Hongxu Yang

Background: Temporomandibular joint osteoarthritis (TMJ OA) is characterized by articular cartilage degeneration involving chondrocyte loss and matrix degradation. However, the underlying mechanisms remain elusive. Hemoglobin (HB) has been identified in chondrocytes and is closely associated with cell survival. Objective: This study aims to investigate the role of HB in condylar cartilage degeneration during the early stage of TMJ OA and evaluate the potential protective effects of hydroxyurea (HU), a therapeutic agent used for sickle cell disease, suggesting its promise as a novel therapeutic strategy against OA. Methods: The TMJ OA model was established in 6‐week‐old female rats using unilateral anterior crossbite (UAC). ATDC5 chondrocytes were exposed to fluid flow shear stress (FFSS) to mimic OA‐like changes in vitro. Histological and immunohistochemical staining, real‐time PCR, and Western blot analysis were performed to detect the morphological and molecular changes in condylar cartilage. Results: The results showed a significant downregulation of HBA and HBB alongside cartilage degeneration in the UAC group, characterized by decreased cartilage thickness, degraded cartilage matrix, and increased matrix metalloproteinases (MMPs). FFSS‐stimulated chondrocytes also exhibited decreased HBA and HBB expression. Notably, HU treatment enhanced γ‐globin gene ( HBG ) expression, mitigated FFSS‐stimulated chondrocyte degeneration in vitro, and attenuated TMJ OA progression in vivo. Specifically, HU ameliorated the degenerative phenotype in cartilage, increasing matrix content and reducing matrix‐degrading enzyme expression. Conclusions: These findings identify HB subunits as novel molecular targets for OA therapy, with HU demonstrating significant chondroprotective efficacy. The study provides mechanistic insights into OA pathogenesis and highlights a promising repurposing therapeutic strategy for clinical intervention.

Subchondral bone loss is an important pathological feature of early-stage temporomandibular joint (TMJ) osteoarthritis (OA). Previous studies focused mainly on the bone resorption by osteoclasts in early-stage OA, but the bone formation feature has not drawn enough attention. Sema4D/Plexin-B1 is a pair of molecules expressed by osteoclast/osteoblast, which is capable of inhibiting bone formation by osteoblasts. The present study found that subchondral bone loss in early-stage TMJ OA was accompanied by up-regulated expression of Sema4D in cartilage and subchondral bone and Plexin-B1 in subchondral bone. Reducing Sema4D level could inhibit the subchondral bone loss and cartilage degeneration of early-stage TMJ OA. In vitro, results revealed that Sema4D could reduce the expression of osteocalcin (OCN) and alkaline phosphatase (ALP), and increase the migrating capability of Plexin-B1-positive osteoblasts. Our results revealed that elevated Sema4D expression in early-stage TMJ OA might decrease the bone formation activity of osteoblasts in the subchondral bone by binding to Plexin-B1 expressed by osteoblasts. Inhibiting Sema4D/Plexin-B1 signaling in the early-stage OA holds promise as a strategy for new therapeutic approaches to osteoarthritis.

The temporomandibular joint (TMJ), which is biomechanically related to dental occlusion, is often insulted by osteoarthritis (OA). This study was conducted to clarify the relationship between Indian hedgehog (Ihh) and parathyroid hormone receptor 1 (PTH1R) signaling in modulating the enhanced chondrocyte terminal differentiation in dental stimulated TMJ osteoarthritic cartilage. A gain- and loss-of-function strategy was used in an in vitro model in which fluid flow shear stress (FFSS) was applied, and in an in vivo model in which the unilateral anterior cross-bite (UAC) stimulation was adopted. Ihh and PTH1R signaling was modulated through treating the isolated chondrocytes with inhibitor/activator and via deleting Smoothened (Smo) and/or Pth1r genes in mice with the promoter gene of type 2 collagen (Col2-CreER) in the tamoxifen-inducible pattern. We found that both FFSS and UAC stimulation promoted the deep zone chondrocytes to undergo terminal differentiation, while cells in the superficial zone were robust. We demonstrated that the terminal differentiation process in deep zone chondrocytes promoted by FFSS and UAC was mediated by the enhanced Ihh signaling and declined PTH1R expression. The FFSS-promoted terminal differentiation was suppressed by administration of the Ihh inhibitor or PTH1R activator. The UAC-promoted chondrocytes terminal differentiation and OA-like lesions were rescued in Smo knockout, but were enhanced in Pth1r knockout mice. Importantly, the relieving effect of Smo knockout mice was attenuated when Pth1r knockout was also applied. Our data suggest a chondrocyte protective effect of suppressing Ihh signaling in TMJ OA cartilage which is dependent on PTH1R signaling.

