650394 treatment, indicating enhanced extracellular matrix preservation. Immunohistochemical profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) further revealed that while ACLT surgery dramatically upregulated MMP-13 expression while suppressing collagen type II production, therapeutic intervention with GSK650394 effectively reversed these metabolic imbalances. Quantitative evaluations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H, K-L) consistently demonstrated that GSK650394 treatment significantly lowered synovitis and OARSI scores while normalizing MMP-13 and collagen type II expression patterns. These collective findings establish that pharmacological inhibition of SGK1 via GSK650394 exerts comprehensive therapeutic effects against OA progression through dual mechanisms of inflammation suppression and cartilage protection, highlighting its potential as a disease-modifying therapeutic agent for osteoarthritis management.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eContemporary research has established a robust association between synovial inflammation, fibrotic transformation, and the pathogenesis of osteoarthritis (OA) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Histologically, synovial membranes display a distinct bilayer organization. The synovial intima, which directly interfaces with the articular cavity, contains two principal cell populations: phagocytic macrophages and matrix-producing synovial fibroblasts [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This superficial layer contrasts with the deeper subintimal region, which consists of dense connective tissue permeated by an extensive microvascular network [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].The progression of osteoarthritis induces profound architectural reorganization of synovial tissue, manifesting as a triad of interconnected pathological changes: synovial intimal hyperplasia driven by dysregulated cellular proliferation, progressive fibrotic transformation of the superficial synovial compartment, and aberrant neovascularization within the extracellular matrix microenvironment[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These coordinated structural alterations synergistically promote the development of a dysfunctional synovial phenotype that actively contributes to joint degeneration in OA.\u003c/p\u003e \u003cp\u003eEmerging evidence highlights the pivotal role of M1-polarized macrophage activation in synovium-mediated joint degeneration [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Immunohistochemical analyses of OA synovial specimens demonstrate significant upregulation of M1-specific markers (CD86 and iNOS), with expression levels showing strong positive correlation with standardized synovitis scoring systems. Comparative studies utilizing different OA animal models reveal distinct temporal patterns of synovial inflammation. Although the anterior cruciate ligament transection (ACLT) model initially produces less severe synovitis than collagenase-induced models, the inflammatory response exhibits progressive escalation, reaching maximal intensity at 8 weeks post-operation. This chronological progression suggests that synovial microenvironment dysregulation actively contributes to early-stage cartilage degradation, rather than representing a secondary consequence of advanced disease [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePathological evaluation of ACLT-induced OA models reveals significant synovial immune cell infiltration, marked upregulation of key inflammatory cytokines (TNF-α, IL-1β, and IL-6), and faithful recapitulation of human OA histopathological characteristics. These observations collectively support the critical role of synovial macrophage polarization and the ensuing chronic inflammatory response in driving OA pathogenesis, establishing a mechanistic link between synovial microenvironment dysregulation and progressive joint degeneration.\u003c/p\u003e \u003cp\u003eSerum/glucocorticoid-regulated kinase 1 (SGK1) is a pleiotropic enzyme that orchestrates diverse biological processes, including cellular survival, ion homeostasis, metabolic adaptation, and inflammatory regulation [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. As a stress-responsive kinase, its expression exhibits significant tissue-specific variability under the control of multiple physiological and pathological stimuli. In renal physiology, SGK1 serves as a critical modulator of electrolyte balance through its regulatory effects on tubular ion channels and transporters [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Beyond its renal functions, SGK1 has emerged as a key mediator of mechanical stress-induced inflammatory activation in cardiac fibroblasts [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], while also promoting oncogenic processes through enhancement of tumor cell survival, metastatic potential (via epithelial-mesenchymal transition), and proliferative capacity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, SGK1 plays a pivotal role in immune cell regulation, particularly in monocyte/macrophage biology. During atherogenesis, it facilitates monocyte migration and matrix metalloproteinase-9 (MMP-9) transcriptional activation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Pioneering work by Xi et al. demonstrated that SGK1 critically regulates macrophage recruitment and activation in hypoxia-induced pulmonary hypertension, with SGK1-deficient mice exhibiting significantly reduced perivascular and pulmonary macrophage infiltration compared to wild-type counterparts [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Mechanistically, SGK1 orchestrates immunomodulation through two principal molecular cascades: first, by phosphorylating the ubiquitin ligase NEDD4 to promote Th17/Th2 polarization while concurrently inhibiting Treg differentiation, thereby establishing a pro-inflammatory milieu; second, through enhancing SMAD2/3-dependent TGF-β signal transduction, which exacerbates fibrotic pathogenesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These dual mechanisms collectively underscore SGK1's pivotal role in bridging inflammatory and fibrotic pathways in disease progression. Furthermore, SGK1 enhances NF-κB transcriptional activity, leading to upregulated expression of various fibro-inflammatory mediators, including connective tissue growth factor.