Chondrocyte-specific STAT3 knockout attenuates osteoarthritis progression via inflammation regulation
CaiZhang1
LeiXing1
LuChen2
LiminLi2
XiaoliHou2
MingjianBei3
QiSun4
JingyuanGao
MD
1✉Phone+86-0315-15131528090Emailgaojingyuan2009@126.comFamingTian
MD. Ph.D.
1✉,2,5Phone+86-0315-8816230Emailtianfaming@ncst.edu.cn1North China University of Science and Technology Affiliated Hospital73 Jianshe South Road, Lubei District Tangshan063000TangshanChina
2School of Public HealthNorth China University of Science and Technology063210TangshanChina
3Department of Orthopedic SurgeryBeijing Ji Shui Tan Hospital, Capital Medical University102208BeijingChina
4Department of Orthopedic SurgeryThe First Hospital of Hebei Medical University050023ShijiazhuangChina
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North China University of Science and Technology21 Bohai Avenue, Caofeidian District Tangshan063200ChinaCai Zhang1, Lei Xing1, Lu Chen2, Limin Li2, Xiaoli Hou2, Mingjian Bei3, Qi Sun4, Jingyuan Gao1*, Faming Tian1,2*
1. North China University of Science and Technology Affiliated Hospital, Tangshan, 063000, China;
2. School of Public Health, North China University of Science and Technology, Tangshan, 063210, China
3. Department of Orthopedic Surgery, Beijing Ji Shui Tan Hospital Affiliated to Capital Medical University, Beijing, 102208, China.
4. Department of Orthopedic Surgery, The First Hospital of Hebei Medical University, Shijiazhuang, 050023, China.
*Corresponding author:
Jingyuan Gao, MD
North China University of Science and Technology Affiliated Hospital
73 Jianshe South Road, Lubei District
Tangshan, 063000, China
Tel: +86-0315-15131528090
E-mail: gaojingyuan2009@126.com
Faming Tian, MD. Ph.D.
North China University of Science and Technology
21 Bohai Avenue, Caofeidian District
Tangshan, 063200, China
Tel: +86-0315-8816230
E-mail: tianfaming@ncst.edu.cn
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Abstract
Objective
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To investigate the role and mechanism of chondrocyte-specific knockout of signal transducer and activator of transcription 3 (STAT3) in medial meniscus instability (DMM)-induced osteoarthritis (OA) in mice. Methods: A tamoxifen-induced conditional knockout (cKO) mouse model (COL-II-Cre+/STAT3flox/flox) was established, and OA was induced by DMM surgery. Littermate (STAT3flox/flox) mice served as controls. Samples were collected at 4 and 8 weeks post-surgery. Cartilage pathology was evaluated using safranin-O-green staining and Mankin scoring. Subchondral bone changes were assessed by micro-computed tomography (CT). Enzyme-linked immunosorbent assay (ELISA) was performed to determine the serum interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α). Immunohistochemistry was performed to detect Aggrecan, COL-II, STAT3, p-STAT3, IL-6, TNF-α, matrix metalloproteinase-13 (MMP13), and A Disintegrin And Metalloproteinase with Thrombospondin motifs-4 (ADAMTS-4) expressions in the cartilage. Gene expression was analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). For in vitro studies, primary chondrocytes from control mice were transduced with Cre recombinase to generate STAT3-deficient cells and stimulated with IL-1β to establish an OA model. The gene and protein expression were examined by qRT-PCR and Western blotting. Results: Compared with control DMM mice, cKO-DMM mice exhibited significantly reduced cartilage degeneration and lower serum levels of IL-1β, IL-6, and TNF-α (P < 0.05). In cartilage tissues, cKO-DMM mice showed increased COL-II and Aggrecan expression, along with downregulation of IL-1β, IL-6, TNF-α, MMP13, ADAMTS-4, JAK2, and the p-STAT3/STAT3 ratio (P < 0.05). Chondrocyte apoptosis was also reduced. Micro-CT analysis demonstrated that STAT3 knockout attenuated subchondral osteosclerosis at 8 weeks post-DMM, with significant reductions in BV/TV and BMD (P < 0.001). In vitro, STAT3 deletion alleviated IL-1β–induced COL-II and Aggrecan loss, while suppressing IL-6, TNF-α, and JAK2/STAT3 pathway activation (P < 0.05).Conclusion
Chondrocyte-specific STAT3 knockout effectively mitigates DMM-induced OA progression in mice. The protective mechanism involves suppression of inflammatory cytokine release, downregulation of MMP13/ADAMTS-4 and JAK2/STAT3 signaling, and the preservation of cartilage integrity.
