HIF-1α protects articular cartilage in osteoarthritis by activating autophagy

HIF-1α protects articular cartilage in osteoarthritis by activating autophagy | 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 HIF-1α protects articular cartilage in osteoarthritis by activating autophagy Xiaolei Chen, Gangning Feng, Xue Lin, xiaoxin He, Yong Yang, Xin Zhao, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3419638/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 Objective Hypoxia-inducible factor-1α (HIF-1α) is known to regulate the energy metabolism and autophagy of chondrocytes under inflammatory and hypoxic conditions. This study aims to investigate the mechanisms by which HIF-1α influences cartilage injury through autophagy and oxidative stress pathways following the onset of osteoarthritis (OA). Methods Human knee joint samples were categorized into the OA group and the control group (CON) for radiological and pathological assessments, along with proteomic analysis to elucidate the interplay between osteoarthritis, HIF-1α, and autophagy. Chondrocytes were stimulated with IL-1β to establish an OA model, and these cells were subsequently divided into the control group (CON), IL-1β group (OA), IL-1β + LW6 group, IL-1β + DMOG100 group, and IL-1β + DMOG200 group. Immunofluorescence and western blot analyses were employed to measure the expression levels of HIF-1α, ROS, and LC3 to clarify the association between HIF-1α and autophagy. In addition, mice were categorized into the control group (CON), model group (DMM), and treatment group (DMM + DMOG). Immunohistochemistry, immunofluorescence, and RT-qPCR were conducted to assess the expression levels of HIF-1α, LC3, MMP-13, COL2, β-catenin, and HIF-2α. Micro-CT was utilized to evaluate subchondral bone morphology to elucidate the relationship between HIF-1α and cartilage injury, as well as its underlying mechanisms. Results Osteoarthritic cartilage exhibited elevated levels of HIF-1α, reduced LC3 expression, and increased ROS levels. Inhibition of HIF-1α using LW6 led to further reductions in LC3 levels and increased ROS production. Conversely, the activation of HIF-1α with DMOG significantly elevated HIF-1α levels, increased LC3 expression, reduced ROS levels, decreased MMP-13 levels, enhanced COL2 expression, decreased β-catenin levels, and lowered HIF-2α expression, resulting in a reduced severity of articular cartilage injury. Conclusion After the onset of osteoarthritis, low or physiologically elevated levels of HIF-1α may not adequately activate autophagy. Maintaining HIF-1α at elevated levels can protect articular cartilage by inhibiting oxidative stress and enhancing autophagy. Osteoarthritis chondrocytes hypoxia-inducible factor-1α autophagy oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Osteoarthritis (OA) is a degenerative joint disease that seriously affects the quality of life of patients. It can not only lead to joint pain, deformity and dysfunction [1] but also significantly increase the risk of cardiovascular events, lower extremity deep venous thromboembolism, hip fracture and all-cause mortality[2-4]. At present, there are more than 300 million OA patients in the world [5], and the prevalence of knee OA in people over 60 in China is 27.6%. The possible pathogenic factors of OA mainly include degeneration and calcification of articular cartilage, synovial inflammation, osteophyte formation, subchondral cysts, subchondral bone sclerosis, vascular invasion and increased permeability of the osteochondral interface[6, 7]. Articular cartilage is a highly differentiated tissue that lacks blood vessels and nerves [8] and mainly receives nutrition and oxygen from synovial fluid and subchondral bone in the form of diffusion [9]. Articular chondrocytes are in a hypoxic environment for a long time. The oxygen concentration at the surface of healthy articular cartilage is approximately 6%-10%, and the oxygen concentration at the deepest part is only 1%-6%[10]. With the progression of osteoarthritis, the degree of hypoxia of articular chondrocytes is further increased due to the stimulation of inflammatory factors and the increase in synovial oxygen consumption. The energy source of articular chondrocytes is mainly anaerobic metabolism, and HIF-1α is the key factor that induces the conversion from oxidative metabolism to glycolytic metabolism and can maintain the anaerobic glycolysis process of cells[11]. Therefore, HIF-1α plays an important role in the metabolism of chondrocytes. In addition, HIF-1α can also promote the differentiation of mesenchymal stem cells along the chondrocyte pathway by activating SOX-9, enhance the gene expression of type II collagen and proteoglycan [12], prevent the catabolism of chondrocytes by maintaining the low Wnt/β-catenin signaling pathway [13], and protect chondrocytes by promoting the secretion of extracellular matrix. Some studies have found that the level of autophagy in OA cartilage is reduced [14]. Autophagy can regulate the adaptation of chondrocytes to inflammatory and hypoxic environments and improve the vitality of chondrocytes. HIF-1α can promote autophagy through the mTOR pathway[15], thus playing a role in cartilage protection. The classical Wnt/β-catenin signaling pathway plays an important role in the pathogenesis of OA [16-18], and studies have shown that the expression of β-catenin is increased in the cartilage of OA mice and that the activation of β-catenin in the cartilage of adult mice leads to the formation and development of OA-like phenotypes [19]. After autophagy is activated, β-catenin is inhibited, and the bone and articular cartilage of mice are protected[20]. However, it is not known whether increased HIF-1α in mouse cartilage protects articular cartilage and prevents the development of osteoarthritis by activating autophagy and affecting chondrocyte differentiation. In this study, we investigated whether HIF-1α and autophagy are associated with the pathological progression and imaging findings of OA by specifically examining damaged cartilage from human OA patients compared with relatively undamaged cartilage from the same patient. We intervened in the expression of HIF-1α using the HIF-1α inhibitor LW6 and the HIF-1α degradation inhibitor DMOG to study the relationship between different expression levels of HIF-1α and autophagy and cartilage damage. We determined the mechanism by which HIF-1α affects the pathogenesis and progression of OA and explored the therapeutic effect of DMOG in osteoarthritis. Materials and Methods 1. Ethical Review This study was approved by the Ethics Committee of Ningxia Medical University General Hospital, and all research procedures complied with the standards of the Helsinki Declaration. Human articular cartilage samples were obtained from osteoarthritis (OA) patients undergoing total knee arthroplasty, with informed consent from patients and approval from the Ethics Committee of Ningxia Medical University General Hospital (Approval No: 2023-25). All animal experimental procedures were conducted following the guidelines of the National Institutes of Health's "Guide for the Care and Use of Laboratory Animals" and were approved by the Ethics Committee of Ningxia Medical University (Approval No: IACUC-NYLAC-2020-115). 2. Patient Characteristics We collected samples from the tibial plateau of 12 patients who underwent total knee arthroplasty (TKA) between September 25, 2022, and December 25, 2022, at Ningxia Medical University General Hospital. The patients were between 60 and 70 years old and diagnosed with knee osteoarthritis (KOA), with significant narrowing of the medial joint space compared to the lateral compartment and no joint dislocation. The diagnosis of OA was based on the criteria of the American College of Rheumatology, and patients with secondary OA due to trauma, connective tissue diseases, or other conditions were excluded. The removed cartilage and subchondral bone were categorized into damaged regions (OA group) and relatively undamaged regions (control group) based on the region and degree of damage (Figure1A-B). 3. Animal Grouping and Treatment A total of 24 healthy female C57BL/6 mice (age: 8 weeks; weight: 18-22 g) were obtained from the Experimental Animal Center of Ningxia Medical University. Mice were allowed free access to food and water, and the experimental environment was SPF grade (temperature: 22±1°C; humidity: 55%; light/dark cycle: 12/12 hours). Animal experiments were conducted following ARRIVE guidelines. Mice were randomly divided into three groups (n=8 per group) (Figure2): the sham surgery group (CON), destabilization of the medial meniscus (DMM) group, and DMM+DMOG group. Among them, 5 were used for tissue sectioning, and 3 were used for qPCR experiments. OA was induced in mice by destabilization of the medial meniscus (DMM). Surgical procedure: After anesthetizing the mice with an intraperitoneal injection of 1% pentobarbital (60 mg/kg), the skin and joint capsule were incised, and the anterior horn of the medial meniscus was transected to induce instability, or sham surgery was performed without transection. Mice in the DMM+DMOG group received daily intraperitoneal injections of DMOG (25 mg/kg) [21], while mice in the sham surgery and DMM groups received daily injections of an equal volume of saline. After 8 weeks, the mice were euthanized, blood was removed by enucleation, and the right knee joints were harvested for subsequent experiments. 4. Cell Isolation and Culture Five healthy female C57BL/6 mice, aged 8 weeks, were selected. After anesthesia with pentobarbital sodium, aseptic procedures were performed under a microscope to isolate cartilage from the femoral and tibial ends of the mouse knee joints. The isolated cartilage was minced and digested with 2 mg/ml (0.1%) collagenase II at 37°C to obtain a single-cell suspension. The cells were then seeded in culture dishes precoated with DMEM supplemented with 10% fetal bovine serum (FBS) and placed in a cell culture incubator at 37°C with 5% CO2. The culture medium was changed every 3 days, and cell confluence was observed daily. Passages were performed when necessary, using cells between the 2nd and 5th passages for subsequent experiments. All cells tested negative for mycoplasma contamination. 5. Cell Viability Assay The cell viability of chondrocytes was assessed using the Cell Counting Kit-8 (CCK-8) assay. Chondrocytes were seeded in a 96-well plate at a density of 3000 cells per well with six replicates per group. Cells were treated separately with IL-1β, LW6, and DMOG for 24 hours. After removing the culture medium, 100 μL of 10% CCK-8 solution was added to each well and incubated at 37°C for 1 hour. Absorbance at 460 nm was measured using a microplate reader. 6. Immunofluorescence For cell experiments, chondrocytes were seeded on 12 mm round glass coverslips in 24-well plates at a density of 20,000 cells per well. After cells adhered to the coverslips, they were treated with drugs for 24 hours. Cells were then washed with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 1% BSA at room temperature for 30 minutes. For paraffin sections, deparaffinization and dehydration were performed in xylene, antigen retrieval was performed using 0.1% trypsin at 37°C for 15 minutes, and endogenous peroxidase activity was quenched by incubating in 3% hydrogen peroxide solution for 15 minutes. Sections were blocked with goat serum for 30 minutes, incubated with primary antibodies overnight at 4°C, and washed with PBS. Secondary antibodies were then applied at room temperature in the dark for 2 hours. Finally, cell nuclei were counterstained with DAPI, and coverslips were mounted. The following primary antibodies were used: anti-HIF-1alpha (Abcam, ab189494, 1:200), anti-LC3 (Proteintech, 14972-1-AP, 1:200), anti-β-catenin (Proteintech, 51067-1-AP, 1:200), anti-MMP13 (Abcam, ab39012, 1:300), and anti-COLⅡ (Abcam, ab34712, 1:300). Sample images were acquired under constant acquisition settings using a fluorescence microscope, and the number of positive cells and total fluorescence intensity (IntDen) were quantified using ImageJ software. 7. Immunohistochemistry Tissues were fixed in 4% paraformaldehyde buffer and decalcified in EDTA buffer (20% EDTA, pH 7.4) for 2 weeks. After dehydration in graded ethanol, tissues were embedded in paraffin, sectioned, and used for H&E staining, Safranin-O-Fast Green staining, and immunohistochemistry (IHC). For IHC, after deparaffinization and hydration of the sections, antigen retrieval was performed using 0.1% trypsin at 37°C for 30 minutes, followed by blocking endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes at room temperature. Sections were then incubated with 5% goat serum for 30 minutes at 37°C to block nonspecific binding, followed by overnight incubation with primary antibodies at 4°C. After washing with PBS, the sections were incubated with the appropriate secondary antibodies at room temperature for 1 hour. Subsequently, DAPI was used for nuclear counterstaining. 8. Western blot Chondrocytes were plated at a density of 5 × 10^5 cells per well in 6-well plates and treated with drugs for 24 hours. After washing with PBS, cells were lysed in RIPA lysis buffer (80 μL per well) on ice. After centrifugation and determination of protein concentration, 30 μg of protein from each sample was loaded onto SDS‒PAGE gels and transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies at room temperature for 1 hour. The following primary antibodies were used: anti-HIF-1alpha (Abcam, ab189494, 1:1000) and anti-LC3 (Proteintech, 14972-1-AP, 1:1000). Protein bands were visualized using chemiluminescence, and images were acquired using a gel imaging system. The grayscale values of each band were measured using ImageJ software. 9. Micro-CT After removing the surrounding skin and muscle, the right knee joints of the mice were fixed in 4% paraformaldehyde at room temperature for 48 hours. Microcomputed tomography (μ-CT) scanning was performed using a SkyScan 1176 scanner (Kontich, Belgium) with a resolution of 9 micrometers per pixel, an exposure time of 900 ms, a voltage of 50 kV, and a current of 500 μA. Data analysis was performed using CTAn and DataViewer software (Bruker MicroCT, Kontich, Belgium). Image reconstruction was performed using NRecon software (Bruker MicroCT, Kontich, Belgium). The selected region was between the subchondral plate of the tibia and the articular cartilage surface of the tibia joint. The measured parameters included bone volume fraction (BV/TV, %), trabecular separation (Tb.Sp, mm), and trabecular thickness (Tb.th, mm). 