\u003c/p\u003e \u003cp\u003eThe pathological significance of SGK1 is underscored by its elevated expression across multiple fibrotic disorders, encompassing pulmonary fibrosis, diabetic nephropathy, hepatic cirrhosis, hypertensive cardiac remodeling, peritoneal fibrosis, and Crohn's disease [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our current findings extend this spectrum to osteoarthritis (OA), suggesting that SGK1 inhibition may confer joint protection through macrophage phenotype modulation. Experimental evidence demonstrates that SGK1 gene silencing significantly attenuates LPS-induced expression of M1 polarization markers (CD86 and iNOS), highlighting its crucial role in innate immune regulation.\u003c/p\u003e \u003cp\u003eFrom a therapeutic standpoint, SGK1 inhibitors demonstrate a novel dual-action mechanism by concurrently suppressing M1 macrophage-mediated synovial inflammation and preventing fibrotic tissue remodeling. This combined pharmacological effect disrupts both the inflammatory cascade and pathological extracellular matrix reorganization, thereby attenuating cartilage degeneration. Such bifunctional properties establish SGK1 as a highly promising therapeutic target for osteoarthritis, offering a comprehensive approach that addresses the intertwined inflammatory and fibrotic pathways driving disease progression.\u003c/p\u003e \u003cp\u003eEmerging evidence highlights the pivotal role of the JAK-STAT signaling pathway in modulating inflammatory responses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Transcriptomic analysis revealed that SGK1 gene silencing markedly reduced JAK and STAT protein phosphorylation in LPS-stimulated RAW 264.7 cells, suggesting SGK1 may serve as an upstream regulator of this critical inflammatory pathway.\u003c/p\u003e \u003cp\u003eThis study has certain limitations that warrant acknowledgment. Firstly, our investigation was conducted solely using in vitro experiments and in vivo rat models, highlighting the necessity for extensive clinical trials to validate these findings. Additionally, this study did not delve into the specific mechanisms underlying the M1 polarization of synovial macrophages, a critical scientific question that will serve as the focal point of our future research endeavors.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current investigation provides novel insights into the functional involvement of SGK1 in osteoarthritis pathogenesis. Our results demonstrate that SGK1 suppression attenuates M1 phenotype polarization in synovial macrophages by modulating the JAK1-STAT3 pathway. This intervention effectively mitigates synovial inflammatory responses and fibrotic changes, while concurrently slowing articular cartilage degradation, collectively contributing to the amelioration of OA progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These observations significantly broaden the therapeutic prospects of SGK1-targeted interventions for osteoarthritis management.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Orthopaedic Laboratory of the USTC for providing technical guidance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhichao Yang: Writing \u0026ndash; Original Draft, Review \u0026amp; Editing, Visualization, Verification, Methodology, Formal Analysis, Conceptualization. Ming Wei、\u0026nbsp;Yang Liu and Wenwei Li: Software, Methodology, Conceptualization. Zhaoyu Li and Liang Yan: Formal Analysis, Data Curation. Yang Lv and Zezhong Guo: Formal Analysis, Data Curation. Wei Zhou and Feng Li: Writing \u0026ndash; Review \u0026amp; Editing, Supervision, Resources, Project Administration, Funding Acquisition, Conceptualization. Wei Huang: Writing \u0026ndash; Review \u0026amp; Editing, Supervision, Resources, Project Administration, Funding Acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study received funding from the National Natural Science Foundation of China (No. 82472503) and the Key Research and Development Program of Anhui Province (No. 2023s07020008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eThe article contains the original contributions discussed in this study; for further questions, please contact the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of the First Affiliated Hospital of USTC approved all experimental protocols. and the research complied with their animal ethics standards. (Approval No. 2023-N (A) -106)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest concerning this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePrieto-Alhambra, D., Judge, A., Javaid, M. K., Cooper, C., Diez-Perez, A., \u0026amp; Arden, N. K. (2014). Incidence and risk factors for clinically diagnosed knee, hip and hand osteoarthritis: influences of age, gender and osteoarthritis affecting other joints. Annals of the rheumatic diseases, 73(9), 1659\u0026ndash;1664. https://doi.org/10.1136/annrheumdis-2013-203355\u003c/li\u003e\n\u003cli\u003eHunter, D. J., March, L., \u0026amp; Chew, M. (2020). Osteoarthritis in 2020 and beyond: a Lancet Commission. Lancet (London, England), 396(10264), 1711\u0026ndash;1712. https://doi.org/10.1016/S0140-6736(20)32230-3\u003c/li\u003e\n\u003cli\u003eHunter, D. 