Keywords:
Osteoarthritis
STAT3
signaling pathway
Cre recombinase
cell-specific knockout
Introduction
Osteoarthritis (OA) is a common and multifactorial joint disease characterized by progressive degradation of articular cartilage, remodeling of subchondral bone, and synovial inflammation [1, 2]. It is a leading cause of chronic pain and disability worldwide, especially among the elderly, and poses a substantial socioeconomic burden [3]. Epidemiological data indicate that OA prevalence continues to rise in parallel with population aging and increasing obesity rates [4–7]. Despite extensive research, the molecular mechanisms driving OA onset and progression remain incompletely defined [8–10], underscoring the urgent need for novel therapeutic strategies.
Chondrocytes, the sole cellular component of articular cartilage, are essential for maintaining cartilage structure and function [10]. They regulate extracellular matrix (ECM) synthesis, which provides mechanical strength and joint lubrication. In OA, chondrocytes acquire a catabolic phenotype, marked by excessive production of matrix-degrading enzymes such as matrix metalloproteinases (MMPs) [11, 12] and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) [13], together with increased secretion of pro-inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), ultimately leading to ECM breakdown and cartilage destruction [14].
Signal transducer and activator of transcription 3 (STAT3) is a central transcription factor that regulates cell proliferation, survival, differentiation, and inflammatory responses [15]. In OA, STAT3 has gained attention as a potential therapeutic target due to its dual role in cartilage biology [16]. Although STAT3 activation supports chondrocyte proliferation and survival during development and repair, persistent activation by cytokines such as IL-1β and IL-6 contributes to OA pathogenesis [11, 12, 17]. Specifically, chronic STAT3 signaling promotes MMP and ADAMTS expression while suppressing ECM synthesis [18–20]. Moreover, emerging evidence suggests that STAT3 may influence the epigenetic regulation of chondrocytes, adding another layer of complexity to its role in OA progression [18].
Here, we investigated the impact of chondrocyte-specific STAT3 deletion on the progression of destabilization of the medial meniscus (DMM)-induced OA in mice [21, 22] focusing on two key time points: 4 weeks [23] and 8 weeks [24]. We further explored the downstream molecular mechanisms and validated the functional role of STAT3 deletion in an in vitro OA chondrocyte model. Collectively, this study aims to elucidate the pathological role of STAT3 in OA and highlight its potential as a therapeutic target for this debilitating disease.
Materials and Methods
Experimental animals and diet
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All animal procedures were approved by the Experimental Animal Ethics Committee of North China University of Science and Technology and conducted in strict accordance with institutional and national guidelines. STAT3 floxed mice (strain: B6-STAT3tm1Xyfu/Nju) were obtained from Jackson Laboratory. To generate chondrocyte-specific STAT3 knockout (cKO) mice, COL-II-Cre+ male mice were crossed with STAT3flox/flox females, producing F1 COL-II-Cre+/STAT3flox/+ heterozygotes. These were backcrossed with STAT3flox/flox mice, and offspring with genotypes COL-II-Cre+/STAT3flox/flox (cKO) and COL-II-Cre−/STAT3flox/flox littermate controls (LCs) were identified by PCR genotyping of tail DNA (primer sequences listed in Supplementary Table 1). Mice were maintained in a specific pathogen-free (SPF) facility under controlled conditions (24 ± 2°C, 30–40% humidity, 12-h light/dark cycle), with 3–5 animals per cage and ad libitum access to standard chow and sterile water. Male mice aged 10 weeks, litter- and sex-matched, were selected for experiments.Animal group management