10. Liquid Chromatography-Tandem Mass Spectrometry (LC‒MS/MS) and Label-Free Quantitative Tissue Proteomics A mixture of cartilage tissue samples from 12 patients, including 12 OA group samples and 12 control group samples, was prepared in a 4:1 ratio for LC‒MS/MS and label-free quantitative tissue proteomics. Proteins were extracted, reduced, and alkylated following the manufacturer’s instructions. Proteins were then digested into peptides using trypsin and subjected to high-pH reversed-phase peptide fractionation. Subsequently, LC‒MS/MS-based data acquisition was performed, followed by database searching to identify proteins. Bioinformatic analysis was carried out to select differentially expressed proteins (L-DEPs). 11. RNA Extraction and Quantitative RT‒qPCR Total RNA from cells was extracted using TRIzol reagent (TaKaRa, Japan) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of each RNA sample using a reverse transcription reaction kit (TaKaRa, Japan). Quantitative real-time polymerase chain reaction (qPCR) was performed using the SYBR Green detection kit (Roche Diagnostics) on a MiniOpticon real-time PCR system (Bio-Rad). β-actin was used as the reference gene for quantification. The 2(-ΔΔCt) method was used for statistical analysis, and all reactions were performed in triplicate. Specific primer sequences were as follows（Table1）: Table I. Primer sequences used in RT‑qPCR Gene Sequence Hif-1α Forward 5/- CTGCCACCACTGATGAAT-3/ Reverse 5/- TGCCACTGTATGCTGATG-3/ LC3 Forward 5/- GAGCGAGTTGGTCAAGAT-3/ Reverse 5/- TCATAGATGTCAGCGATGG-3/ MMP-13 Forward 5/- TGACCTCCACAGTTGACA-3/ Reverse 5/- CAGGCACTCCACATCTTG-3/ HIF-2α Forward 5/- CTAACAGGACACAGCATCT-3/ Reverse 5/- CCGACTTGAGGTTGACAG-3/ β-catenin Forward 5/- AAGCCACAGGATTACAAGAA-3/ Reverse 5/- CCAATGTCCAGTCCAAGAT-3/ 12. Statistical Analysis All data are presented as the mean ± standard deviation. Statistical analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, Inc.). After assessing data for homogeneity of variance, comparisons were made using one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test. Statistical significance was considered when P < 0.05. RESULTS 1. Upregulation of HIF-1α and Decreased Autophagy Levels in Osteoarthritis Cartilage To investigate the expression of HIF-1α and autophagy in osteoarthritis (OA) cartilage at different pathological stages, we initially compared weight-bearing anteroposterior X-ray images, supine position magnetic resonance imaging scans, and intraoperative photographs of articular cartilage appearance in 12 eligible patients. We measured cartilage thickness in the damaged and nondamaged areas and assessed joint function using the Osteoarthritis Research Society International (OARSI) score and pain using the Visual Analog Scale (VAS) score. Radiological assessment was performed using the Kellgren-Lawrence (K-L) grading system. All 12 patients had a K-L grade of 4, an OARSI score of 14.92±0.99, and a VAS score of 6.33±1.07. Cartilage thickness (medial femoral MF 0.50±0.44 mm, medial tibial MT 0.43±0.38 mm, lateral femoral LF 2.24±0.49 mm, lateral tibial LT 2.57±0.53 mm) showed significant differences between the inner damaged and outer nondamaged areas, and cartilage thickness was negatively correlated with OARSI and VAS scores (Figure 1). In an unlabeled analysis of 12 samples, we compared the OA group with the control group and identified 292 differentially expressed proteins. Among these, 268 were upregulated, while 24 were downregulated. A volcano plot and heatmap revealed significant upregulation of HIF-1α (Figure 3A-B). To further explore the functional implications of these differentially expressed proteins, we performed functional enrichment analysis. KEGG analysis results indicated enrichment of differentially expressed genes in mitochondrial autophagy processes (Figure 3C). Gene Ontology (GO) database analysis highlighted biological processes associated with osteoarthritis, autophagy, and oxidative stress (Figure 3D). These findings suggest a close association between HIF-1α, autophagy, and oxidative stress in osteoarthritis. To validate the correlation between HIF-1α and autophagy in osteoarthritis cartilage, we conducted H&E staining and Safranin O-Fast Green staining to observe changes in articular cartilage morphology. We evaluated cartilage morphology using the MANKIN score (Figure 4A-B). The results showed an OA area score of 13.25±0.75 and a control area score of 4.17±0.94, with pathological scores consistent with radiological scores but inversely related to functional scores and cartilage thickness. Immunohistochemical staining for HIF-1α, LC3, MMP13, and COL II in chondrocytes was performed, and the ratio of positive cells to the total cell count was calculated (Figure 4C-K). In the OA area, HIF-1α (39.08±7.70%) and MMP13 (82.83±5.92%) were significantly upregulated compared to the control area, while LC3 (10.8±5.59%) and COL II (20.33±8.52%) were significantly downregulated compared to the control area (LC3: 51.9±10.2%, COL II: 76.42±6.78%). Additionally, we observed hypertrophic chondrocytes and a significant decrease in cell numbers in the OA area. These findings reflect that under conditions of abnormal stress, hypoxia, and infiltration of inflammatory factors, hypoxia-inducible factor HIF-1α is upregulated in osteoarthritis, but autophagy levels decrease, and type II collagen significantly decreases. This ultimately leads to hypertrophic chondrocytes, apoptosis, accelerated extracellular matrix degradation, cartilage thinning or disappearance, tidemark calcification, and subchondral bone plate thickening, all of which are characteristic features of osteoarthritis. 2. HIF-1α Influence on Autophagy Through Oxidative Stress To delve into the impact of HIF-1α expression levels on autophagy, we conducted in vitro experiments employing human chondrocytes. We induced an osteoarthritis (OA) chondrocyte injury model by intervening with IL-1β. We used the HIF-1α inhibitor LW6 to suppress HIF-1α expression and the HIF-1α degradation inhibitor DMOG to increase HIF-1α expression. To determine the appropriate intervention dosages, we subjected chondrocytes to various concentrations of IL-1β (10, 50, 100, 200, and 500 ng/ml), LW6 (5, 10, 50, 100, and 200 μg/ml), and DMOG (10, 50, 100, 200, and 500 μg/ml) for 24 hours, followed by cell viability assessments using the CCK-8 assay. The results revealed that cell viability significantly decreased at an IL-1β concentration of 200 ng/ml, LW6 concentration of 100 μg/ml, and DMOG concentration of 500 μg/ml. Consequently, we selected IL-1β at 100 ng/ml, LW6 at 50 μg/ml, and DMOG at 100 μg/ml and 200 μg/ml for subsequent experiments (Figure 5). Following 24 hours of IL-1β intervention in chondrocytes, we performed immunofluorescence detection and calculated the total fluorescence intensity of the positive areas (IntDen). In the OA group, in comparison to the control group, HIF-1α expression increased by 3.8-fold, ROS expression increased by 2.6-fold, and LC3 decreased by 0.54-fold, consistent with the observed trend in human tissue specimens. Subsequently, after inhibiting HIF-1α expression with LW6, we observed that HIF-1α expression decreased to 0.4-fold of the control group, ROS expression increased to 2.9-fold, and LC3 expression decreased to 0.46-fold. This suggests that within the osteoarthritis microenvironment, reducing HIF-1α levels inhibits autophagy by intensifying oxidative stress. To further explore the influence of HIF-1α expression levels on autophagy, we maintained HIF-1α aggregation with DMOG at concentrations of 100 μg/ml and 200 μg/ml. In comparison to the control group, we noted that HIF-1α expression increased to 7.1-fold and 13.5-fold, ROS expression increased to 2.3-fold and 1.64-fold, and LC3 expression significantly increased to 1.15-fold and 2.30-fold. To validate HIF-1α and LC3 expression levels, we conducted Western blotting, with results consistent with the immunofluorescence data. This indicates that within the osteoarthritis microenvironment, inhibiting HIF-1α degradation and maintaining its aggregation and high expression state can suppress oxidative stress and boost autophagy. Furthermore, this effect strengthens within a certain range as the HIF-1α concentration increases. In conclusion, maintaining elevated levels of HIF-1α can enhance mitochondrial autophagy by diminishing cellular oxidative stress (Figure 6). 3. Inhibition of HIF-1α Degradation Preserves Joint Cartilage We explored the impact of HIF-1α on cartilage degeneration through animal experiments (Figure 2). Histological analysis of animal tissue sections was conducted using HE staining and Safranin-O/Fast Green staining, and Mankin's scoring system was employed to assess joint cartilage degeneration. In the control group, mouse joint surfaces remained intact and continuous, with a normal number of chondrocytes. In the DMM group, joint surfaces were discontinuous, chondrocyte numbers were reduced, and there was noticeable loss of transparent and calcified cartilage. In the DMM+DMOG group, the degree of joint cartilage damage fell between the other two groups. Mankin's scores and histological analysis revealed that, compared to the control group (1.67±0.52 points), mice in the DMM group exhibited significantly increased scores, reaching 13.00±0.89 points, with evident cartilage surface damage. However, in the DMOG group, the scores were notably reduced to 8.50±0.55 points compared to the OA group, and cartilage surface damage was also alleviated (Figure 7A-C). 4. Inhibition of HIF-1α Degradation Protects Joint Cartilage by Activating Autophagy To elucidate the role of HIF-1α in cartilage degeneration, we initially employed immunohistochemistry and immunofluorescence techniques to assess HIF-1α protein expression. We observed relatively low expression of HIF-1α in normal cartilage, with positive cell rates of 6.58±1.32% and 3.34±2.85%, respectively. In the DMM group, which represented OA, there was a significant increase in HIF-1α expression, with rates of 67.09±4.32% and 15.32±3.80%. Notably, in the DMM+DMOG group, where DMOG inhibited HIF-1α degradation, the positive rates further increased to 92.92±3.22% and 65.64±4.52%. This illustrates the effective inhibition of HIF-1α degradation in cartilage due to DMOG treatment (Figure 7D, Figure 8A). To establish the link between in vivo HIF-1α levels and autophagy, we examined LC3 protein expression (Figure 7F). LC3 exhibited a certain level of expression in normal joint cartilage, with an immunohistochemistry positive rate of 69.46±5.85%. During OA degeneration, LC3 expression decreased in cartilage to 10.50±2.25%, potentially due to factors such as local inflammatory infiltration and severe hypoxia. In the DMOG group, LC3 levels surged to 77.33±5.40%, indicating that sustained inhibition of HIF-1α degradation effectively elevated autophagic activity in cartilage cells, approaching levels observed in normal cartilage. To delve deeper into the interplay between HIF-1α, autophagy, and cartilage damage, we conducted immunohistochemistry and immunofluorescence assays targeting matrix metalloproteinase MMP13 (Figure 7J, Figure 8B). In normal cartilage, MMP13 showed low expression, with rates of 10.37±3.40% and 2.29±1.67%. Conversely, in the DMM group, MMP13 expression significantly increased to 64.61±4.05% and 56.53±10.47%. Subsequently, in the DMOG intervention group, MMP13 levels decreased to 52.76±4.33% and 46.20±7.61%. This implies that osteoarthritis led to an acceleration of extracellular matrix degradation, and inhibiting HIF-1α degradation reduced the degree of matrix breakdown. To ascertain the extent of cartilage damage, we conducted immunohistochemistry and immunofluorescence examinations targeting type II collagen, COL Ⅱ (Figure 7H, Figure 8C). COL Ⅱ displayed high expression in normal cartilage, with rates of 78.56±4.98% and 79.08±8.00%. However, in the DMM group, there was a significant reduction, with rates of 11.02±4.12% and 7.30±3.93%. In the DMOG intervention group, COL Ⅱ expression increased to 27.63±5.02% and 30.92±4.86%. This underscores that changes in HIF-1α, LC3, and MMP13 were mirrored by corresponding variations in COL Ⅱ expression, and this trend was inversely correlated with autophagy. In summary, sustaining elevated HIF-1α levels can enhance mitochondrial autophagy by reducing cellular oxidative stress, subsequently slowing down extracellular matrix degradation and bolstering COLⅡ expression, thereby safeguarding joint cartilage. 5. HIF-1α's Potential Influence on Subchondral Bone Remodeling Through the Wnt/β-Catenin Signaling Pathway Osteoarthritis development and progression encompass not only changes within cartilage but also significant alterations in the subchondral bone. In human knee joints, we observed elevated levels of HIF-1α in regions of severe cartilage damage (Figure 4C, Figure 4E). To investigate the implications of this finding, we conducted micro-CT scans of mouse knee joints (Figure 9B-C). The results indicated that the DMM group exhibited modest increases in bone volume fraction (BV/TV), trabecular separation (Tb.sp), and trabecular thickness (Tb.Th) compared to the control group. In the DMOG group, these increases were slightly attenuated, although statistical significance was not achieved. Consequently, it appears that the morphological changes in subchondral bone only weakly correlate with those in cartilage. To delve into the underlying factors contributing to this distinction, we utilized immunohistochemistry to examine the expression of β-catenin, a protein implicated in osteoarthritis (Figure 9A). We observed that β-catenin levels were elevated in the DMM group (87.32±3.63%) compared to the control group (39.54±2.39%). Conversely, in the DMOG intervention group, β-catenin expression decreased (57.43±7.38%), aligning with reduced cartilage damage. This suggests that HIF-1α might exert its influence on subchondral bone remodeling, potentially through the Wnt/β-catenin signaling pathway, which in turn affects cartilage health. 