Detection and Evaluation of Serological Biomarkers to Predict Osteoarthritis in Anterior Cruciate Ligament Transection Combined Medial Meniscectomy Rat Model

Biomarkers are essential tools in osteoarthritis (OA) research, clinical trials, and drug development. Detecting and evaluating biomarkers in OA research can open new avenues for researching and developing new therapeutics. In the present report, we have explored the serological detection of various osteoarthritis-related biomarkers in the preclinical model of OA. In this surgical OA model, we disrupted the medial tibial cartilage’s integrity via anterior cruciate ligament transection combined with medial meniscectomy (ACLT+MMx) of a single joint of Wistar rats. The progression of OA was verified, as shown by the microscopic deterioration of cartilage and the increasing cartilage degeneration scoring from 4 to 12 weeks postsurgery. The concentration of serological biomarkers was measured at two timepoints, along with the complete blood count and bone electrolytes, with biochemical analysis further conducted. The panel evaluated inflammatory biomarkers, bone/cartilage biomarkers, and lipid metabolic pathway biomarkers. In chronic OA rats, we found a significant reduction of total vitamin D3 and C-telopeptide fragments of type II (CTX-II) levels in the serum as compared to sham-operated rats. In contrast, the serological levels of adiponectin, leptin, and matrix metallopeptidase (MMP3) were significantly enhanced in chronic OA rats. The inflammatory markers, blood cell composition, and biochemical profile remained unchanged after surgery. In conclusion, we found that a preclinical model of single-joint OA with significant deterioration of the cartilage can lead to serological changes to the cartilage and metabolic-related biomarkers without alteration of the systemic blood and biochemical profile. Thus, this biomarker profile provides a new tool for diagnostic/therapeutic assessment in OA scientific research.

Hesperetin Exhibits Anti-Inflammatory Effects on Chondrocytes via the AMPK Pathway to Attenuate Anterior Cruciate Ligament Transection-Induced Osteoarthritis

This study aimed to determine whether hesperetin (HPT) has chondroprotective effects against the TNF-α-induced inflammatory response of chondrocytes and related mechanisms and clarify the impact of HPT on osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). Under tumor necrosis factor-α (TNF-α) stimulation, rat chondrocytes were treated with or without HPT. The CCK-8 assay was used to detect viability and cytotoxicity. RT-qPCR and Western blot were used to examine the expression of aggrecan, collagen type II, and inflammatory and proliferative genes/proteins in chondrocytes. Flow cytometry was used to check the cell cycle to determine whether HPT protects chondrocytes against the inhibitory effect of TNF-α on chondrocyte proliferation. In addition, RNA sequencing was used to discover possible molecular targets and pathways and then validate these pathways with specific protein phosphorylation levels. Finally, immunofluorescence staining was used to examine the phosphorylation of the AMP-activated protein kinase (AMPK) pathway. The results showed that HPT restored the upregulation of interleukin 1β (IL-1β), PTGS2, and MMP-13 induced by TNF-α. In addition, HPT reversed the degradation of the extracellular matrix of chondrocytes induced by TNF-α. HPT also reversed the inhibitory effect of TNF-α on chondrocyte proliferation. RNA sequencing revealed 549 differentially expressed genes (DEGs), of which 105 were upregulated and 444 were downregulated, suggesting the potential importance of the AMPK pathway. Progressive analysis showed that HPT mediated the repair of TNF-α-induced chondrocyte damage through the AMPK signaling pathway. Thus, local treatment of HPT can improve OA induced by ACLT. These findings indicated that HPT has significant protective and anti-inflammatory effects on chondrocytes through the AMPK signaling pathway, effectively preventing cartilage degradation. Given the various beneficial effects of HPT, it can be used as a potential natural drug to treat OA.

Chondroprotective Effects of a Histone Deacetylase Inhibitor, Panobinostat, on Pain Behavior and Cartilage Degradation in Anterior Cruciate Ligament Transection-Induced Experimental Osteoarthritic Rats

Osteoarthritis (OA) is the most common articular degenerative disease characterized by chronic pain, joint inflammation, and movement limitations, which are significantly influenced by aberrant epigenetic modifications of numerous OA-susceptible genes. Recent studies revealed that both the abnormal activation and differential expression of histone deacetylases (HDACs) might contribute to OA pathogenesis. In this study, we investigated the chondroprotective effects of a marine-derived HDAC inhibitor, panobinostat, on anterior cruciate ligament transection (ACLT)-induced experimental OA rats. The intra-articular administration of 2 or 10 µg of panobinostat (each group, n = 7) per week from the 6th to 17th week attenuates ACLT-induced nociceptive behaviors, including secondary mechanical allodynia and weight-bearing distribution. Histopathological and microcomputed tomography analysis showed that panobinostat significantly prevents cartilage degeneration after ACLT. Moreover, intra-articular panobinostat exerts hypertrophic effects in the chondrocytes of articular cartilage by regulating the protein expressions of HDAC4, HDAC6, HDAC7, runt-domain transcription factor-2, and matrix metalloproteinase-13. The study indicated that HDACs might have different modulations on the chondrocyte phenotype in the early stages of OA development. These results provide new evidence that panobinostat may be a potential therapeutic drug for OA.

miR-29b is Involved in Cartilage Autophagy and Muscle Atrophy in a Rat Model of Knee Osteoarthritis

Background Knee osteoarthritis (KOA) is a progressively degenerative form of arthritis characterized by chondrocyte apoptosis and cartilage degeneration. KOA also involves limb muscle atrophy, especially in the quadriceps muscles. However, there are limited options for the treatment of KOA. miR-29b stimulates apoptosis in the chondrocytes from patients with KOA and muscle atrophy in other models. Therefore, we investigated the therapeutic effect of miR-29b in cartilage autophagy and muscle atrophy. Methods Ten rats comprised the control cohort without anterior cruciate ligament transection. The treatment group (KOA induced in the right knee via anterior cruciate ligament transection) was divided into a model untreated group and a miR-29b-antagomir group (miR-29b antagomir injected 1 wk before surgery). Results Real-time polymerase chain reaction revealed successful downregulation of miR-29b using antagomir in the joints and muscles. A weight-bearing test showed that miR-29b downregulation affected joint function. Enzyme-linked immunosorbent assays demonstrated that downregulating miR-29b reduced pro-inflammatory cytokine expression. Immunohistochemistry revealed that miR-29b depletion enhanced autophagy by activating LC3 and beclin-1 in the cartilage. Autophagy was stimulated by the activation of MAPK and mTOR signaling. Depletion of miR-29b ameliorated the decrease in the weight of the quadriceps and the quadriceps weight/body weight ratio of the rats. Hematoxylin–eosin and periodic acid–Schiff staining showed that miR-29b downregulation inhibited muscular atrophy. Immunofluorescence showed that miR-29b downregulation affected IGF/PI3K/AKT signaling. Conclusions This study demonstrated the therapeutic effect of miR-29b on autophagy in the cartilage and on muscle atrophy in a rat model for KOA, highlighting the potential of miR-29b as a therapeutic target for KOA.