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Ten-week-old male cKO mice (COL-II-Cre+/STAT3flox/flox) and their LC counterparts (STAT3flox/flox) were randomly assigned to experimental groups (n = 5/group): (1) destabilization of the medial meniscus (DMM) surgery group and (2) sham surgery/normal feeding control group (Sham). OA was induced by unilateral DMM surgery in the DMM groups. From the second postoperative day, all cKO mice (regardless of grouping) received intraperitoneal tamoxifen (TAM; Sigma-Aldrich, T5648; 20 mg/mL dissolved in corn oil, C8267) at 40 mg/kg/day for 5 consecutive days to induce STAT3 deletion [25]. After surgery, animals were housed under SPF conditions until sacrifice. Tissue and serum samples were collected at 4 and 8 weeks postoperatively (Fig. 1A).Cell culture
Primary chondrocytes were isolated from the tibial plateau cartilage of 10-week-old male STAT3flox/flox mice. Cartilage fragments (approximately 1 mm³) were digested with 0.25% trypsin (AC15L821, Liji Biology, China) at 37°C for 15 min, followed by 3 mg/mL type II collagenase (17101015, Gibco, USA) digestion for 4 h at 37°C with gentle mixing every 30 min. The digested suspension was filtered through a 40-µm strainer and centrifuged at 300 ×g for 5 min at 4°C. The pellet was resuspended in high-glucose DMEM (PYG0073, Boster, China) supplemented with 10% fetal bovine serum (FBS; AC03L156, Life iLab) and 100 U/mL penicillin–streptomycin (AC03L332, Liji Biology). The cells were seeded in T25 flasks and cultured at 37°C in 5% CO₂. Non-adherent cells were removed after 48 h, and the medium was refreshed every 2–3 days. The cells were then passaged with trypsin at 80–90% confluence, and third-passage (P3) chondrocytes with optimal growth were used for subsequent experiments [26].
Cell processing and grouping
Third-passage chondrocytes were seeded into a 6-well plate at 2 × 10⁵ cells/cm². Once the cells reached 80–90% confluence, they were divided into four groups of 3 each, as follows: Ctrl-Vehicle: DMEM only; Ctrl-IL-1β: DMEM with 10 ng/mL IL-1β (KGD1258, KeyGEN BioTECH, China); cKO-Vehicle: DMEM with 8 µM TAT-Cre fusion protein (cell-penetrating Cre recombinase; M0298S, NEB, USA) for 24 h to induce STAT3 deletion [27]. cKO-IL-1β: TAT-Cre treatment for 24 h followed by DMEM with 10 ng/mL IL-1β for an additional 24 h Following treatments, the cells were harvested for total RNA and protein extraction and stored at − 80°C for downstream analysis (Fig. 1B).
Histology
Right knee joint tissues were fixed in 4% paraformaldehyde (BL539A, Biosharp, China) for 48 h, followed by decalcification in 10% Na₂EDTA (CE4971, Coolaber, China, pH 7.4) for 7 weeks. The samples were then dehydrated through graded ethanol, cleared in xylene, embedded in paraffin, and sectioned into 5-µm serial coronal slices with a rotary microtome. The sections were stained with safranin-O/fast green (G1371, Solarbio, China) and imaged under an optical microscope (BX53, Olympus, Japan) at 200× and 400× magnifications. The degree of cartilage destruction was graded independently by two blinded observers using Mankin’s histopathological scoring system [28] to ensure objectivity and reproducibility.
Immunohistochemistry and Immunocytochemistry
Following dewaxing (xylene) or dehydration (graded ethanol), antigen retrieval was performed using 0.05% trypsin. The endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min at room temperature The sections were incubated overnight at 4°C with the following primary antibodies: Collagen type II (COL-II; 1:50, BA0533, Boster), MMP-13 (1:200, AF5355, Affinity, China), aggrecan (AGG; 1:200, 13880-1-AP, Proteintech, China), ADAMTS-4 (1:200, DF6986, Affinity), IL-1β (1:200, 26048-1-AP, Proteintech), IL-6 (1:200, 26404-1-AP, Proteintech), TNF-α (1:200, 17590-1-AP, Proteintech), JAK2 (1:200, AF6022, Affinity), p-STAT3 (1:100, 60479-1-IG, Proteintech), and STAT3 (1:100, 10253-2-AP, Proteintech). The following day, samples underwent secondary antibody incubation (2414D1020, ZSBG-Bio, China), DAB chromogenic reaction, and hematoxylin counterstaining. For quantitative analysis, the tibial plateau cartilage was defined as the region of interest (ROI). Image J software was used to calculate the percentage of positively stained area (Area%) for each target protein.