6. HIF-1α May Mitigate Cartilage Calcification and Subchondral Bone Sclerosis via Competitive Suppression of HIF-2α Given the competitive interplay between HIF-1α and HIF-2α in the formation of the HIF-1 complex, along with HIF-2α's propensity for inducing ossification, we explored their roles in cartilage calcification. We conducted qPCR analysis of cartilage, which revealed the following mRNA expression alterations compared to the control group: in the DMM group, HIF-1α mRNA expression increased by a factor of 6.09±0.9; LC3 expression plummeted to only 0.37±0.1 of its baseline; MMP13 expression surged to 7.0±1 times its original level; β-catenin expression spiked to 7.0±1.4 times its baseline; and HIF-2α expression rose by a factor of 5.3±0.9. In the DMOG+DMM group, HIF-1α mRNA expression escalated to 10.9±1.5 times its original level; LC3 expression increased to 1.22±0.2 times its baseline; MMP13 expression decreased to 4.31±0.8 times its original level; β-catenin expression decreased to 4.25±0.6 times its baseline; and HIF-2α expression was reduced to 2.51±0.7 times its original level. These findings lead us to hypothesize that HIF-1α may exert its influence by competitively inhibiting HIF-2α, consequently mitigating cartilage calcification and subchondral bone sclerosis. Discussion Osteoarthritis (OA) is a disease primarily characterized by the degeneration and destruction of joint cartilage, resulting from various causes[22]. Typical OA manifestations include cartilage surface damage, reduced chondrocyte population, thinning of transparent cartilage due to wear and tear, and an increase in calcified cartilage thickness, among other changes. Due to the avascular and nonneural characteristics of joint cartilage[23], chondrocytes endure long-term metabolic and repair processes in a hypoxic environment. In cases of OA, chondrocyte hypoxia intensifies due to inflammatory factor stimulation and heightened synovial oxygen consumption [24]. Our proteomic analysis of human knee joint cartilage samples revealed a significant increase in HIF-1α protein expression and a decrease in autophagy levels in regions with severe cartilage damage. Although OA is a systemic disease and the medial and lateral compartments of the knee joint share the same synovial cavity, we observed opposite changes in HIF-1α and autophagy. This finding piqued our interest, and after observing human joint cartilage samples and mouse knee joint cartilage, we found that after the onset of OA, HIF-1α was upregulated, while LC3 was downregulated. This led to an upregulation of MMP13, which is responsible for cartilage matrix degradation, and a decrease in the major cartilage component, COL II. Therefore, we hypothesize that HIF-1α affects cartilage repair through its influence on autophagy. To explore this relationship further, we intervened with chondrocytes in vitro using IL-1β and found that HIF-1α and ROS expression increased while LC3 decreased. This aligns with the manifestations of OA, primarily due to inflammation-induced local hypoxia and damage, which elevated cellular oxidative stress, thereby inhibiting mitochondrial autophagy. Some studies have suggested that HIF-1α acts as a protective agent, alleviating cell apoptosis and death in OA by promoting mitochondrial autophagy within OA cartilage[25]. Subsequently, we intervened with HIF-1α, initially using the HIF-1α inhibitor LW6. We observed that as HIF-1α decreased, ROS levels increased further, and LC3 levels decreased. This demonstrated that although HIF-1α expression is upregulated after OA, inhibiting HIF-1α expression does not respond to cellular oxidative stress and cannot reverse the decline in autophagy. Thus, we speculate that there may be an effective expression threshold between HIF-1α and the inflammatory response. Since HIF-1α is degraded under the action of prolyl hydroxylases (PHDs), we used the PHD inhibitor DMOG to maintain HIF-1α at a higher level. We found that after DMOG intervention, chondrocytes showed a significant increase in HIF-1α, a noticeable decrease in ROS, and a significant increase in LC3. Therefore, we infer that maintaining a high level of HIF-1α can accelerate ROS clearance, thereby enhancing mitochondrial autophagy capability. Current research suggests that HIF-1α enables bone cells to adapt to a hypoxic microenvironment and promotes their growth and development. It plays a significant role in regulating various physiological and pathological responses in the body, with particular attention to its role in vascular and bone formation. In cartilage tissue, HIF-1α has the capacity to promote cartilage formation and is a critical regulatory factor in the early differentiation of chondrocytes [26]. Hypoxia-inducible factors directly affect the survival of chondrocytes. In human bone marrow cells, under low oxygen conditions, HIF-1α can effectively induce chondrocytes, enabling them to adapt to the hypoxic microenvironment of the growth plate between bones. Martin et al . [27] found that cartilage degeneration is associated with the accumulation of metabolites resulting from trauma, chronic injury, and prolonged hypoxia. To validate the impact of maintaining high HIF-1α expression on osteoarthritic (OA) cartilage, we induced OA in mice using destabilization of the medial meniscus (DMM) surgery, which is widely used in mouse OA studies. Compared to the anterior cruciate ligament transection (ACLT) method, DMM surgery closely mimics the natural progression of human OA due to its longer OA cycle. We injected DMOG daily into mice for eight weeks. The final experimental results showed that under the continuous influence of high HIF-1α expression, autophagy remained highly expressed. The critical cartilage-degrading protein MMP13 decreased in content, and the main collagen protein, COL II, in the extracellular matrix of cartilage cells increased. Type II collagen is closely associated with the occurrence and development of OA[28], as it nourishes cartilage cells and maintains their structural stability. During the progression of OA, increased extracellular matrix degradation, reduced mRNA expression of type II collagen, and excessive apoptosis of cartilage cells all contribute to OA progression[29]. We used micro-CT to examine the subchondral bone, and while there were no significant differences between groups, there were subtle differences. The subchondral bone-related parameters of the DMOG-treated group were closer to those of the control group. This may be due to the insufficient sample size we used and the substantial intragroup variations. We believe a more plausible explanation is the sustained high expression of HIF-1α's suppression of abnormal bone mineralization [30]. In our experiment, we detected a decrease in β-catenin expression in the DMOG-treated group. β-catenin is a key upstream factor in the Wnt signaling pathway[31] and is involved in chondrocyte osteogenic differentiation, cartilage calcification, and subchondral bone sclerosis processes[32]. According to the literature[33], hypoxia-inducible factors HIF-1α and HIF-2α each exert their biological effects by forming HIF-1 heterodimers with HIF-1β. HIF-1α primarily induces autophagy and vascular formation, while HIF-2α induces chondrocyte differentiation and promotes osteogenesis [34, 35]. Therefore, we speculate that HIF-1α may inhibit HIF-2α-mediated chondrocyte osteogenic differentiation through competitive binding to HIF-1β. This will be investigated in our next study. This research, through the exploration of proteomics and biological information, discovered a correlation between HIF-1α activation and autophagy in OA cartilage. This phenomenon has been validated in clinical sample experiments. Subsequent research indicates that maintaining high HIF-1α expression can protect osteoarthritic chondrocytes through oxidative stress regulation of mitochondrial autophagy and inhibit abnormal mineralization of subchondral bone. This provides new effective strategies for understanding the pathogenesis of OA and its early prevention and treatment. Abbreviations HIF-1α,Hypoxia-Inducible Factor-1α;OA, osteoarthritis; RT‑qPCR,reverse transcription‑quantitative polymerase chain reaction; H&E, haematoxylin and eosin; OARSI, Osteoarthritis Research Society International；ROS, reactive oxygen species;PHD2, prolyl hydroxylase 2；MMP, Mitochondrial membrane potential;DMOG, dimethyloxalylglycine；IL‑1β, interleukin‑1β;MMP3, matrix metalloproteinases 13; SOX9, SRY‑box transcription factor 9; CCK‑8, Cell Counting Kit‑8; KEGG, Kyoto Encyclopedia of Genes and Genomes；LC3, light chain 3；COL Ⅱ，Collagen TypeⅡ；HIF-2α,Hypoxia-Inducible Factor-2α；DMM，destabilization of the medial meniscus surgery；mTOR，Mechanistic Target Of Rapamycin；TKA，total knee arthroplasty；KOA，knee osteoarthritis；FBS, fetal bovine serum;IHC, immunohistochemistry;VAS, Visual Analog Scale;K-L, Kellgren-Lawrence grading system;GO, Gene Ontology Declarations Acknowledgements The authors thank Servicebio Biotechnology Company（Wu han, China），Chengdu Lilai Biotechnology Company（Cheng du, China）and Applied Protein technology Biotechnology Company(Shang hai, China) for providing technical services. Funding The present study was supported by by the following grants： National Natural Science Foundation of China - (No.U22A20285)； National Natural Science Foundation of China （No. 82360319）； Key R&D Project of Autonomous Region (No.2023BEG02018)； Key R&D Project of Autonomous Region（2022BEG03126）； Scientific Research Project of Ningxia Universities (No. NYG-2022033)； Key R&D Project of Autonomous Region (No.2021BEG02037)； National Natural Science Foundation of China - (No.82160433)； Ningxia Medical University General Hospital "Medical Engineering Special" (No. NYZYYG-001)； Scientific Research Project of Ningxia Universities (No.XZ2020014)； Autonomous Region Major Scientific and Technological Achievements Transformation Project (No. 2023CJE09037)； Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors' contributions QHJ and DX designed the experiment. XLC,GNF, XXH and YZ conducted the experiment. XL,YY,LM,HW and ZDL analysed the data, XLC,GNF,XL,JBYdrafted and revised the manuscript. QHJ, DX and XLC confirm the authenticity of all the raw data. All authors read and approved the final manuscript. Ethics approval and consent to participate All experiments were approved by the Animal Experiment Ethics Committee of Ningxia Medical University (Yinchuan, China; Approval no. IACUC-NYLAC-2020-115)，and the Ethics Committee of Ningxia Medical University General Hospital (Approval No: 2023-25). All experiments were performed under the standard ethical principles of animal and human experiments. Patient consent for publication All experiments involving human beings have been explained in detail to the patients and obtained their permission, signed informed consent, and the patients agreed to donate the bone tissue removed during the operation to us for research. Competing interests The authors declare that they have no competing interests. References Bijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update with relevance for clinical practice. Lancet. 2011;377(9783):2115–26. Turkiewicz A, Kiadaliri AA, Englund M. Cause-specific mortality in osteoarthritis of peripheral joints. Osteoarthritis Cartilage. 2019;27(6):848–54. Zeng C, Bennell K, Yang Z, Nguyen U, Lu N, Wei J, et al. Risk of venous thromboembolism in knee, hip and hand osteoarthritis: a general population-based cohort study. Ann Rheum Dis. 2020;79(12):1616–24. Wang Y, Nguyen U, Lane NE, Lu N, Wei J, Lei G, et al. Knee osteoarthritis, potential mediators, and risk of all-cause mortality: data from the osteoarthritis initiative. Arthritis Care Res (Hoboken). 2021;73(4):566–73. Safiri S, Kolahi AA, Smith E, Hill C, Bettampadi D, Mansournia MA, et al. Global, regional and national burden of osteoarthritis 1990–2017: a systematic analysis of the global burden of disease study 2017. Ann Rheum Dis. 2020;79(6):819–28. Poulet B, Staines KA. New developments in osteoarthritis and cartilage biology. Curr Opin Pharmacol. 2016;28:8–13. Glyn-Jones S, Palmer AJ, Agricola R, Price AJ, Vincent TL, Weinans H, et al. Osteoarthritis. Lancet. 2015;386(9991):376–87. Kuettner KE, Aydelotte MB, Thonar EJ. Articular cartilage matrix and structure: a minireview. J Rheumatol Suppl. 1991;27:46–8. Coimbra IB, Jimenez SA, Hawkins DF, Piera-Velazquez S, Stokes DG. Hypoxia inducible factor-1 alpha expression in human normal and osteoarthritic chondrocytes. Osteoarthritis Cartilage. 2004;12(4):336–45. Kiaer T, Gronlund J, Sorensen KH. Subchondral po2, pco2, pressure, ph, and lactate in human osteoarthritis of the hip. Clin Orthop Relat Res. 1988(229):149–55. Pfander D, Cramer T, Schipani E, Johnson RS. Hif-1alpha controls extracellular matrix synthesis by epiphyseal chondrocytes. J Cell Sci. 2003;116(Pt 9):1819–26. Taheem DK, Jell G, Gentleman E. Hypoxia inducible factor-1alpha in osteochondral tissue engineering. Tissue Eng Part B Rev. 2020;26(2):105–15. Bouaziz W, Sigaux J, Modrowski D, Devignes CS, Funck-Brentano T, Richette P, et al. Interaction of hif1alpha and beta-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice. Proc Natl Acad Sci U S A. 2016;113(19):5453–8. Feng L, Feng C, Wang CX, Xu DY, Chen JJ, Huang JF, et al. Circulating microrna let–7e is decreased in knee osteoarthritis, accompanied by elevated apoptosis and reduced autophagy. Int J Mol Med. 2020;45(5):1464–76. Bohensky J, Leshinsky S, Srinivas V, Shapiro IM. Chondrocyte autophagy is stimulated by hif-1 dependent ampk activation and mtor suppression. Pediatr Nephrol. 2010;25(4):633–42. Oh H, Chun CH, Chun JS. Dkk-1 expression in chondrocytes inhibits experimental osteoarthritic cartilage destruction in mice. Arthritis Rheum. 2012;64(8):2568–78. Blom AB, Brockbank SM, van Lent PL, van Beuningen HM, Geurts J, Takahashi N, et al. Involvement of the wnt signaling pathway in experimental and human osteoarthritis: prominent role of wnt-induced signaling protein 1. Arthritis Rheum. 2009;60(2):501–12. Bouaziz W, Funck-Brentano T, Lin H, Marty C, Ea HK, Hay E, et al. Loss of sclerostin promotes osteoarthritis in mice via beta-catenin-dependent and -independent wnt pathways. Arthritis Res Ther. 2015;17(1):24. Zhu M, Tang D, Wu Q, Hao S, Chen M, Xie C, et al. Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice. J Bone Miner Res. 2009;24(1):12–21. Ma L, Liu Y, Zhao X, Li P, Jin Q. Rapamycin attenuates articular cartilage degeneration by inhibiting beta-catenin in a murine model of osteoarthritis. Connect Tissue Res. 2019;60(5):452–62. Hu S, Zhang C, Ni L, Huang C, Chen D, Shi K, et al. Stabilization of hif-1α alleviates osteoarthritis via enhancing mitophagy. Cell Death Dis. 2020;11(6). Sandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3(2):107–13. Sandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3(2):107–13. Svalastoga E, Kiaer T. Oxygen consumption, diffusing capacity and blood flow of the synovial membrane in osteoarthritic rabbit knee joints. Acta Vet Scand. 