Objective To investigate the regulatory role and molecular mechanisms of TSPAN9-mediated mitocytosis in an interleukin-1β (IL-1β)-induced rat chondrocyte senescence model, and to identify novel therapeutic targets for osteoarthritis (OA). MethodsPrimary knee articular chondrocytes were isolated from the cartilage of knee joints harvested from 1-week-old male Sprague-Dawley rats and maintained in culture. Cell viability was assessed using the cell counting kit 8 (CCK-8) assay, and cell-cycle distribution was analyzed by flow cytometry to determine the optimal concentration and exposure time of IL-1β for model induction, thereby establishing an in vitro chondrocyte senescence model [early-OA (E-OA), middle-OA (M-OA), late-OA (L-OA)] and grouped. Cellular senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining. Real-time quantitative PCR (qRT-PCR) was used to quantify the mRNA expression levels of senescence markers [cyclin-dependent kinase inhibitor 2A (Cdkn2a) and Cdkn1a], extracellular matrix (ECM) catabolic-anabolic genes [collagen type Ⅱ alpha 1 chain (Col2a1), Aggrecan (Acan), matrix metalloproteinase 13 (MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5)], and key mitocytosis-related genes [kinesin family member 5B (KIF5B), TSPAN9, and TSPAN4]. Mitochondrial ultrastructure and mitocytosis-related morphological features were examined by transmission electron microscopy, and changes in mitochondrial membrane potential were assessed using the JC-1 fluorescent probe. To activate TSPAN9 expression, chondrocytes were transduced with a TSPAN9-overexpressing lentivirus (LV-TSPAN9). The experiments included four groups: control group, M-OA group, lentivirus negative control group, and LV-TSPAN9 group. After confirming overexpression efficiency, differences in cellular senescence, mitochondrial homeostasis, and key ECM metabolic indices were compared among the groups. Results Using CCK-8 assays and cell-cycle analysis, the staged rat chondrocyte senescence model induced by IL-1β (5 ng/mL), and defined 12, 24, and 48 hours as the E-OA, M-OA, and L-OA senescence phenotypes were successfully established, respectively. Model validation demonstrated that, with prolonged induction, the proportion of SA-β-gal-positive cells increased markedly, mitochondrial membrane potential progressively declined (P<0.05), and mitochondrial ultrastructural damage became increasingly severe. qRT-PCR showed progressive upregulation of the senescence markers Cdkn2a and Cdkn1a, as well as the ECM catabolic genes MMP-13 and ADAMTS5 (P<0.05), together with sustained downregulation of the ECM anabolic genes Col2a1 and Acan (P<0.05). Notably, the key mitocytosis-related genes KIF5B, TSPAN4, and TSPAN9 displayed a biphasic pattern, with early compensatory upregulation followed by decompensatory decline at the middle-to-late stages. In the M-OA model, TSPAN9 overexpression markedly reversed these pathological changes, restored mitochondrial membrane potential (P<0.05), ameliorated the senescent phenotype and ECM metabolic imbalance, and significantly upregulated the expression of mitocytosis-related genes and proteins (P<0.05), with transmission electron microscope revealing morphological structures suggestive of mitocytosis-related events. Conclusion Impaired mitocytosis is an important pathological mechanism underlying IL-1β-induced chondrocyte senescence, and TSPAN9 overexpression may delay chondrocyte senescence by promoting mitocytosis in concert with KIF5B, thereby facilitating the clearance of damaged mitochondria and restoring intracellular homeostasis.
Objective To explore the diagnostic efficacy of electrochemical detection of synovial fluid leukocyte esterase (LE) based on glucosyl ester for periprosthetic joint infection (PJI) in rabbits. Methods The enzyme kinetic parameters of the LE-catalyzed reaction based on glucosyl esters were determined electrochemically, and the charge-LE concentration relationship was plotted. Forty-eight healthy New Zealand rabbits were randomly divided into a sham-operation group (blank group), a knee joint metal prosthesis implantation group (control group), and a knee joint metal prosthesis implantation with PJI group (experimental group), with 16 rabbits in each group. The experimental group underwent intra-articular injection of 1×105 CFU/mL Staphylococcus aureus suspension at 7 days after right knee joint prosthesis implantation. The control group underwent intra-articular injection of 1 mL of sterile saline at 7 days after right knee joint prosthesis implantation. The blank group underwent right knee joint capsule incision and suture, followed by intra-articular injection of 1 mL of sterile saline 7 days later. The general condition of the animals was observed after operation. At 28 days after operation, imaging examination, microbiological examination, hematological test, and electrochemical detection of synovial fluid LE were performed. The area (AUC) under the receiver operating characteristic curve (ROC) was used to evaluate the sensitivity and specificity of LE-based detection of PJI. Results The Michaelis constant of the enzymatic reaction of LE catalyzing glucosyl ester was 7.335 mmol/L, and the maximum reaction rate of the enzymatic reaction was 63.750 μmol·L?1·min?1. The calibration curve regression equation for the charge difference (ΔQ)-LE concentration was C(LE)=0.7115ΔQ?14.75, with a determination coefficient R2 of 0.988 4 (95%CI: 0.5698, 0.8532, P<0.001). All animals survived to the end of the experiment. During the period, the experimental group showed signs of joint infection, and the microbiological test results were all positive. The other two groups had no related infection manifestations and the tests were negative. Imaging examination showed that the experimental prosthesis was stable in position, soft tissue swelling around the joint and mild deformation in the joint. There was no obvious swelling in the soft tissue around the joints in the other two groups, and the joints were in good alignment. Synovial fluid and hematological tests revealed that the experimental group exhibited significantly higher levels of LE, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell count (WBC), neutrophil count (Neu), and monocytes count (Mon) compared to both the control group and the blank group (P<0.05). Notably, ESR and Mon levels were significantly higher in the control group compared to the blank group (P<0.05). The mentioned indicators, with an AUC>0.9 (P<0.05), possess good diagnostic value for PJI. Among them, CPR and Neu exhibit the highest sensitivity, reaching 93.8%, while LE, ESR, and Mon demonstrate the highest specificity, reaching 100%. Conclusion Both synovial fluid LE and hematological testing exhibit good diagnostic efficacy in the rabbit PJI model, with the method based on electrochemical quantitative detection of glucose-based esters for LE demonstrating higher specificity, sensitivity, and accuracy.