Micro-computed tomography (micro-CT) analysis
Subchondral bone microarchitecture was assessed using a high-resolution micro-CT scanner (SkyScan 1176; Bruker, Billerica, MA, USA). The ROI was defined as the trabecular bone region of the tibial subchondral bone, excluding the cortical shell. The following morphometric parameters were quantified: trabecular number (Tb.N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th, mm), bone mineral density (BMD), bone volume over total volume (BV/TV), and structure model index (SMI).
Enzyme-linked immunosorbent assay (ELISA)
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Blood was collected via enucleation following anesthesia with 7% chloral hydrate. Samples were allowed to clot for 30 min at room temperature and centrifuged at 3000 ×g for 15 min at 4°C to isolate serum. The levels of TNF-α (RXW202412M-6, Ruixin Bio, China), IL-6 (RXW203049M-6, Ruixin Bio), and IL-1β (RXW203063M-6, Ruixin Bio) were measured using commercial ELISA kits according to the kit instructions. Absorbance was read at 450 nm using a microplate reader, and cytokine concentrations were determined with reference to a standard curve.Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from liquid nitrogen–ground articular cartilage or cultured cells using TRNzol Universal reagent (DP424, TIANGEN, China). RNA was purified via chloroform/isopropanol precipitation, and purity was confirmed by A260/A280 ratios of 1.8–2.0. First-strand cDNA was synthesized from 1 µg RNA using the First-Strand cDNA Synthesis Kit (ZR108, ZOMANBIO, China). qRT-PCR was performed on an FTC-3000 system (Funglyn Biotech, Canada) using 2× HQ Probe qPCR Mix (ZF601, ZOMANBIO) with specific primers (sequences in Supplementary Table 2). Cycling conditions were: 95°C for 30 s; 40 cycles of 95°C for 10 s, 56°C for 10 s, and 72°C for 30 s. The targeted genes included COL-II, AGG, IL-6, TNF-α, JAK2, and STAT3, with GAPDH serving as the reference gene. The relative expressions were calculated using the 2−∆∆Cq method against the Ctrl-Sham/Ctrl-Vehicle group.
Western blotting
Cells were lysed on ice with RIPA buffer (R0010, Solarbio) supplemented with protease and phosphatase inhibitors. Lysates were centrifuged at 12,000 ×g for 15 min at 4°C, and the supernatants were collected. Protein concentrations were determined using the BCA assay (P0012S, Biyuntian, China). Equal amounts of protein (30 µg) were separated by 10% SDS–PAGE and transferred onto PVDF membranes (Millipore, USA) by wet transfer (250 mA, 90 V, 120 min). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies against TNF-α (1:1000, 17590-1-AP, Proteintech), p-STAT3 (1:1000, 60479-1-IG, Proteintech), STAT3 (1:1000, 10253-2-AP, Proteintech), and COL-II (1:1000, BA0533, Boster). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Boster) for 2 h at 37°C. Signals were developed using ECL reagent, and images were acquired with a Bio-Rad gel imaging system (Bio-Rad, USA). Densitometric analysis was performed with ImageJ (NIH, USA). The target protein levels were normalized to GAPDH (1:50,000, A00227-HRP, Boster) and expressed as the ratio of target protein to GAPDH.
Statistical analysis
Data are presented as the mean ± SD. Statistical analyses were conducted using SPSS 22.0 (IBM, USA). One-way analysis of variance (ANOVA) was applied for group comparisons; when significant (P < 0.05), the LSD post-hoc test was used. Statistical significance was set at P < 0.05. Significance in figures is denoted as: *P < 0.05, **P < 0.01, ***P < 0.001.
Results
Chondrocyte-specific knockout of STAT3 significantly alleviated cartilage degradation in OA
At 4 and 8 weeks, both the Ctrl-Sham and cKO-Sham groups exhibited normal cartilage morphology: smooth and intact surfaces, regular chondrocyte distribution, uniform matrix staining, and continuous tidemarks. At 4 weeks post-DMM surgery, the Ctrl-DMM group displayed clear cartilage injury, characterized by reduced matrix staining (indicating proteoglycan loss) and decreased chondrocyte numbers. In contrast, the cKO-DMM group showed only mild degeneration, with slightly weakened staining and a minor reduction in chondrocytes. At 8 weeks post-DMM surgery, degeneration in the Ctrl-DMM group worsened, showing extensive matrix loss, chondrocyte depletion, and structural disruption of the cartilage layer. In comparison, the cKO-DMM group exhibited only focal superficial cartilage injury, a slight reduction in staining, and minor chondrocyte loss, significantly less severe than in the Ctrl-DMM group.