A switch from autophagy to apoptosis is implicated in chondrocytes during the osteoarthritis (OA) progression with currently unknown mechanism(s). In this study we utilized a flow fluid shear stress (FFSS) model in cultured chondrocytes and a unilateral anterior crossbite (UAC) animal model. We found that both FFSS and UAC actively induced endoplasmic reticulum stress (ERS) in the temporomandibular joints (TMJ) chondrocytes, as demonstrated by dramatic increases in expression of HSPA5, p-EIF2AK3, p-ERN1 and ATF6. Interestingly, both FFSS and UAC activated not only pro-death p-EIF2AK3-mediated ERS-apoptosis programs but also pro-survival p-ERN1-mediated autophagic flux in chondrocytes. Data from FFSS demonstrated that MTORC1, a downstream of p-ERN1, suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis. Data from UAC model demonstrated that at early stage both the p-ERN1 and p-EIF2AK3 were activated and MTORC1 was inhibited in TMJ chondrocytes. At late stage, MTORC1-p-EIF2AK3-mediated ERS apoptosis were predominant, while p-ERN1 and autophagic flux were inhibited. Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of MTORC1 expression selectively in chondrocytes in mice promoted chondrocyte autophagy and suppressed apoptosis, and reduced TMJ cartilage loss induced by UAC. In contrast, MTORC1 activation by TMJ local administration of MHY1485 or genetic deletion of Tsc1, an upstream MTORC1 suppressor, resulted in opposite effects. Collectively, our results establish that aberrant mechanical loading causes cartilage degeneration by activating, at least in part, the MTORC1 signaling which modulates the autophagy and apoptosis programs in TMJ chondrocytes. Thus, inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA. Abbreviations : ACTB: actin beta; ATF6: activating transcription factor 6; BECN1: beclin 1; BFL: bafilomycin A₁; CASP12: caspase 12; CASP3: caspase 3; DAPI: 4ʹ,6-diamidino-2-phenylindole; DDIT3: DNA-damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FFSS: flow fluid shear stress; HSPA5/GRP78/BiP: heat shock protein 5; LAMP2: lysosome-associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; OA: osteoarthritis; PRKAA1/2/AMPK1/2: protein kinase, AMP-activated, alpha 1/2 catalytic subunit; RPS6: ribosomal protein S6; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: thapsigargin; TMJ: temporomandibular joints; TSC1/2: tuberous sclerosis complex 1/2; UAC: unilateral anterior crossbite; UPR: unfolded protein response; XBP1: x-box binding protein 1.

The present study aimed to analyze novel mechanisms underlying Nrf2-mediated anti-apoptosis in periodontal ligament stem cells (PDLSCs) in the periodontitis oxidative microenvironment. We created an oxidative stress model with H₂O₂-treated PDLSCs. We used real-time PCR, Western blotting, TUNEL staining, fluorogenic assay and transfer genetics to confirm the degree of oxidative stress and apoptosis as well as the function of nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrated that with upregulated levels of reactive oxygen species (ROS) and malondialdehyde (MDA), the effect of oxidative stress was obvious under H₂O₂ treatment. Oxidative molecules were altered after the H₂O₂ exposure, whereby the signaling of Nrf2 was activated with an increase in its downstream effectors, heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1) and γ-glutamyl cysteine synthetase (γ-GCS). Additionally, the apoptosis levels gradually increased with oxidative stress by the upregulation of caspase-9, caspase-3, Bax and c-Fos levels in addition to the downregulation of Bcl-2. However, there was no alterations in levels of caspase-8. The enhanced antioxidant effect could not mitigate the occurrence of apoptosis. Furthermore, Nrf2 overexpression effectively improved the anti-oxidative levels and increased cell proliferation. At the same time, overexpression effectively restrained TUNEL staining and decreased the molecular levels of caspase-9, caspase-3, Bax and c-Fos, but not that of caspase-8. In contrast, silencing the expression of Nrf2 levels had the opposite effect. Collectively, Nrf2 alleviates PDLSCs via its effects on regulating oxidative stress and anti-intrinsic apoptosis by the activation of oxidative enzymes.

The present study aimed to analyze novel mechanisms underlying Nrf2-mediated anti-apoptosis in periodontal ligament stem cells (PDLSCs) in the periodontitis oxidative microenvironment. We created an oxidative stress model with H2O2-treated PDLSCs. Herein, we used real-time PCR, western blotting, TUNEL staining, fluorogenic assay and transfer genetics to confirm the degree of oxidative stress and apoptosis as well as the Nrf2 function. Surprisingly, we demonstrated that with up-regulated ROS and MDA levels, the effect of oxidative stress was obvious under H2O2 treatment. Anti-oxidative molecules were changed after the H2O2 exposure, whereby the anti-oxidative signaling of Nrf2 was activated with the increase of its downstream effectors, HO-1, NQO1 and γ-GCS. Additionally, the apoptosis levels gradually increased with oxidative stress and changes in the caspase-9, caspase-3, Bax and c-Fos levels, but not with caspase-8 and down-regulated Bcl-2. The enhanced antioxidant effect could not resist the occurrence of apoptosis. Furthermore, Nrf2 overexpression effectively improved the anti-oxidative levels and increased cell proliferation. At the same time, overexpression effectively restrained TUNEL staining and decreased the molecular levels of caspase-9, caspase-3, et al, but not that of caspase-8. By contrast, silencing the expression Nrf2 levels had the opposite effect. Collectively, Nrf2 alleviates PDLSCs via its effects on anti-oxidative and anti-intrinsic apoptosis by the activation of anti-oxidative enzymes.