1989;30(2):121–5. Lu J, Peng Y, Zou J, Wang J, Lu S, Fu T, et al. Hypoxia inducible factor-1alpha is a regulator of autophagy in osteoarthritic chondrocytes. Cartilage. 2021;13(2_suppl):1030S-1040S. Ryu JH, Yang S, Shin Y, Rhee J, Chun CH, Chun JS. Interleukin-6 plays an essential role in hypoxia-inducible factor 2alpha-induced experimental osteoarthritic cartilage destruction in mice. Arthritis Rheum. 2011;63(9):2732–43. Martin JA, Brown T, Heiner A, Buckwalter JA. Post-traumatic osteoarthritis: the role of accelerated chondrocyte senescence. Biorheology. 2004;41(3–4):479–91. Lane AR, Harkey MS, Davis HC, Luc-Harkey BA, Stanley L, Hackney AC, et al. Body mass index and type 2 collagen turnover in individuals after anterior cruciate ligament reconstruction. J Athl Train. 2019;54(3):270–5. An QD, Li YY, Zhang HX, Lu J, Yu XD, Lin Q, et al. Inhibition of bromodomain-containing protein 4 ameliorates oxidative stress-mediated apoptosis and cartilage matrix degeneration through activation of nf-e2-related factor 2-heme oxygenase-1 signaling in rat chondrocytes. J Cell Biochem. 2018;119(9):7719–28. Amir MS, Chiba N, Seong CH, Kusuyama J, Eiraku N, Ohnishi T, et al. Hif-1alpha plays an essential role in bmp9-mediated osteoblast differentiation through the induction of a glycolytic enzyme, pdk1. J Cell Physiol. 2022;237(4):2183–97. Yao Q, Wu X, Tao C, Gong W, Chen M, Qu M, et al. Osteoarthritis: pathogenic signaling pathways and therapeutic targets. Signal Transduct Target Ther. 2023;8(1):56. Li W, Xiong Y, Chen W, Wu L. Wnt/beta-catenin signaling may induce senescence of chondrocytes in osteoarthritis. Exp Ther Med. 2020;20(3):2631–8. Zhang FJ, Luo W, Lei GH. Role of hif-1alpha and hif-2alpha in osteoarthritis. Joint Bone Spine. 2015;82(3):144–7. Saito T, Kawaguchi H. Hif-2alpha as a possible therapeutic target of osteoarthritis. Osteoarthritis Cartilage. 2010;18(12):1552–6. Zhang XA, Kong H. Mechanism of hifs in osteoarthritis. Front Immunol. 2023;14:1168799. Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3419638","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":238691592,"identity":"8fc3ade4-6985-4818-ad32-05c514099335","order_by":0,"name":"Xiaolei Chen","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolei","middleName":"","lastName":"Chen","suffix":""},{"id":238691593,"identity":"c29e604b-cec9-4fc9-aea6-0936c2c34747","order_by":1,"name":"Gangning Feng","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gangning","middleName":"","lastName":"Feng","suffix":""},{"id":238691595,"identity":"c5c1786d-899b-4159-a990-6339015b5560","order_by":2,"name":"Xue Lin","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Lin","suffix":""},{"id":238691598,"identity":"569e2338-ca9b-4b17-9434-c4ced6d45baa","order_by":3,"name":"xiaoxin He","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"xiaoxin","middleName":"","lastName":"He","suffix":""},{"id":238691601,"identity":"3feff09d-b0af-4a8a-b160-9133ef3d30d1","order_by":4,"name":"Yong Yang","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Yang","suffix":""},{"id":238691603,"identity":"b035be41-da95-4489-a3b1-aa7d259676e5","order_by":5,"name":"Xin Zhao","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhao","suffix":""},{"id":238691606,"identity":"c861d407-0661-4e07-acb2-3563ef6420cc","order_by":6,"name":"Jiangbo Yan","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiangbo","middleName":"","lastName":"Yan","suffix":""},{"id":238691607,"identity":"7502c95b-4c02-409f-b8df-b21cb8659766","order_by":7,"name":"Long Ma","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Ma","suffix":""},{"id":238691609,"identity":"7e817edd-a8c4-4df0-8b1e-0925cfbd0aad","order_by":8,"name":"Yong Zhou","email":"","orcid":"","institution":"DaXing Hospital of Xi'an","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhou","suffix":""},{"id":238691610,"identity":"fc825306-bbee-4652-9fab-1dff782fb282","order_by":9,"name":"Hui Wang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":238691611,"identity":"fcab4eb0-0b6e-4274-b5f9-217047834680","order_by":10,"name":"Zhidong Lu","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhidong","middleName":"","lastName":"Lu","suffix":""},{"id":238691612,"identity":"bc87858b-c1f2-4423-91d9-cc616a7e208d","order_by":11,"name":"Di Xue","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Xue","suffix":""},{"id":238691613,"identity":"f24cc01f-ff92-4f1b-8965-8eeb1b78a832","order_by":12,"name":"Qunhua Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBAC9mYwdQDIamx8kFAhISdPSAsjXAvP4cMGH85YGBs2ENLSANMikZYmObOtIhHExq+lnffgbd4ddxL7G3IMpHnnSSQwNjA/fHQDr8P4kq15zzxLnHHgjIEx7zaJPHYGNmPjHLxaeMykedsOJzYc7DFIBmopZmzgYZMmSsv8wzwGh3nnSCQ2HCCgRRCmZcMxtsTGmQ1EaJFm5jG2nNt22HjjGebDDB+OSRgbNhPwCx//GcMbb9sOy867/7D9R0JNnZw8e/PDx/i0gIAEKpeZgHIsWkbBKBgFo2AUoAEA8SZOWbsERLsAAAAASUVORK5CYII=","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qunhua","middleName":"","lastName":"Jin","suffix":""}],"badges":[],"createdAt":"2023-10-07 16:59:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3419638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3419638/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44539422,"identity":"151c4ec3-a1c7-44cb-9425-431a0710112a","added_by":"auto","created_at":"2023-10-12 21:05:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4221488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiological Presentation and Functional Scores of Knee Osteoarthritis in Humans. \u003c/strong\u003e(A) Representative images of knee osteoarthritis patients: weight-bearing anteroposterior X-ray images, supine position magnetic resonance imaging (MRI) scans, and intraoperative photographs of articular cartilage appearance. (B) Bone tissue excised during total knee arthroplasty, categorized by cartilage damage location and severity into OA group (black) and Con group (green). (C) Preoperative OARSI scores (0-14 points), pain Visual Analogue Scale (VAS) scores (0-10 points), and Kellgren-Lawrence (K-L) grading (0-4 grades) for patients (n=12). (D) Preoperative measurement of cartilage thickness in knee joint MRI of the damaged area (OA group) and non-damaged area (CON group) of affected limbs (n=12); significant difference between the two groups (*P\u0026lt;0.05 vs CON，CON, medial femur and medial tibia；MF, Medial femur; MT, Medial tibia；LF, Lateral femur; LT, Lateral tibia)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/363be7c6a4906069a814e909.png"},{"id":44539421,"identity":"dd2927c6-c6a5-45cb-9dc7-8eec75894869","added_by":"auto","created_at":"2023-10-12 21:05:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagram of the Animal Experiment Strategy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty 8-week-old mice were randomly allocated into three groups, each consisting of 10 mice: CON, DMM, and DMM+DMOG. The DMM and DMM+DMOG groups underwent DMM surgery on the right knee joint. Post-surgery, all mice received daily intraperitoneal injections of DMOG or an equivalent volume of PBS. The mice were euthanized on the 57th day post-surgery for subsequent examinations.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/a22818bac0af8f525bd308d0.png"},{"id":44539196,"identity":"b17029d6-801a-4d3f-945a-a7f8ed522456","added_by":"auto","created_at":"2023-10-12 20:57:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":702911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLabel-free proteomic quantitative analysis. \u003c/strong\u003e(A-B) display volcano plots and heatmaps for differential analysis, while (C-D) depict the results of KEGG and GO analyses for differentially expressed genes\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/83aea0ca92f7350e69b78dc2.png"},{"id":44538574,"identity":"a843cd73-b664-46dc-a2c0-6f8218ad20b7","added_by":"auto","created_at":"2023-10-12 20:49:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4452133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological Presentation of Human Joint Tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) H-E staining and Safranin O-Fast Green staining, scale bar: 500 μm; (B) Mankin's cartilage scoring based on staining in (A); (C) Representative immunohistochemical images of HIF-1α in cartilage and subchondral bone, scale bar: 100 μm; (D-E) Quantitative analysis of HIF-1α immunohistochemistry; (F) Representative immunohistochemical image of LC3 in cartilage, scale bar: 100 μm; (G) Quantitative analysis of LC3 immunohistochemistry; (H) Representative immunohistochemical image of MMP13 in cartilage, scale bar: 100 μm; (I) Quantitative analysis of MMP13 immunohistochemistry; (J) Representative immunohistochemical image of COL2 in cartilage, scale bar: 100 μm; (K) Quantitative analysis of COL2 immunohistochemistry.\" (*P\u0026lt;0.05 vs CON).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/fc98c6075b5af9f56f6585d0.png"},{"id":44538571,"identity":"e9eb6f52-20c4-4286-b76d-76b599d00202","added_by":"auto","created_at":"2023-10-12 20:49:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell Viability Assessment after 24-hour Intervention on Chondrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Viability assay of IL-1β (10, 50, 100, 200, 500ng/ml), with the optimal concentration highlighted in red.(B) Viability assay of DMOG (10, 50, 100, 200, 500μg/ml), with the optimal concentration highlighted in red.(C) Viability assay of LW6 (5, 10, 50, 100, 200μg/ml), with the optimal concentration highlighted in red. (*P\u0026lt;0.05 vs 0 ng/ml and 0 μg/ml).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/d57f81f19b4db012cad105ea.png"},{"id":44538573,"identity":"0dceca5f-aed0-4431-9919-b8aa5cdc24c0","added_by":"auto","created_at":"2023-10-12 20:49:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1538527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between HIF-1α Expression, Oxidative Stress, and Autophagy in Chondrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing intervention with DMOG and LW6, chondrocytes were divided into 5 groups:\u003c/p\u003e\n\u003cp\u003e(A) Representative immunofluorescence images of HIF-1α expression in chondrocytes. Scale bar: 100μm.(B) Representative immunofluorescence images of ROS (Reactive Oxygen Species) probe in chondrocytes. Scale bar: 100μm.(C) Representative immunofluorescence images of LC3 in chondrocytes. Scale bar: 100μm.(D-F) Quantitative analysis (IntDen) of total fluorescence intensity of HIF-1α, ROS, and LC3.(G) Western blot analysis of HIF-1α and LC3 protein levels.(H-I) Quantification of HIF-1α and LC3 expression in chondrocytes. (*P\u0026lt;0.05 vs CON; LW6, IL1β+LW6\u003c/p\u003e\n\u003cp\u003eGroup；DG1, IL1β+DMOG 100 μg/ml group；DG2，IL1β+DMOG 200 μg/ml group; DMOG, IL1β+DMOG 200 μg/ml group.）\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/f8fbb3d07152026b77532cb4.png"},{"id":44539199,"identity":"6b068686-4769-4c48-97c2-48dd2929e8cb","added_by":"auto","created_at":"2023-10-12 20:57:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5230028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMouse Articular Cartilage Pathology and Immunohistochemistry.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) H-E staining and Safranin O-Fast Green staining.(C) MANKIN'S scoring of cartilage based on staining.(D) Representative immunohistochemical image of HIF-1α in cartilage.(E) Quantitative analysis of HIF-1α immunohistochemistry in cartilage.(F) Representative immunohistochemical image of LC3 in cartilage.(G) Quantitative analysis of LC3 immunohistochemistry in cartilage.(H)Representative immunohistochemical image of COL II in cartilage.(I) Quantitative analysis of COL II immunohistochemistry in cartilage.(J) Representative immunohistochemical image of MMP13 in cartilage.(K) Quantitative analysis of MMP13 immunohistochemistry in cartilage. (*P\u0026lt;0.05 vs CON，scale bar 50 μm)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/4242b166a1a861900010b3ad.png"},{"id":44538581,"identity":"7903d22c-ea9c-478c-9faf-2f44fc264698","added_by":"auto","created_at":"2023-10-12 20:49:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1646359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMouse Articular Cartilage Immunofluorescence.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative immunofluorescence image of HIF-1α in cartilage;(B) Representative immunofluorescence image of MMP13 in cartilage;(C) Representative immunofluorescence image of COL II in cartilage;(D-F) Quantitative analysis of the ratio of positive cells (HIF-1α, MMP13, COL II) to total cells in cartilage.(*P\u0026lt;0.05 vs CON，scale bar 50 μm, 5 μm；DMOG,DMM+DMOG group)\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/c2732c07fa93a2e4220073d3.png"},{"id":44539197,"identity":"492583d0-60e4-406a-89a6-ad40f8dbeba2","added_by":"auto","created_at":"2023-10-12 20:57:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1740732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Cartilage and Subchondral Bone Mineralization-Related Parameters in Mouse Joints.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative immunohistochemical image of β-catenin, scale bar 50 μm.(B) 3D imaging of subchondral bone using micro-CT.(C) Sagittal and coronal cross-sectional views of the medial tibial plateau.(D) Quantitative analysis of β-catenin.(E) Ratio of subchondral bone volume (BV) to tissue volume (TV).(F) Separation degree of subchondral bone trabeculae.(G) Thickness of subchondral bone trabeculae. (H) Quantitative analysis of mRNA expression levels of HIF-1α, LC3, MMP-13, β-catenin, and HIF-2α in cartilage using qPCR. (*P\u0026lt;0.05 vs CON)\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/fc9ce28b614f1c8e37cd217a.png"},{"id":44538578,"identity":"ee8ac749-5c58-498f-a428-b3cbafb7a8a3","added_by":"auto","created_at":"2023-10-12 20:49:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1770822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism of Osteoarthritis Pathogenesis Mediated by HIF-1α.