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Mankin’s histological scores confirmed these observations: at both 4 and 8 weeks, scores in the cKO-DMM group were significantly lower than those in the Ctrl-DMM group (P < 0.05) (Fig. 2).Chondrocyte-specific knockout of STAT3 alleviated microstructural degeneration of subchondral bone in OA
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At 4 weeks, DMM mice exhibited significant subchondral bone abnormalities compared with sham mice, including reduced bone volume fraction (BV/TV) and bone mineral density (BMD) (P < 0.05). By 8 weeks, the DMM group displayed the opposite phenotype, with significantly increased BV/TV, BMD, and trabecular thickness (Tb.Th), along with decreased SMI (P < 0.05), consistent with subchondral bone sclerosis. Notably, in the 8-week cKO-DMM group, BV/TV and BMD were significantly lower than in the Ctrl-DMM group (P < 0.001), indicating that STAT3 deletion effectively suppressed late-stage subchondral bone sclerosis (Fig. 3).Chondrocyte-specific knockout of STAT3 attenuated inflammatory responses in a murine model of OA
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Immunohistochemistry analysis revealed (Fig. 4A) that when compared with the Ctrl-Sham group, the protein expressions of the key pro-inflammatory factors IL-1β, IL-6, and TNF-α in the articular cartilages of the Ctrl-DMM group were significantly increased (Fig. 4B), suggesting that the progression of OA was accompanied by local abnormal upregulation of inflammatory mediators such as IL-1β, IL-6, and TNF-α, which may further activate the JAK2/STAT3-signaling pathway in chondrocytes and promote STAT3 phosphorylation.The qRT-PCR results further confirmed (Fig. 4C) that, when compared with the sham operation group at 4 weeks, the mRNA expressions of IL-1β, IL-6, and TNF-α in the chondrocytes of the DMM surgery-induced OA model group (4 weeks Ctrl-DMM) were also significantly increased.
Notably, in chondrocyte-specific STAT3 knockout mice (4-week cKO-DMM group), when compared with the Ctrl-DMM group, the mRNA levels of these three inflammatory factors were significantly inhibited (P < 0.05), suggesting that chondrocyte STAT3 deletion did not significantly affect the inflammatory state of the peripheral circulation at this point of time (Fig. 4D). However, at 8 weeks after surgery, the concentrations of the abovementioned serum inflammatory factors in the cKO-DMM group were significantly lower than those in the Ctrl-DMM group (P < 0.05).
Loss of STAT3 in chondrocytes consistently inhibits the activation of the JAK2/STAT3-signaling pathway
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Immunohistochemistry analysis (Fig. 5A) revealed that when compared with the Ctrl-Sham group, the basal expression of STAT3 protein in the chondrocytes of the cKO-Sham group was significantly decreased (P < 0.05), confirming that the STAT3 knockout model effectively reduced the target gene expression. Crucially, when compared with the Ctrl-DMM group, the protein expressions of JAK2, STAT3, and its phosphorylated form p-STAT3 in the articular cartilages of the cKO-DMM group were all significantly inhibited (Fig. 5B).The qRT-PCR results revealed the following: (Fig. 5C) 4 weeks after DMM surgery, the mRNA expressions of JAK2 and STAT3 in the cartilage tissues of the Ctrl-DMM group were significantly upregulated, whereas chondrocyte-specific STAT3 knockout (cKO-DMM group) effectively reversed this phenomenon, and the transcription levels of JAK2 and STAT3 were comprehensively downregulated relative to the Ctrl-DMM group (P < 0.05). Thus, the pathological progression of OA is closely associated with the abnormal persistent activation of the JAK2/STAT3-signaling pathway, and cartilage cell-targeted STAT3 deletion can efficiently block the activation cascade of this pathway. This effect demonstrated a highly consistent stability at the 4-week and 8-week time points, thereby enhancing the biological reliability of the conclusion.