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SGK1 enhances Th9 cell differentiation and airway inflammation through NF-\u0026kappa;B signaling pathway in asthma. \u003cem\u003eCell and tissue research\u003c/em\u003e, \u003cem\u003e382\u003c/em\u003e(3), 563\u0026ndash;574. https://doi.org/10.1007/s00441-020-03252-3\u003c/li\u003e\n\u003cli\u003eArtunc, F., \u0026amp; Lang, F. (2014). Mineralocorticoid and SGK1-sensitive inflammation and tissue fibrosis. \u003cem\u003eNephron. Physiology\u003c/em\u003e, \u003cem\u003e128\u003c/em\u003e(1-2), 35\u0026ndash;39. https://doi.org/10.1159/000368267\u003c/li\u003e\n\u003cli\u003eHarrison D. A. (2012). The Jak/STAT pathway. \u003cem\u003eCold Spring Harbor perspectives in biology\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(3), a011205. https://doi.org/10.1101/cshperspect.a011205\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Small interfering RNA sequence\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eSense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eAntisense\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003esi-NC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e5\u0026apos;-UUCUCCGAACGU GUCACGUTT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e5\u0026apos;-ACGUGACACGUU CGGAGAATT-3\u0026apos;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003esi-SGK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e5\u0026apos;-GUCCAAUCCUCA UGCUAAATT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e5\u0026apos;-UUUAGCAUGAGG AUUGGACTT-3\u0026apos;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Primer sequences of the genes\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5732%;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42.8571%;\"\u003e\n \u003cp\u003eForward primer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.5696%;\"\u003e\n \u003cp\u003eReverse prime\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5732%;\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42.8571%;\"\u003e\n \u003cp\u003e5\u0026apos;-GGAAAGCTGTGGCGTGATGG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.5696%;\"\u003e\n \u003cp\u003e5\u0026apos;-AGCTCTGGGATGACCTTGCC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5732%;\"\u003e\n \u003cp\u003eTNF-a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42.8571%;\"\u003e\n \u003cp\u003e5\u0026apos;-AGCTCTGGGATGACCTTGCC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.5696%;\"\u003e\n \u003cp\u003e5\u0026apos;-CAGAGCAATGACTCCAAAGT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5732%;\"\u003e\n \u003cp\u003e1L-1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42.8571%;\"\u003e\n \u003cp\u003e5\u0026apos;-GGCCTTGGGCCTCAAAGGAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.5696%;\"\u003e\n \u003cp\u003e5\u0026apos;-GCTTGGGATCCACACTCTCCA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5732%;\"\u003e\n \u003cp\u003e1L-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 42.8571%;\"\u003e\n \u003cp\u003e5\u0026apos;-TCTGGGAAATCGTGGAAATGAG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.5696%;\"\u003e\n \u003cp\u003e5\u0026apos;-TCTCTGAAGGACTCTGGCTTTGTC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"​​Osteoarthritis, M1 polarization, Synovial fibrosis, SGK1, JAK-STAT signaling, Targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-6880630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6880630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSynovial inflammation and fibrosis constitute significant pathological characteristics of osteoarthritis, with their progression being intricately linked to the M1 polarization of synovial macrophages and subsequent synovial fibrosis. Despite this understanding, the precise molecular mechanisms remain elusive. In the present study, we employed both a lipopolysaccharide (LPS)-induced inflammation model and an anterior cruciate ligament transection (ACLT)-induced osteoarthritis rat model to elucidate the role of serum and glucocorticoid-regulated kinase 1 (SGK1) in this context. RNA sequencing analysis revealed that the knockdown of SGK1 markedly suppressed the activity of the JAK-STAT signaling pathway in macrophages. Furthermore, in vitro experiments demonstrated that the silencing of SGK1 led to a reduction in the release of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, and diminished the migratory and invasive capabilities of fibroblast-like synoviocytes (FLS). Mechanistically, the silencing of SGK1 was found to inhibit the expression of M1 polarization markers, specifically iNOS and CD86, by suppressing JAK1-STAT3 phosphorylation. In an ACLT-induced osteoarthritis (OA) rat model, intra-articular administration of an SGK1 inhibitor significantly attenuated synovitis and fibrosis. Histological analyses revealed an up-regulation of collagen II expression and a down-regulation of MMP13, indicating a chondroprotective effect. Collectively, these findings suggest that SGK1 modulates macrophage M1 polarization and synovial fibrosis via the JAK1-STAT3 signaling pathway, and that targeted inhibition of SGK1 may represent a novel therapeutic strategy for OA management. This study thus provides a theoretical foundation for the development of anti-OA pharmacological interventions targeting SGK1.\u003c/p\u003e","manuscriptTitle":"Targeting SGK1 Mitigates Synovial Fibrosis via Suppressing M1 Polarization to Alleviate Osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 16:55:01","doi":"10.21203/rs.3.rs-6880630/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"437b42d4-f37a-40fd-955f-c6d9ec8ba012","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-22T14:08:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 16:55:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6880630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6880630","identity":"rs-6880630","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}