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcessive mechanical stress on the knee joint, in conjunction with the activation of the mTOR pathway by inflammatory mediators and local hypoxia, instigates a transient surge in HIF-1α levels. Within the cellular nucleus, HIF-1α engages in competitive binding with HIF-2α, forming the HIF-1 complex alongside HIF-1β and HRE, thus facilitating its biological functionalities. Elevated HIF-1 levels induce an upregulation of LC3 on the mitochondrial membrane and the formation of mitochondrial autophagosomes, subsequently activating the mitochondrial autophagy pathway. This, in turn, results in a reduction in MMP-13 levels and an increase in COL II content within the cartilage. The introduction of exogenous DMOG effectively inhibits the degradation of HIF-1α, thereby maintaining HIF-1α at a heightened level, sustaining the autophagic process. Ultimately, this cascade of events translates into diminished cartilage damage, slowing down the progression of osteoarthritis.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/bae2970666a95d2a9f881877.png"},{"id":44665083,"identity":"78f09464-80e7-4551-815c-b6d6c4d1e746","added_by":"auto","created_at":"2023-10-16 03:52:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9263768,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3419638/v1/6f6391eb-bf17-4c7a-8a2c-247841005cfc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"HIF-1α protects articular cartilage in osteoarthritis by activating autophagy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a degenerative joint disease that seriously affects the quality of life of patients. It can not only lead to joint pain, deformity and dysfunction\u0026nbsp;[1]\u0026nbsp;but also significantly increase the risk of cardiovascular events, lower extremity deep venous thromboembolism, hip fracture and all-cause mortality[2-4]. At present, there are more than 300 million OA patients in the world\u0026nbsp;[5], and the prevalence of knee OA in people over 60 in China is 27.6%. The possible pathogenic factors of OA mainly include degeneration and calcification of articular cartilage, synovial inflammation, osteophyte formation, subchondral cysts, subchondral bone sclerosis, vascular invasion and increased permeability of the osteochondral interface[6, 7]. Articular cartilage is a highly differentiated tissue\u0026nbsp;that\u0026nbsp;lacks blood vessels and nerves\u0026nbsp;[8]\u0026nbsp;and mainly receives nutrition and oxygen from synovial fluid and subchondral bone in the form of diffusion\u0026nbsp;[9]. Articular chondrocytes are in a hypoxic environment for a long time. The oxygen concentration at the surface of healthy articular cartilage is\u0026nbsp;approximately\u0026nbsp;6%-10%, and the oxygen concentration at the deepest part is only 1%-6%[10]. With the\u0026nbsp;progression\u0026nbsp;of osteoarthritis, the degree of hypoxia of articular chondrocytes is further increased due to the stimulation of inflammatory factors and the increase\u0026nbsp;in\u0026nbsp;synovial oxygen consumption. The energy source of articular chondrocytes is mainly anaerobic metabolism, and HIF-1\u0026alpha; is the key factor\u0026nbsp;that induces\u0026nbsp;the conversion from oxidative metabolism to glycolytic metabolism and can maintain the anaerobic glycolysis process of cells[11]. Therefore, HIF-1\u0026alpha; plays an important role in the metabolism of chondrocytes. In addition, HIF-1\u0026alpha; can also promote the differentiation of mesenchymal stem cells along the chondrocyte pathway by activating SOX-9, enhance the gene expression of type\u0026nbsp;II\u0026nbsp;collagen and proteoglycan\u0026nbsp;[12], prevent the catabolism of chondrocytes by maintaining the low Wnt/\u0026beta;-catenin signaling pathway\u0026nbsp;[13], and protect chondrocytes by promoting the secretion of extracellular matrix.\u003c/p\u003e\n\u003cp\u003eSome studies have found that the level of autophagy in OA cartilage is reduced\u0026nbsp;[14]. Autophagy can regulate the adaptation of chondrocytes to inflammatory and hypoxic\u0026nbsp;environments\u0026nbsp;and improve the vitality of chondrocytes. HIF-1\u0026alpha; can promote autophagy through\u0026nbsp;the mTOR\u0026nbsp;pathway[15], thus playing a role in cartilage protection. The classical Wnt/\u0026beta;-catenin signaling pathway plays an important role in the pathogenesis of OA\u0026nbsp;[16-18], and studies have shown that the expression of \u0026beta;-catenin is increased in\u0026nbsp;the\u0026nbsp;cartilage of OA mice and\u0026nbsp;that\u0026nbsp;the activation of \u0026beta;-catenin in\u0026nbsp;the\u0026nbsp;cartilage of adult mice leads to the formation and development of OA-like phenotypes\u0026nbsp;[19]. After autophagy is activated, \u0026beta;-catenin is inhibited,\u0026nbsp;and the bone and articular cartilage of mice\u0026nbsp;are\u0026nbsp;protected[20]. However, it is not known whether increased HIF-1\u0026alpha; in mouse cartilage protects articular cartilage and prevents the development of osteoarthritis by activating autophagy and affecting chondrocyte differentiation.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated whether HIF-1\u0026alpha; and autophagy are associated with the pathological progression and imaging findings of OA by specifically examining damaged cartilage from human OA patients compared with relatively undamaged cartilage from the same patient. We intervened in the expression of HIF-1\u0026alpha; using the HIF-1\u0026alpha; inhibitor LW6 and the HIF-1\u0026alpha; degradation inhibitor DMOG to study the relationship between different expression levels of HIF-1\u0026alpha; and autophagy and cartilage damage. We determined the mechanism by which HIF-1\u0026alpha; affects the pathogenesis and progression of OA and explored the therapeutic effect of DMOG in osteoarthritis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e1. Ethical Review\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Ningxia Medical University General Hospital, and all research procedures complied with the standards of the Helsinki Declaration. Human articular cartilage samples were obtained from osteoarthritis (OA) patients undergoing total knee arthroplasty, with informed consent from patients and approval from the Ethics Committee of Ningxia Medical University General Hospital (Approval No: 2023-25). All animal experimental procedures were conducted following the guidelines of the National Institutes of Health\u0026apos;s \u0026quot;Guide for the Care and Use of Laboratory Animals\u0026quot; and were approved by the Ethics Committee of Ningxia Medical University (Approval No: IACUC-NYLAC-2020-115).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2. Patient Characteristics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe collected samples from the tibial plateau of 12 patients who underwent total knee arthroplasty (TKA) between September 25, 2022, and December 25, 2022, at Ningxia Medical University General Hospital. The patients were between 60 and 70 years old and diagnosed with knee osteoarthritis (KOA), with significant narrowing of the medial joint space compared to the lateral compartment and no joint dislocation. The diagnosis of OA was based on the criteria of the American College of Rheumatology, and patients with secondary OA due to trauma, connective tissue diseases, or other conditions were excluded. The removed cartilage and subchondral bone were categorized into damaged regions (OA group) and relatively undamaged regions (control group) based on the region and degree of damage (Figure1A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3. Animal Grouping and Treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 24 healthy female C57BL/6 mice (age: 8 weeks; weight: 18-22 g) were obtained from the Experimental Animal Center of Ningxia Medical University. Mice were allowed free access to food and water, and the experimental environment was SPF grade (temperature: 22\u0026plusmn;1\u0026deg;C; humidity: 55%; light/dark cycle: 12/12 hours). Animal experiments were conducted following ARRIVE guidelines. Mice were randomly divided into three groups (n=8 per group) (Figure2):\u0026nbsp;the sham\u0026nbsp;surgery group (CON), destabilization of the medial meniscus (DMM) group, and DMM+DMOG\u0026nbsp;group. Among\u0026nbsp;them, 5 were used for tissue\u0026nbsp;sectioning, and 3 were used for qPCR experiments. OA was induced in mice by destabilization of the medial meniscus (DMM). Surgical procedure: After anesthetizing the mice with\u0026nbsp;an\u0026nbsp;intraperitoneal injection of 1% pentobarbital (60 mg/kg), the skin and joint capsule were incised, and the anterior horn of the medial meniscus was transected to induce instability, or sham surgery was performed without transection. Mice in the DMM+DMOG group received daily intraperitoneal injections of DMOG (25 mg/kg)\u0026nbsp;[21], while mice in the sham surgery and DMM groups received daily injections of an equal volume of saline. After 8 weeks,\u0026nbsp;the\u0026nbsp;mice were euthanized, blood was removed by\u0026nbsp;enucleation, and the right knee joints were harvested for subsequent experiments.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4. Cell Isolation and Culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive\u0026nbsp;healthy female C57BL/6 mice, aged 8 weeks, were selected. After anesthesia with pentobarbital sodium, aseptic procedures were performed under a microscope to isolate cartilage from the femoral and tibial ends of the mouse knee joints. The isolated cartilage was minced and digested with 2 mg/ml (0.1%) collagenase II at 37\u0026deg;C to obtain a single-cell suspension. The cells were then seeded in culture dishes\u0026nbsp;precoated\u0026nbsp;with DMEM supplemented with 10% fetal bovine serum (FBS) and placed in a cell culture incubator at 37\u0026deg;C with 5% CO2. The culture medium was changed every 3 days, and cell confluence was observed daily. Passages were performed when necessary, using cells between the 2nd and 5th passages for subsequent experiments. All cells tested negative for mycoplasma contamination.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e5. Cell Viability Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell\u0026nbsp;viability of chondrocytes was assessed using the Cell Counting Kit-8 (CCK-8) assay. Chondrocytes were seeded in a 96-well plate at a density of 3000 cells per well with six replicates per group. Cells were treated separately with IL-1\u0026beta;, LW6, and DMOG for 24 hours. After removing the culture medium, 100 \u0026mu;L of 10% CCK-8 solution was added to each well and incubated at 37\u0026deg;C for 1 hour. Absorbance at 460 nm was measured using a microplate reader.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e6. Immunofluorescence\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cell experiments, chondrocytes were seeded on 12 mm round glass coverslips in 24-well plates at a density of 20,000 cells per well. After cells adhered to the coverslips, they were treated with drugs for 24 hours. Cells were then washed with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 1% BSA at room temperature for 30 minutes.\u003c/p\u003e\n\u003cp\u003eFor paraffin sections, deparaffinization and dehydration were performed in xylene, antigen retrieval was\u0026nbsp;performed\u0026nbsp;using 0.1% trypsin at 37\u0026deg;C for 15 minutes, and endogenous peroxidase activity was quenched by incubating in 3% hydrogen peroxide solution for 15 minutes. Sections were blocked with goat serum for 30 minutes, incubated with primary antibodies overnight at 4\u0026deg;C,\u0026nbsp;and washed\u0026nbsp;with PBS. Secondary antibodies were then applied at room temperature in the dark for 2 hours. Finally, cell nuclei were counterstained with DAPI, and coverslips were mounted.\u003c/p\u003e\n\u003cp\u003eThe following primary antibodies were used:\u0026nbsp;anti-HIF-1alpha (Abcam, ab189494, 1:200),\u0026nbsp;anti-LC3 (Proteintech, 14972-1-AP, 1:200),\u0026nbsp;anti-\u0026beta;-catenin (Proteintech, 51067-1-AP, 1:200),\u0026nbsp;anti-MMP13 (Abcam, ab39012, 1:300),\u0026nbsp;and anti-COLⅡ (Abcam, ab34712, 1:300). Sample images were acquired under constant acquisition settings using a fluorescence microscope, and the number of positive cells and total fluorescence intensity (IntDen) were quantified using ImageJ software.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e7. Immunohistochemistry\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were fixed in 4% paraformaldehyde buffer and decalcified in EDTA buffer (20% EDTA, pH 7.4) for 2 weeks. After dehydration in graded ethanol, tissues were embedded in paraffin, sectioned, and used for H\u0026amp;E staining, Safranin-O-Fast Green staining, and immunohistochemistry (IHC). For IHC, after deparaffinization and hydration of the sections, antigen retrieval was performed using 0.1% trypsin at 37\u0026deg;C for 30 minutes, followed by blocking endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes at room temperature. Sections were then incubated with 5% goat serum for 30 minutes at 37\u0026deg;C to block\u0026nbsp;nonspecific\u0026nbsp;binding, followed by overnight incubation with primary antibodies at 4\u0026deg;C. After washing with PBS, the sections were incubated with the appropriate secondary antibodies at room temperature for 1 hour. Subsequently, DAPI was used for nuclear counterstaining.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e8. Western blot\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChondrocytes were plated at a density of 5 \u0026times; 10^5 cells per well in 6-well plates and treated with drugs for 24 hours. After washing with PBS, cells were lysed in RIPA lysis buffer (80 \u0026mu;L per well) on ice. After centrifugation and determination of protein concentration, 30 \u0026mu;g of protein from each sample was loaded onto SDS‒PAGE gels and transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C, followed by incubation with secondary antibodies at room temperature for 1 hour.\u003c/p\u003e\n\u003cp\u003eThe following primary antibodies were used:\u0026nbsp;anti-HIF-1alpha (Abcam, ab189494, 1:1000)\u0026nbsp;and anti-LC3 (Proteintech, 14972-1-AP, 1:1000). Protein bands were visualized using chemiluminescence, and images were acquired using a gel imaging system. The grayscale values of each band were measured using\u0026nbsp;ImageJ\u0026nbsp;software.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e9. Micro-CT\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter removing the surrounding skin and muscle, the right knee joints of\u0026nbsp;the\u0026nbsp;mice were fixed in 4% paraformaldehyde at room temperature for 48 hours.