STAT3 deletion delays cartilage degeneration by regulating matrix metabolic homeostasis
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Immunohistochemistry analysis (Fig. 6A) revealed that when compared with the 4-week sham group, the expressions of matrix degrading enzymes MMP13 and ADAMTS-4 in the cartilage tissue of the 4-week DMM group were significantly increased (P < 0.05); while the expressions of the above catabolic factors in the cKO-DMM group were significantly lower than those in the Ctrl-Sham group (P < 0.05).Detection of matrix anabolic markers (Fig. 6B) indicated that: COL-II and AGG were uniformly and strongly positively expressed in the articular cartilage of the sham group; the expressions of COL-II and AGG in the DMM group were significantly weakened; importantly, the immunopositive signals of both in the cKO-DMM group were significantly stronger than those in the Ctrl-DMM group (P < 0.05).
The qRT-PCR results further verified (Fig. 6C): the mRNA expression trends of COL-II and AGG were highly consistent with the protein levels. These data collectively revealed that, in the pathological process of OA, MMP13/ADAMTS-4 synergistically mediated the abnormal degradation of ECM, whereas chondrocyte-specific STAT3 deletion can remodel the matrix metabolic homeostasis by inhibiting the expression of catabolic factors (MMPs/ADAMTS family) and maintaining the levels of anabolic markers (COL-II/AGG), thereby effectively delaying the structural destruction of articular cartilage.
STAT3 depletion blocks the IL-6/STAT3 positive feedback loop to maintain chondrocyte homeostasis
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Cell phenotype verification: Alcian blue staining revealed typical blue deposition of glycosaminoglycan (GAG); immunocytochemistry detection confirmed that COL-II was located in the cytoplasm, consistent with the matrix secretion phenotype of chondrocyte (Fig. 7A).The qRT-PCR analysis (Fig. 7B) revealed that, when compared with the vehicle group, the mRNA of pro-inflammatory pathway genes (IL-6, TNF-α, JAK2, and STAT3) in the IL-1β stimulation group was significantly upregulated, whereas the matrix synthesis genes (COL-II, AGG) were synchronously downregulated. STAT3 deletion (cKO-IL-1β group) significantly inhibited the expression of inflammatory factors (IL-6, TNF-α) and partially reversed the transcriptional inhibition of matrix genes.
Protein level verification (Figs. 7C–D): IL-1β stimulation induced a significant increase in IL-6 and TNF-α expression secretion by chondrocytes, accompanied by a simultaneous increase in the STAT3 total protein and phosphorylated STAT3 (p-STAT3) expression, whereas the expression of COL-II protein decreased. The cKO-IL-1β group revealed a comprehensive inhibition of the IL-6/STAT3-signaling axis and significantly delayed the degradation of COL-II protein [29].
Discussion
We demonstrated that chondrocyte-specific knockout of STAT3 significantly ameliorates OA pathology in the DMM-induced mouse model.
First, histological analysis revealed improved knee joint morphology and reduced cartilage loss in the cKO group, suggesting that STAT3 in chondrocytes plays a crucial role in maintaining cartilage integrity. Notably, the positive expression of COL-II and AGG was increased in the cKO group, indicating that STAT3 depletion promotes ECM synthesis in chondrocytes. These results align with previous studies reporting the inhibitory effect of STAT3 activation on ECM synthesis [30–33]. Chronic activation of proinflammatory cytokines has been shown to suppress COL-II synthesis via STAT3 signaling [31]. Moreover, STAT3 can directly inhibit COL-II/AGG transcription by competitively binding the SOX9 promoter and recruiting HDAC3 to form a transcriptional repression complex. Thus, the enhanced matrix synthesis observed in our study may reflect the release of SOX9 transcriptional activity and reversal of epigenetic repression following STAT3 deletion [16].
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Secondly, systemic and local inflammatory responses were attenuated in the cKO group. The serum levels of IL-1β, IL-6, and TNF-α were significantly reduced, consistent with the literature [11, 12]. As the JAK2/STAT3-signaling axis is strongly activated by IL-6 [34, 35], the observed downregulation of JAK2, STAT3, and p-STAT3 in the cKO group suggests that STAT3 deletion disrupts this feedback loop, reducing the production of cytokines [36]. This interpretation is supported by a systematic review by Chen et al. [37], which highlighted the central role of the JAK2/STAT3 pathway in OA progression and its therapeutic potential. Additional evidence suggests that STAT3 deficiency may suppress macrophage M1 polarization and synovial fibroblast activation via altered exosomal miRNA cargo (e.g., let-7a-5p enrichment) [38]. Furthermore, STAT3 activation in subchondral bone endothelial cells has been implicated in promoting type H vessel proliferation and inflammatory infiltration through VEGF signaling, processes that may also be alleviated by STAT3 inhibition [39].Third, we observed decreased expression of MMP13 and ADAMTS-4 in the cKO group. These enzymes are major mediators of cartilage ECM degradation in OA [40]. Our data therefore suggest that STAT3 regulates matrix catabolism, and that its deletion protects cartilage by suppressing MMP and ADAMTS expression. Previous studies have similarly reported a positive correlation between STAT3 activation and matrix-degrading enzyme expression [41].
Finally, chondrocyte-specific STAT3 deletion exerted time-dependent effects on subchondral bone remodeling. In early OA (4 weeks post-DMM), bone mass decreased (reflected by reduced BV/TV and BMD and increased SMI), and STAT3 deletion did not significantly improve microstructural deterioration, which is consistent with earlier reports [42, 43]. One possible explanation is that STAT3 supports early bone homeostasis by promoting MSC differentiation into osteoblasts, and its absence at this stage does not prevent bone loss [44]. In contrast, in late OA (8 weeks post-DMM), STAT3 deletion significantly inhibited subchondral bone sclerosis, as reflected by reduced BV/TV and BMD. This may be attributed to delayed chondrocyte senescence, reduced inflammatory mediator release [16], and subsequent suppression of type H vessel formation in subchondral bone [39]. Together, these results suggest that STAT3 in chondrocytes has a stage-specific role in OA progression: contributing to early bone homeostasis but promoting pathological bone remodeling in advanced disease.
However, there are some limitations to our study. The murine model used in our study may not fully reproduce all aspects of human OA. Differences exist between mice and humans owing to the differences in genetic background, joint mechanics, and disease progression [45]. Moreover, we focused only on the specific STAT3 gene knockout phenomenon associated with chondrocytes; OA is a complex disease involving multiple cell types within the joint, such as synoviocytes [46, 47], osteoblasts [48], and osteoclasts [49]. Future studies should therefore explore the role of STAT3 in other cell types and its interactions in the context of OA.
Figure Legends:
Figure 1. Schematic depiction of the experimental group designing and modeling. (A) Schematic representation of animal experiment grouping and modeling. *normal feeding group. (B) Schematic representation of cell experiment grouping and treatment. *normal Ctrl group.
Figure 2. Chondrocyte-specific knockout of STAT3 significantly alleviated cartilage degradation in OA. (A) Safranin-O-green staining was performed to observe the morphological changes in the right knee joint cartilage at 4 weeks and 8 weeks (scale bar = 200 µm/100 µm). The regions rich in proteoglycans are indicated in red, and the collagen matrix is indicated in green. (B) The articular cartilage was scored using the Mankin score.
Figure 3. Chondrocyte-specific knockout of STAT3 alleviated microstructural degeneration of subchondral bone in OA. (A) Representative three-dimensional reconstructed images of subchondral bone at 4 weeks and 8 weeks, illustrating the overall morphology, structural traits, and spatial distribution of subchondral bone during this period. (B) The results of the quantitative analysis of subchondral bone by micro-computed tomography (micro-CT) at 4 weeks and 8 weeks. The evaluation indicators include: bone mineral density, BMD; bone volume/total volume, BV/TV; trabecular number, Tb.N; trabecular thickness, Tb.Th; trabecular separation, Tb.Sp; and structure model index, SMI.
Figure 4 Chondrocyte-specific knockout of STAT3 attenuated inflammatory responses in a murine model of OA. (A) Representative immunohistochemistry (IHC) staining images of IL-1β, IL-6, and TNF-α in the mouse knee joint sections at 4 and 8 weeks after surgery (scale bar = 50 µm). (B) Semi-quantitative analysis results of IL-1β, IL-6, and TNF-α IHC staining intensity in Figure (A) using Image J software. (C) mRNA expressions of IL-1β, IL-6, and TNF-α in the articular cartilage tissues at 4 and 8 weeks after surgery were detected by qRT-PCR. (D) The concentrations of TNF-α, IL-6, and IL-1β in the serum at 4 and 8 weeks after surgery, as detected by enzyme-linked immunosorbent assay (ELISA).
Figure 5. Loss of STAT3 in chondrocytes consistently inhibits the activation of the JAK2/STAT3-signaling pathway. (A) Representative immunohistochemistry staining images of JAK2, STAT3, and p-STAT3 in murine knee joints at 4 and 8 weeks after surgery (scale bar = 50 µm). (B) Semi-quantitative analysis results of the protein expression in (A) based on ImageJ software. (C) qRT-PCR detection results of JAK2 and STAT3 mRNA in articular cartilage tissues at 4 and 8 weeks after surgery.