\u0026nbsp;Microcomputed\u0026nbsp;tomography (\u0026mu;-CT) scanning was performed using a SkyScan 1176 scanner (Kontich, Belgium) with a resolution of 9 micrometers per pixel, an exposure time of 900 ms, a voltage of 50 kV, and a current of 500 \u0026mu;A. Data analysis was performed using CTAn and DataViewer software (Bruker MicroCT, Kontich, Belgium). Image reconstruction was\u0026nbsp;performed\u0026nbsp;using NRecon software (Bruker MicroCT, Kontich, Belgium). The selected region was between the subchondral plate of the tibia and the articular cartilage surface of the tibia joint. The measured parameters included bone volume fraction (BV/TV, %), trabecular separation (Tb.Sp, mm), and trabecular thickness (Tb.th, mm).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e10. Liquid Chromatography-Tandem Mass Spectrometry (LC‒MS/MS) and Label-Free Quantitative Tissue Proteomics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of cartilage tissue samples from 12 patients, including 12 OA group samples and 12 control group samples, was prepared in a 4:1 ratio for LC‒MS/MS and label-free quantitative tissue proteomics. Proteins were extracted, reduced, and alkylated following\u0026nbsp;the manufacturer\u0026rsquo;s\u0026nbsp;instructions. Proteins were then digested into peptides using trypsin and subjected to high-pH reversed-phase peptide fractionation. Subsequently, LC‒MS/MS-based data acquisition was performed, followed by database searching to identify proteins. Bioinformatic analysis was carried out to select differentially expressed proteins (L-DEPs).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e11. RNA Extraction and Quantitative RT‒qPCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA from cells was extracted using TRIzol reagent (TaKaRa, Japan) according to the manufacturer\u0026apos;s instructions. cDNA was synthesized from 1 \u0026mu;g of each RNA sample using a reverse transcription reaction kit (TaKaRa, Japan). Quantitative real-time polymerase chain reaction (qPCR) was performed using the SYBR Green detection kit (Roche Diagnostics) on a MiniOpticon real-time PCR system (Bio-Rad). \u0026beta;-actin was used as the reference gene for quantification. The 2(-\u0026Delta;\u0026Delta;Ct) method was used for statistical analysis, and all reactions were performed in triplicate. Specific primer sequences were as follows（Table1）:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable I. Primer sequences used in RT‑qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.11392405063291%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.45207956600362%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eHif-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.11392405063291%\" valign=\"top\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.45207956600362%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;CTGCCACCACTGATGAAT-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.59090909090909%\" valign=\"top\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"63.40909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;TGCCACTGTATGCTGATG-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eLC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.11392405063291%\" valign=\"top\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.45207956600362%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;GAGCGAGTTGGTCAAGAT-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.59090909090909%\" valign=\"top\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"63.40909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;TCATAGATGTCAGCGATGG-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eMMP-13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.11392405063291%\" valign=\"top\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.45207956600362%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;TGACCTCCACAGTTGACA-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.59090909090909%\" valign=\"top\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"63.40909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;CAGGCACTCCACATCTTG-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eHIF-2\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.11392405063291%\" valign=\"top\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.45207956600362%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;CTAACAGGACACAGCATCT-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.59090909090909%\" valign=\"top\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"63.40909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;CCGACTTGAGGTTGACAG-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.43399638336347%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026beta;-catenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.11392405063291%\" valign=\"top\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50.45207956600362%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;AAGCCACAGGATTACAAGAA-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.59090909090909%\" valign=\"top\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"63.40909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e5/-\u0026nbsp;CCAATGTCCAGTCCAAGAT-3/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e12. Statistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as the mean \u0026plusmn; standard deviation. Statistical analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, Inc.). After assessing data for homogeneity of variance, comparisons were made using one-way analysis of variance (ANOVA) followed by Tukey\u0026apos;s post hoc multiple comparison test. Statistical significance was considered when P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eUpregulation of HIF-1\u0026alpha; and Decreased Autophagy Levels in Osteoarthritis Cartilage\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the expression of HIF-1\u0026alpha; and autophagy in osteoarthritis (OA) cartilage at different pathological stages, we initially compared weight-bearing anteroposterior X-ray images, supine position magnetic resonance imaging scans, and intraoperative photographs of articular cartilage appearance in 12 eligible patients. We measured cartilage thickness in the damaged and\u0026nbsp;nondamaged\u0026nbsp;areas and assessed joint function using the Osteoarthritis Research Society International (OARSI) score and pain using the Visual Analog Scale (VAS) score. Radiological assessment was performed using the Kellgren-Lawrence (K-L) grading system. All 12 patients had a K-L grade of 4, an OARSI score of 14.92\u0026plusmn;0.99, and a VAS score of 6.33\u0026plusmn;1.07. Cartilage thickness (medial femoral MF 0.50\u0026plusmn;0.44 mm, medial tibial MT 0.43\u0026plusmn;0.38 mm, lateral femoral LF 2.24\u0026plusmn;0.49 mm, lateral tibial LT 2.57\u0026plusmn;0.53 mm) showed significant differences between the inner damaged and outer\u0026nbsp;nondamaged\u0026nbsp;areas, and cartilage thickness was negatively correlated with OARSI and VAS scores (Figure 1).\u003c/p\u003e\n\u003cp\u003eIn an unlabeled analysis of 12 samples, we compared the OA group with the control group and identified 292 differentially expressed proteins. Among these, 268 were upregulated, while 24 were downregulated. A volcano plot and heatmap revealed significant upregulation of HIF-1\u0026alpha; (Figure 3A-B). To further explore the functional implications of these differentially expressed proteins, we performed functional enrichment analysis. KEGG analysis results indicated enrichment of differentially expressed genes in mitochondrial autophagy processes (Figure 3C). Gene Ontology (GO) database analysis highlighted biological processes associated with osteoarthritis, autophagy, and oxidative stress (Figure 3D). These findings suggest a close association between HIF-1\u0026alpha;, autophagy, and oxidative stress in osteoarthritis.\u003c/p\u003e\n\u003cp\u003eTo validate the correlation between HIF-1\u0026alpha; and autophagy in osteoarthritis cartilage, we conducted H\u0026amp;E staining and Safranin O-Fast Green staining to observe changes in articular cartilage morphology. We evaluated cartilage morphology using the\u0026nbsp;MANKIN\u0026nbsp;score (Figure 4A-B). The results showed an OA area score of 13.25\u0026plusmn;0.75 and a control area score of 4.17\u0026plusmn;0.94, with pathological scores consistent with radiological scores but inversely related to functional scores and cartilage thickness. Immunohistochemical staining for HIF-1\u0026alpha;, LC3, MMP13, and COL II in chondrocytes was performed, and the ratio of positive cells to the total cell count was calculated (Figure 4C-K). In the OA area, HIF-1\u0026alpha; (39.08\u0026plusmn;7.70%) and MMP13 (82.83\u0026plusmn;5.92%) were significantly upregulated compared to the control area, while LC3 (10.8\u0026plusmn;5.59%) and COL II (20.33\u0026plusmn;8.52%) were significantly downregulated compared to the control area (LC3: 51.9\u0026plusmn;10.2%, COL II: 76.42\u0026plusmn;6.78%). Additionally, we observed hypertrophic chondrocytes and a significant decrease in cell numbers in the OA area. These findings reflect that under conditions of abnormal stress, hypoxia, and infiltration of inflammatory factors, hypoxia-inducible factor HIF-1\u0026alpha; is upregulated in osteoarthritis, but autophagy levels decrease, and type II collagen significantly decreases. This ultimately leads to hypertrophic chondrocytes, apoptosis, accelerated extracellular matrix degradation, cartilage thinning or disappearance, tidemark calcification, and subchondral bone plate thickening, all of which are characteristic features of osteoarthritis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2. HIF-1\u0026alpha; Influence on Autophagy Through Oxidative Stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve into the impact of HIF-1\u0026alpha; expression levels on autophagy, we conducted in vitro experiments employing human chondrocytes. We induced an osteoarthritis (OA) chondrocyte injury model by intervening with IL-1\u0026beta;. We used the HIF-1\u0026alpha; inhibitor LW6 to suppress HIF-1\u0026alpha; expression and the HIF-1\u0026alpha; degradation inhibitor DMOG to increase HIF-1\u0026alpha; expression. To determine the appropriate intervention dosages, we subjected chondrocytes to various concentrations of IL-1\u0026beta; (10, 50, 100, 200,\u0026nbsp;and\u0026nbsp;500 ng/ml), LW6 (5, 10, 50, 100,\u0026nbsp;and\u0026nbsp;200 \u0026mu;g/ml), and DMOG (10, 50, 100, 200,\u0026nbsp;and\u0026nbsp;500 \u0026mu;g/ml) for 24 hours, followed by cell viability assessments using the CCK-8 assay. The results revealed that cell viability significantly decreased at an IL-1\u0026beta; concentration of 200 ng/ml, LW6 concentration of 100 \u0026mu;g/ml, and DMOG concentration of 500 \u0026mu;g/ml. Consequently, we selected IL-1\u0026beta; at 100 ng/ml, LW6 at 50 \u0026mu;g/ml, and DMOG at 100 \u0026mu;g/ml and 200 \u0026mu;g/ml for subsequent experiments (Figure 5).\u003c/p\u003e\n\u003cp\u003eFollowing 24 hours of IL-1\u0026beta; intervention in chondrocytes, we performed immunofluorescence detection and calculated the total fluorescence intensity of the positive areas (IntDen). In the OA group, in comparison to the control group, HIF-1\u0026alpha; expression increased by 3.8-fold, ROS expression increased by 2.6-fold,\u0026nbsp;and\u0026nbsp;LC3 decreased\u0026nbsp;by\u0026nbsp;0.54-fold, consistent with the observed trend in human tissue specimens. Subsequently, after inhibiting HIF-1\u0026alpha; expression with LW6, we observed that HIF-1\u0026alpha; expression decreased to 0.4-fold of the control group, ROS expression increased to 2.9-fold, and LC3 expression decreased to 0.46-fold. This suggests that within the osteoarthritis microenvironment, reducing HIF-1\u0026alpha; levels inhibits autophagy by intensifying oxidative stress.\u003c/p\u003e\n\u003cp\u003eTo further explore the influence of HIF-1\u0026alpha; expression levels on autophagy, we maintained HIF-1\u0026alpha; aggregation with DMOG at concentrations of 100 \u0026mu;g/ml and 200 \u0026mu;g/ml. In comparison to the control group, we noted that HIF-1\u0026alpha; expression increased to 7.1-fold and 13.5-fold, ROS expression increased to 2.3-fold and 1.64-fold, and LC3 expression significantly increased to 1.15-fold and 2.30-fold. To validate HIF-1\u0026alpha; and LC3 expression levels, we conducted Western blotting, with results consistent with the immunofluorescence data. This indicates that within the osteoarthritis microenvironment, inhibiting HIF-1\u0026alpha; degradation and maintaining its aggregation and high expression state can suppress oxidative stress and boost autophagy. Furthermore, this effect strengthens within a certain range as the HIF-1\u0026alpha; concentration increases. In conclusion, maintaining elevated levels of HIF-1\u0026alpha; can enhance mitochondrial autophagy by diminishing cellular oxidative stress (Figure 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3. Inhibition of HIF-1\u0026alpha; Degradation Preserves Joint Cartilage\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe explored the impact of HIF-1\u0026alpha; on cartilage degeneration through animal experiments (Figure 2). Histological analysis of animal tissue sections was conducted using HE staining and Safranin-O/Fast Green staining, and Mankin\u0026apos;s scoring system was employed to assess joint cartilage degeneration. In the control group, mouse joint surfaces remained intact and continuous, with a normal number of chondrocytes. In the DMM group, joint surfaces were discontinuous, chondrocyte numbers were reduced, and there was noticeable loss of transparent and calcified cartilage. In the DMM+DMOG group, the degree of joint cartilage damage fell between the other two groups. Mankin\u0026apos;s scores and histological analysis revealed that, compared to the control group (1.67\u0026plusmn;0.52 points), mice in the DMM group exhibited significantly increased scores, reaching 13.00\u0026plusmn;0.89 points, with evident cartilage surface damage. However, in the DMOG group, the scores were notably reduced to 8.50\u0026plusmn;0.55 points compared to the OA group, and cartilage surface damage was also alleviated (Figure 7A-C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4. Inhibition of HIF-1\u0026alpha; Degradation Protects Joint Cartilage by Activating Autophagy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the role of HIF-1\u0026alpha; in cartilage degeneration, we initially employed immunohistochemistry and immunofluorescence techniques to assess HIF-1\u0026alpha; protein expression. We observed relatively low expression of HIF-1\u0026alpha; in normal cartilage, with positive cell rates of 6.58\u0026plusmn;1.32% and 3.34\u0026plusmn;2.85%, respectively. In the DMM group, which represented OA, there was a significant increase in HIF-1\u0026alpha; expression, with rates of 67.09\u0026plusmn;4.32% and 15.32\u0026plusmn;3.80%. Notably, in the DMM+DMOG group, where DMOG inhibited HIF-1\u0026alpha; degradation, the positive rates further\u0026nbsp;increased\u0026nbsp;to 92.92\u0026plusmn;3.22% and 65.64\u0026plusmn;4.52%. This illustrates the effective inhibition of HIF-1\u0026alpha; degradation in cartilage due to DMOG treatment (Figure 7D, Figure 8A).