Figure 6 STAT3 deletion delays cartilage degeneration by regulating matrix metabolic homeostasis. (A) Representative immunohistochemistry staining of MMP13, ADAMTS-4, type II collagen (COL-II), and aggrecan (AGG) in murine knee joints at 4 and 8 weeks after surgery (scale bar = 50 µm). (B) Semi-quantitative analysis of the protein expression intensity in (A) based on Image J software. (C) qRT-PCR detection results of COL-II and AGG mRNA in articular cartilage tissues at 4 and 8 weeks after surgery.
Figure 7. STAT3 depletion blocks the IL-6/STAT3-positive feedback loop to maintain chondrocyte homeostasis. (A) Phenotypic identification of primary chondrocytes: Alcian blue staining (GAG deposition, blue) and COL-II immunocytochemistry (brown, cytoplasmic localization); scale bar = 50 µm. (B) qRT-PCR detection of IL-6, TNF-a, JAK2, STAT3, COL-II, and AGG mRNA expression in chondrocytes stimulated by IL-1β. (C) Representative protein blotting results: IL-6, TNF-α, STAT3, p-STAT3, COL-II, and internal reference GAPDH. (D) Semi-quantitative analysis of the protein band gray value based on Image J (n = 3).
Abbreviations
OA
Osteoarthritis
DMM
Dorsal Medial Meniscus Transection
Ctrl
Control
LC
Littermate Control
cKO
Conditional knockout
IHC
Immunohistochemistry
ICC
Immunocytochemistry
qRT-PCR
Quantitative Real-Time Polymerase Chain Reaction
ELISA
Enzyme-Linked Immunosorbent Assay
IL-1β
Interleukin-1β
IL-6
Interleukin-6
TNF-α
Tumor Necrosis Factor-α
JAK2
Janus Kinase 2
STAT3
Signal Transducer and Activator of Transcription 3
p-STAT3
Phosphorylation signal transduction and transcriptional activation factor 3
AGG
Aggrecan
GAG
glycosaminoglycan
COL-II
Type II collagen
MMP13
Matrix Metalloproteinase 13
ADAMTS-4
A Disintegrin and Metalloproteinase with Thrombospondin Motifs 4
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
ROI
Region of Interest
Tb.N
Trabecular Number
Tb.Sp
Trabecular Spacing
Tb.Pf
Trabecular Pattern Factor
Tb.Th
Trabecular Thickness
BV/TV
Bone Volume over Total Volume
SMI
Structure Model Index
FBS
Foetal Bovine Serum
Na2EDTA
Ethylenediamine tetraacetic acid disodium
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Funding
Natural Science Foundation of Hebei Province (H2022209054); Basic Scientific Research Fundation of Universities in Hebei Province (JYG2021005); National Natural Science Foundation of China (NSFC 81874029).
Acknowledgments
Not applicable
A
Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Statement
Ethical approval and consent to participate
A
A
All experiments were conducted by the Institutional Animal Care and Use Committee of North China University of Science and Technology.Agree to publish
Agree
Competing interests
The authors declare no conflicts of interest
A
Author Contribution
C-Z and J-G conceived and designed the study, supervised the project, interpreted the data, critically revised the manuscript for important intellectual content, and approved the final manuscript. C-Z and J-G were responsible for the integrity of the overall work. C-Z contributed substantially to the conception of the work, performed all experiments, analyzed and interpreted the data, drafted the manuscript, and approved the final version. L-L and L-C confirmed the authenticity of all original data. C-Z and L-C made significant contributions to data acquisition, statistical analysis, interpretation of results, and approval of the final manuscript. M-B and Q-S participated in data collection, validation of experimental results, and approved the final manuscript. L-X and X-H performed safranin-O-green staining experiments, analyzed and interpreted histological data, and approved the final manuscript. F-T facilitated data acquisition by providing resources and technical support, contributed to the review of the manuscript, and approved the final version. All authors have read and approved the final manuscript and agree to be responsible for all aspects of the work.
Electronic Supplementary Material
Below is the link to the electronic supplementary material
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Supplementary Material 6
Supplementary Material 8
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