\u003c/p\u003e\n\u003cp\u003eTo establish the link between in vivo HIF-1\u0026alpha; levels and autophagy, we examined LC3 protein expression (Figure 7F). LC3 exhibited a certain level of expression in normal joint cartilage, with an immunohistochemistry positive rate of 69.46\u0026plusmn;5.85%. During OA degeneration, LC3 expression\u0026nbsp;decreased\u0026nbsp;in cartilage to 10.50\u0026plusmn;2.25%, potentially due to factors such as local inflammatory infiltration and severe hypoxia. In the DMOG group, LC3 levels surged to 77.33\u0026plusmn;5.40%, indicating that sustained inhibition of HIF-1\u0026alpha; degradation effectively elevated autophagic activity in cartilage cells, approaching levels observed in normal cartilage.\u003c/p\u003e\n\u003cp\u003eTo delve deeper into the interplay between HIF-1\u0026alpha;, autophagy, and cartilage damage, we conducted immunohistochemistry and immunofluorescence assays targeting matrix metalloproteinase MMP13 (Figure 7J, Figure 8B). In normal cartilage, MMP13 showed low expression, with rates of 10.37\u0026plusmn;3.40% and 2.29\u0026plusmn;1.67%. Conversely, in the DMM group, MMP13 expression significantly\u0026nbsp;increased\u0026nbsp;to 64.61\u0026plusmn;4.05% and 56.53\u0026plusmn;10.47%. Subsequently, in the DMOG intervention group, MMP13 levels decreased to 52.76\u0026plusmn;4.33% and 46.20\u0026plusmn;7.61%. This implies that osteoarthritis led to an acceleration of extracellular matrix degradation, and inhibiting HIF-1\u0026alpha; degradation reduced the degree of matrix breakdown.\u003c/p\u003e\n\u003cp\u003eTo ascertain the extent of cartilage damage, we conducted immunohistochemistry and immunofluorescence examinations targeting type II collagen, COL Ⅱ (Figure 7H, Figure 8C). COL Ⅱ displayed high expression in normal cartilage, with rates of 78.56\u0026plusmn;4.98% and 79.08\u0026plusmn;8.00%. However, in the DMM group, there was a significant reduction, with rates of 11.02\u0026plusmn;4.12% and 7.30\u0026plusmn;3.93%. In the DMOG intervention group, COL Ⅱ expression increased to 27.63\u0026plusmn;5.02% and 30.92\u0026plusmn;4.86%. This underscores that changes in HIF-1\u0026alpha;, LC3, and MMP13 were mirrored by corresponding variations in COL Ⅱ expression, and this trend was inversely correlated with autophagy.\u003c/p\u003e\n\u003cp\u003eIn summary, sustaining elevated HIF-1\u0026alpha; levels can enhance mitochondrial autophagy by reducing cellular oxidative stress, subsequently slowing down extracellular matrix degradation and bolstering COLⅡ expression, thereby safeguarding joint cartilage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e5. HIF-1\u0026alpha;\u0026apos;s Potential Influence on Subchondral Bone Remodeling Through the Wnt/\u0026beta;-Catenin Signaling Pathway\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOsteoarthritis development and progression encompass not only changes within cartilage but also significant alterations in the subchondral bone. In human knee joints, we observed elevated levels of HIF-1\u0026alpha; in regions of severe cartilage damage (Figure 4C, Figure 4E). To investigate the implications of this finding, we conducted\u0026nbsp;micro-CT scans of mouse knee joints (Figure 9B-C). The results indicated that the DMM group exhibited modest increases in bone volume fraction (BV/TV), trabecular separation (Tb.sp), and trabecular thickness (Tb.Th) compared to the control group. In the DMOG group, these increases were slightly attenuated, although statistical significance was not achieved. Consequently, it appears that the morphological changes in subchondral bone only weakly correlate with those in cartilage.\u003c/p\u003e\n\u003cp\u003eTo delve into the underlying factors contributing to this distinction, we utilized immunohistochemistry to examine the expression of \u0026beta;-catenin, a protein implicated in osteoarthritis (Figure 9A). We observed that \u0026beta;-catenin levels were elevated in the DMM group (87.32\u0026plusmn;3.63%) compared to the control group (39.54\u0026plusmn;2.39%). Conversely, in the DMOG intervention group, \u0026beta;-catenin expression decreased (57.43\u0026plusmn;7.38%), aligning with reduced cartilage damage. This suggests that HIF-1\u0026alpha; might exert its influence on subchondral bone remodeling, potentially through the Wnt/\u0026beta;-catenin signaling pathway, which in turn affects cartilage health.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e6. HIF-1\u0026alpha; May Mitigate Cartilage Calcification and Subchondral Bone Sclerosis via Competitive Suppression of HIF-2\u0026alpha;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the competitive interplay between HIF-1\u0026alpha; and HIF-2\u0026alpha; in the formation of the HIF-1 complex, along with HIF-2\u0026alpha;\u0026apos;s propensity for inducing ossification, we\u0026nbsp;explored\u0026nbsp;their roles in cartilage calcification. We conducted qPCR analysis of cartilage, which revealed the following mRNA expression alterations compared to the control group:\u0026nbsp;in\u0026nbsp;the DMM group,\u0026nbsp;HIF-1\u0026alpha; mRNA expression increased by a factor of 6.09\u0026plusmn;0.9;\u0026nbsp;LC3 expression plummeted to only 0.37\u0026plusmn;0.1 of its baseline;\u0026nbsp;MMP13 expression surged to 7.0\u0026plusmn;1 times its original level;\u0026nbsp;\u0026beta;-catenin expression spiked to 7.0\u0026plusmn;1.4 times its baseline;\u0026nbsp;and\u0026nbsp;HIF-2\u0026alpha; expression rose by a factor of 5.3\u0026plusmn;0.9.\u0026nbsp;In the DMOG+DMM group,\u0026nbsp;HIF-1\u0026alpha; mRNA expression escalated to 10.9\u0026plusmn;1.5 times its original level;\u0026nbsp;LC3 expression increased to 1.22\u0026plusmn;0.2 times its baseline;\u0026nbsp;MMP13 expression\u0026nbsp;decreased\u0026nbsp;to 4.31\u0026plusmn;0.8 times its original level;\u0026nbsp;\u0026beta;-catenin expression decreased to 4.25\u0026plusmn;0.6 times its baseline;\u0026nbsp;and\u0026nbsp;HIF-2\u0026alpha; expression\u0026nbsp;was\u0026nbsp;reduced to 2.51\u0026plusmn;0.7 times its original level.\u003c/p\u003e\n\u003cp\u003eThese findings lead us to hypothesize that HIF-1\u0026alpha; may exert its influence by competitively inhibiting HIF-2\u0026alpha;, consequently mitigating cartilage calcification and subchondral bone sclerosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOsteoarthritis (OA) is a disease primarily characterized by the degeneration and destruction of joint cartilage, resulting from various causes[22]. Typical OA manifestations include cartilage surface damage, reduced chondrocyte population, thinning of transparent cartilage due to wear and tear, and an increase in calcified cartilage thickness, among other changes. Due to the avascular and\u0026nbsp;nonneural\u0026nbsp;characteristics of joint cartilage[23], chondrocytes endure long-term metabolic and repair processes in a hypoxic environment. In cases of OA, chondrocyte hypoxia intensifies due to inflammatory\u0026nbsp;factor\u0026nbsp;stimulation and heightened synovial oxygen consumption\u0026nbsp;[24]. Our proteomic analysis of human knee joint cartilage samples revealed a significant increase in HIF-1\u0026alpha; protein expression and a decrease in autophagy levels in regions with severe cartilage damage.\u0026nbsp;Although\u0026nbsp;OA\u0026nbsp;is\u0026nbsp;a systemic disease and the medial and lateral compartments of the knee joint\u0026nbsp;share\u0026nbsp;the same synovial cavity, we observed opposite changes in HIF-1\u0026alpha; and autophagy. This\u0026nbsp;finding\u0026nbsp;piqued our interest, and after observing human joint cartilage samples and mouse knee joint cartilage, we found that after the onset of OA, HIF-1\u0026alpha; was upregulated, while LC3 was downregulated. This led to an upregulation of MMP13,\u0026nbsp;which is\u0026nbsp;responsible for cartilage matrix degradation, and a decrease in the major cartilage component, COL II. Therefore, we hypothesize that HIF-1\u0026alpha; affects cartilage repair through its influence on autophagy. To explore this relationship further, we intervened with chondrocytes in vitro using IL-1\u0026beta; and found that HIF-1\u0026alpha; and ROS expression increased while LC3 decreased. This aligns with the manifestations of OA, primarily due to inflammation-induced local hypoxia and damage, which elevated cellular oxidative stress, thereby inhibiting mitochondrial autophagy. Some studies have suggested that HIF-1\u0026alpha; acts as a protective agent, alleviating cell apoptosis and death in OA by promoting mitochondrial autophagy within OA cartilage[25].\u003c/p\u003e\n\u003cp\u003eSubsequently, we intervened with HIF-1\u0026alpha;, initially using the HIF-1\u0026alpha; inhibitor LW6. We observed that as HIF-1\u0026alpha; decreased, ROS levels increased further, and LC3 levels decreased. This demonstrated that although HIF-1\u0026alpha; expression is upregulated after OA, inhibiting HIF-1\u0026alpha; expression does not respond to cellular oxidative stress and cannot reverse the decline in autophagy. Thus, we speculate that there may be an effective expression threshold between HIF-1\u0026alpha; and the inflammatory response. Since HIF-1\u0026alpha; is degraded under the action of prolyl hydroxylases (PHDs), we used the PHD inhibitor DMOG to maintain HIF-1\u0026alpha; at a higher level. We found that after DMOG intervention, chondrocytes showed a significant increase in HIF-1\u0026alpha;, a noticeable decrease in ROS, and a significant increase in LC3. Therefore, we infer that maintaining a high level of HIF-1\u0026alpha; can accelerate ROS clearance, thereby enhancing mitochondrial autophagy capability.\u003c/p\u003e\n\u003cp\u003eCurrent research suggests that HIF-1\u0026alpha; enables bone cells to adapt to a hypoxic microenvironment and promotes their growth and development. It plays a significant role in regulating various physiological and pathological responses in the body, with particular attention to its role in vascular and bone formation. In cartilage tissue, HIF-1\u0026alpha; has the capacity to promote cartilage formation and is a critical regulatory factor in the early differentiation of chondrocytes\u0026nbsp;[26]. Hypoxia-inducible factors directly affect the survival of chondrocytes. In human bone marrow cells, under low oxygen conditions, HIF-1\u0026alpha; can effectively induce chondrocytes, enabling them to adapt to the hypoxic microenvironment of the growth plate between bones.\u003c/p\u003e\n\u003cp\u003eMartin \u003cem\u003eet al\u003c/em\u003e.\u0026nbsp;[27]\u003csup\u003e\u0026nbsp;\u003c/sup\u003efound that cartilage degeneration is associated with the accumulation of metabolites resulting from trauma, chronic injury, and prolonged hypoxia. To validate the impact of maintaining high HIF-1\u0026alpha; expression on osteoarthritic (OA) cartilage, we induced OA in mice using destabilization of the medial meniscus (DMM) surgery, which is widely used in mouse OA studies. Compared to the anterior cruciate ligament transection (ACLT) method, DMM surgery closely mimics the natural progression of human OA due to its longer OA cycle. We injected DMOG daily into mice for eight weeks. The final experimental results showed that under the continuous influence of high HIF-1\u0026alpha; expression, autophagy remained highly expressed. The critical cartilage-degrading protein MMP13 decreased in content, and the main collagen protein,\u0026nbsp;COL II,\u0026nbsp;in the extracellular matrix of cartilage cells increased. Type II collagen is closely associated with the occurrence and development of OA[28], as it nourishes cartilage cells and maintains their structural stability. During the progression of OA, increased extracellular matrix degradation, reduced mRNA expression of type II collagen, and excessive apoptosis of cartilage cells all contribute to OA progression[29].\u003c/p\u003e\n\u003cp\u003eWe used micro-CT to examine the subchondral bone, and while there were no significant differences between groups, there were subtle differences. The subchondral bone-related parameters of the DMOG-treated group were closer to those of the control group. This may be due to the insufficient sample size we used and the substantial intragroup variations. We believe a more plausible explanation is the sustained high expression of HIF-1\u0026alpha;\u0026apos;s suppression of abnormal bone mineralization [30]. In our experiment, we detected a decrease in \u0026beta;-catenin expression in the DMOG-treated group. \u0026beta;-catenin is a key upstream factor in the Wnt signaling pathway[31] and is involved in chondrocyte osteogenic differentiation, cartilage calcification, and subchondral bone sclerosis processes[32]. According to the literature[33], hypoxia-inducible factors HIF-1\u0026alpha; and HIF-2\u0026alpha; each exert their biological effects by forming HIF-1 heterodimers with HIF-1\u0026beta;. HIF-1\u0026alpha; primarily induces autophagy and vascular formation, while HIF-2\u0026alpha; induces chondrocyte differentiation and promotes osteogenesis [34, 35]. Therefore, we speculate that HIF-1\u0026alpha; may inhibit HIF-2\u0026alpha;-mediated chondrocyte osteogenic differentiation through competitive binding to HIF-1\u0026beta;. This will be investigated in our next study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research, through the exploration of proteomics and biological information, discovered a correlation between HIF-1\u0026alpha; activation and autophagy in OA cartilage. This phenomenon has been validated in clinical sample experiments. Subsequent research indicates that maintaining high HIF-1\u0026alpha; expression can protect osteoarthritic chondrocytes through oxidative stress regulation of mitochondrial autophagy and inhibit abnormal mineralization of subchondral bone. This provides new effective strategies for understanding the pathogenesis of OA and its early prevention and treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHIF-1\u0026alpha;,Hypoxia-Inducible Factor-1\u0026alpha;;OA, osteoarthritis; RT‑qPCR,reverse transcription‑quantitative polymerase chain reaction; H\u0026amp;E, haematoxylin and eosin; OARSI, Osteoarthritis Research Society International；ROS, reactive oxygen species;PHD2, prolyl hydroxylase 2；MMP, Mitochondrial membrane potential;DMOG, dimethyloxalylglycine；IL‑1\u0026beta;, interleukin‑1\u0026beta;;MMP3, matrix metalloproteinases 13; SOX9, SRY‑box transcription factor 9; CCK‑8, Cell Counting Kit‑8; KEGG, Kyoto Encyclopedia of Genes and Genomes；LC3, light chain 3；COL Ⅱ，Collagen TypeⅡ；HIF-2\u0026alpha;,Hypoxia-Inducible Factor-2\u0026alpha;；DMM，destabilization of the medial meniscus surgery；mTOR，Mechanistic Target Of Rapamycin；TKA，total knee arthroplasty；KOA，knee osteoarthritis；FBS, fetal bovine serum;IHC, immunohistochemistry;VAS, Visual Analog Scale;K-L, Kellgren-Lawrence grading system;GO, Gene Ontology\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Servicebio Biotechnology Company（Wu han, China），Chengdu Lilai Biotechnology Company（Cheng du, China）and Applied Protein technology Biotechnology Company(Shang hai, China) for providing technical services.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by by the following grants：\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China - (No.U22A20285)；\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China\u0026nbsp;（No. 82360319）；\u003c/p\u003e\n\u003cp\u003eKey R\u0026amp;D Project of Autonomous Region (No.2023BEG02018)；\u003c/p\u003e\n\u003cp\u003eKey R\u0026amp;D Project of Autonomous Region（2022BEG03126）；\u003c/p\u003e\n\u003cp\u003eScientific Research Project of Ningxia Universities (No. NYG-2022033)；\u003c/p\u003e\n\u003cp\u003eKey R\u0026amp;D Project of Autonomous Region (No.2021BEG02037)；\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China - (No.82160433)；\u003c/p\u003e\n\u003cp\u003eNingxia Medical University General Hospital \u0026quot;Medical Engineering Special\u0026quot; (No. NYZYYG-001)；\u003c/p\u003e\n\u003cp\u003eScientific Research Project of Ningxia Universities (No.XZ2020014)；\u003c/p\u003e\n\u003cp\u003eAutonomous Region Major Scientific and Technological Achievements Transformation Project (No. 2023CJE09037)；\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQHJ and DX designed the experiment. XLC,GNF, XXH and YZ conducted the experiment. XL,YY,LM,HW and ZDL analysed the data, XLC,GNF,XL,JBYdrafted and revised the manuscript. QHJ, DX and XLC confirm the authenticity of all the raw data. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Animal Experiment Ethics Committee of Ningxia Medical University (Yinchuan, China; Approval no. IACUC-NYLAC-2020-115)，and the Ethics Committee of Ningxia Medical University General Hospital (Approval No: 2023-25). All experiments were performed under the standard ethical principles of animal and human experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments involving human beings have been explained in detail to the patients and obtained their permission, signed informed consent, and the patients agreed to donate the bone tissue removed during the operation to us for research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update with relevance for clinical practice. Lancet. 2011;377(9783):2115\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurkiewicz A, Kiadaliri AA, Englund M. Cause-specific mortality in osteoarthritis of peripheral joints. Osteoarthritis Cartilage. 2019;27(6):848\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng C, Bennell K, Yang Z, Nguyen U, Lu N, Wei J, et al. Risk of venous thromboembolism in knee, hip and hand osteoarthritis: a general population-based cohort study. Ann Rheum Dis. 2020;79(12):1616\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Nguyen U, Lane NE, Lu N, Wei J, Lei G, et al. Knee osteoarthritis, potential mediators, and risk of all-cause mortality: data from the osteoarthritis initiative. Arthritis Care Res (Hoboken). 2021;73(4):566\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSafiri S, Kolahi AA, Smith E, Hill C, Bettampadi D, Mansournia MA, et al. Global, regional and national burden of osteoarthritis 1990\u0026ndash;2017: a systematic analysis of the global burden of disease study 2017. Ann Rheum Dis. 2020;79(6):819\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoulet B, Staines KA. New developments in osteoarthritis and cartilage biology. Curr Opin Pharmacol. 2016;28:8\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlyn-Jones S, Palmer AJ, Agricola R, Price AJ, Vincent TL, Weinans H, et al. Osteoarthritis. Lancet. 2015;386(9991):376\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuettner KE, Aydelotte MB, Thonar EJ. Articular cartilage matrix and structure: a minireview. J Rheumatol Suppl. 1991;27:46\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoimbra IB, Jimenez SA, Hawkins DF, Piera-Velazquez S, Stokes DG. Hypoxia inducible factor-1 alpha expression in human normal and osteoarthritic chondrocytes. Osteoarthritis Cartilage. 2004;12(4):336\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiaer T, Gronlund J, Sorensen KH. Subchondral po2, pco2, pressure, ph, and lactate in human osteoarthritis of the hip. Clin Orthop Relat Res. 1988(229):149\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfander D, Cramer T, Schipani E, Johnson RS. Hif-1alpha controls extracellular matrix synthesis by epiphyseal chondrocytes. J Cell Sci. 2003;116(Pt 9):1819\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaheem DK, Jell G, Gentleman E. Hypoxia inducible factor-1alpha in osteochondral tissue engineering. Tissue Eng Part B Rev. 2020;26(2):105\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouaziz W, Sigaux J, Modrowski D, Devignes CS, Funck-Brentano T, Richette P, et al. Interaction of hif1alpha and beta-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice. Proc Natl Acad Sci U S A. 2016;113(19):5453\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng L, Feng C, Wang CX, Xu DY, Chen JJ, Huang JF, et al. Circulating microrna let\u0026ndash;7e is decreased in knee osteoarthritis, accompanied by elevated apoptosis and reduced autophagy. Int J Mol Med. 2020;45(5):1464\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohensky J, Leshinsky S, Srinivas V, Shapiro IM. Chondrocyte autophagy is stimulated by hif-1 dependent ampk activation and mtor suppression. Pediatr Nephrol. 2010;25(4):633\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOh H, Chun CH, Chun JS. Dkk-1 expression in chondrocytes inhibits experimental osteoarthritic cartilage destruction in mice. Arthritis Rheum. 2012;64(8):2568\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlom AB, Brockbank SM, van Lent PL, van Beuningen HM, Geurts J, Takahashi N, et al. Involvement of the wnt signaling pathway in experimental and human osteoarthritis: prominent role of wnt-induced signaling protein 1. Arthritis Rheum. 2009;60(2):501\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouaziz W, Funck-Brentano T, Lin H, Marty C, Ea HK, Hay E, et al. Loss of sclerostin promotes osteoarthritis in mice via beta-catenin-dependent and -independent wnt pathways. Arthritis Res Ther. 2015;17(1):24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu M, Tang D, Wu Q, Hao S, Chen M, Xie C, et al. Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice. J Bone Miner Res. 2009;24(1):12\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa L, Liu Y, Zhao X, Li P, Jin Q. Rapamycin attenuates articular cartilage degeneration by inhibiting beta-catenin in a murine model of osteoarthritis. Connect Tissue Res. 2019;60(5):452\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu S, Zhang C, Ni L, Huang C, Chen D, Shi K, et al. Stabilization of hif-1α alleviates osteoarthritis via enhancing mitophagy. Cell Death Dis. 2020;11(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3(2):107\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3(2):107\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSvalastoga E, Kiaer T. Oxygen consumption, diffusing capacity and blood flow of the synovial membrane in osteoarthritic rabbit knee joints. Acta Vet Scand. 1989;30(2):121\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, Peng Y, Zou J, Wang J, Lu S, Fu T, et al. Hypoxia inducible factor-1alpha is a regulator of autophagy in osteoarthritic chondrocytes. Cartilage. 2021;13(2_suppl):1030S-1040S.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu JH, Yang S, Shin Y, Rhee J, Chun CH, Chun JS. Interleukin-6 plays an essential role in hypoxia-inducible factor 2alpha-induced experimental osteoarthritic cartilage destruction in mice. Arthritis Rheum. 2011;63(9):2732\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin JA, Brown T, Heiner A, Buckwalter JA. Post-traumatic osteoarthritis: the role of accelerated chondrocyte senescence. Biorheology. 2004;41(3\u0026ndash;4):479\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLane AR, Harkey MS, Davis HC, Luc-Harkey BA, Stanley L, Hackney AC, et al. Body mass index and type 2 collagen turnover in individuals after anterior cruciate ligament reconstruction. J Athl Train. 2019;54(3):270\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn QD, Li YY, Zhang HX, Lu J, Yu XD, Lin Q, et al. Inhibition of bromodomain-containing protein 4 ameliorates oxidative stress-mediated apoptosis and cartilage matrix degeneration through activation of nf-e2-related factor 2-heme oxygenase-1 signaling in rat chondrocytes. J Cell Biochem. 2018;119(9):7719\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmir MS, Chiba N, Seong CH, Kusuyama J, Eiraku N, Ohnishi T, et al. Hif-1alpha plays an essential role in bmp9-mediated osteoblast differentiation through the induction of a glycolytic enzyme, pdk1. J Cell Physiol. 2022;237(4):2183\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Q, Wu X, Tao C, Gong W, Chen M, Qu M, et al. Osteoarthritis: pathogenic signaling pathways and therapeutic targets. Signal Transduct Target Ther. 2023;8(1):56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Xiong Y, Chen W, Wu L. Wnt/beta-catenin signaling may induce senescence of chondrocytes in osteoarthritis. Exp Ther Med. 2020;20(3):2631\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang FJ, Luo W, Lei GH. Role of hif-1alpha and hif-2alpha in osteoarthritis. Joint Bone Spine. 2015;82(3):144\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaito T, Kawaguchi H. Hif-2alpha as a possible therapeutic target of osteoarthritis. Osteoarthritis Cartilage. 2010;18(12):1552\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XA, Kong H. Mechanism of hifs in osteoarthritis. Front Immunol. 2023;14:1168799.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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, chondrocytes, hypoxia-inducible factor-1α, autophagy, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-3419638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3419638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eHypoxia-inducible factor-1α (HIF-1α) is known to regulate the energy metabolism and autophagy of chondrocytes under inflammatory and hypoxic conditions. This study aims to investigate the mechanisms by which HIF-1α influences cartilage injury through autophagy and oxidative stress pathways following the onset of osteoarthritis (OA).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman knee joint samples were categorized into the OA group and the control group (CON) for radiological and pathological assessments, along with proteomic analysis to elucidate the interplay between osteoarthritis, HIF-1α, and autophagy. Chondrocytes were stimulated with IL-1β to establish an OA model, and these cells were subsequently divided into the control group (CON), IL-1β group (OA), IL-1β\u0026thinsp;+\u0026thinsp;LW6 group, IL-1β\u0026thinsp;+\u0026thinsp;DMOG100 group, and IL-1β\u0026thinsp;+\u0026thinsp;DMOG200 group. Immunofluorescence and western blot analyses were employed to measure the expression levels of HIF-1α, ROS, and LC3 to clarify the association between HIF-1α and autophagy. In addition, mice were categorized into the control group (CON), model group (DMM), and treatment group (DMM\u0026thinsp;+\u0026thinsp;DMOG). Immunohistochemistry, immunofluorescence, and RT-qPCR were conducted to assess the expression levels of HIF-1α, LC3, MMP-13, COL2, β-catenin, and HIF-2α. Micro-CT was utilized to evaluate subchondral bone morphology to elucidate the relationship between HIF-1α and cartilage injury, as well as its underlying mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOsteoarthritic cartilage exhibited elevated levels of HIF-1α, reduced LC3 expression, and increased ROS levels. Inhibition of HIF-1α using LW6 led to further reductions in LC3 levels and increased ROS production. Conversely, the activation of HIF-1α with DMOG significantly elevated HIF-1α levels, increased LC3 expression, reduced ROS levels, decreased MMP-13 levels, enhanced COL2 expression, decreased β-catenin levels, and lowered HIF-2α expression, resulting in a reduced severity of articular cartilage injury.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAfter the onset of osteoarthritis, low or physiologically elevated levels of HIF-1α may not adequately activate autophagy. Maintaining HIF-1α at elevated levels can protect articular cartilage by inhibiting oxidative stress and enhancing autophagy.\u003c/p\u003e","manuscriptTitle":"HIF-1α protects articular cartilage in osteoarthritis by activating autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-12 20:49:51","doi":"10.21203/rs.3.rs-3419638/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":"621cb7c8-0e39-4b2f-9a71-68b116504a90","owner":[],"postedDate":"October 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T03:44:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-12 20:49:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3419638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3419638","identity":"rs-3419638","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}