Heat shock proteins in osteoarthritis: how they work, contribute to disease, and potential treatments

HSP27, also known as HSPB1, is one of the small heat shock proteins (sHSPs) (25). It is a chaperone protein that is categorized according to its distinct domain organization, rather than ATPase activity (26). HSP27 does not contain an ATP-binding domain as seen in HSP70 or HSP90, but rather has a conserved α-crystallin domain (containing about 80–100 amino acids) in its center (27), and is associated with a variable N-terminal WD/EPF-rich domain and a disordered C-terminal tail (28). The N-terminal domain appears to be involved in the regulation of oligomerization and phosphorylation-dependent dynamic activity (29), and the α-crystallin domain is responsible for dimerization and substrate binding, which is the chaperone core (30). The IXI/V-rich C-terminal domain plays a role in stabilizing the large oligomeric assembly, as well as mediating client protein interaction (31). Phosphorylation at conserved serine residues (Ser15, Ser78, Ser82) results in dissociation of the large 200–700 kDa oligomers into active dimers or tetramers, with a functional effect on its chaperone and cytoprotective properties (32).

HSP40 (DNAJ) proteins are the most diverse family of molecular cochaperones, and are characterized by a conserved ~70 amino acid J-domain required for activation of HSP70 ATPase activity and for the orderly transfer of substrates between the two chaperones during protein folding (33). HSP40s are further grouped into 3 major classes, on the basis of domain architecture. Class I HSP40s (e.g. E. coli DnaJ) are typically composed of 5 domains. These are the N-terminal J-domain which contains the signature HPD motif required for HSP70 interaction; the glycine/phenylalanine-rich (G/F) region which imparts flexibility to the protein; the C-terminal domain 1 (CTD1) which contains a zinc-finger–like region (ZFLR) and mediates client binding; the C-terminal domain 2 (CTD2) which stabilizes the fold of the protein; and a dimerization (D) domain which enables the formation of functional dimers. Class II HSP40s have a similar overall architecture to class I members but lack the ZFLR in CTD1. Members of Class III are the most divergent and retain only the J-domain. In some of these proteins, the J-domain is not at the N-terminus, but at a variable position in the sequence (22). The structural plasticity of HSP40s underlies the functional heterogeneity within this group. HSP40s can function as general folding assistants or specialized cochaperones that target specific sets of substrates and cellular pathways, along with HSP70 (34).

The HSP60 family, also known as chaperonins, is classified on the basis of its conserved structural domain organization that underlies its unique folding mechanism (35). HSP60 forms a double-ring cylindrical complex, typically composed of 14 identical subunits arranged in two heptameric rings stacked back-to-back, creating a central cavity where protein folding occurs (36). Each subunit contains three structurally distinct domains: an equatorial domain, which harbors the ATP-binding site and mediates inter-ring contacts (37); an intermediate domain, which acts as a flexible hinge transmitting conformational changes (38); and an apical domain, which binds non-native polypeptides and interacts with the co-chaperonin HSP10 (GroES) that functions as a lid (39). This highly conserved architecture is shared across species, from bacterial GroEL/GroES systems to eukaryotic mitochondrial HSP60/HSP10 complexes (40), and defines the Group I chaperonins, distinguishing them from the Group II chaperonins (CCT/TRiC) that lack a separate co-chaperonin lid (13).

The Hsp70 family is classified on the basis of its conserved structural domain organization, which defines both its mechanistic cycle and functional versatility (41). Each Hsp70 molecule consists of three principal domains: an N-terminal nucleotide-binding domain (NBD) that hydrolyzes ATP and drives allosteric conformational changes (42); a substrate-binding domain (SBD) composed of a β-sandwich subdomain (SBDβ) forming the peptide-binding groove and an α-helical lid (SBDα) that stabilizes bound substrates (43); and a C-terminal tail that, in cytosolic isoforms, ends with an EEVD motif acting as a docking site for co-chaperones and downstream protein quality control factors (44). This conserved domain framework enables the ATP-driven allosteric cycle characteristic of the Hsp70 chaperone system, coordinating with J-domain proteins (JDPs) for substrate capture and nucleotide-exchange factors (NEFs) for substrate release, thereby maintaining proteostasis across all cellular compartments (45). Glucose-Regulated Protein 78 (GRP78), also known as Binding immunoglobulin Protein (BiP), is a member of the HSP70 family that serves as the principal chaperone in the ER, orchestrating the UPR under stress conditions (46). Structurally, GRP78 shares the conserved domain organization characteristic of the HSP70 family, comprising two major domains including NBD and SBD (47).

HSP90s are also classified according to their subcellular localization and their conserved domain architectures (9). HSP90s have a domain structure that is conserved in all of the different isoforms, and all three domains are essential for function (48). They all consist of a large N-terminal domain (NTD), a middle domain (MD) and a smaller C-terminal domain (CTD) (49). The NTD binds and hydrolyzes ATP, the MD is involved in client interaction and regulation of ATPase activity and the CTD is involved in dimerization (50). Between the NTD and MD is a charged linker region that allows for flexibility between the two domains and also regulates interactions between the domains (51). The mammalian cytosolic HSP90 proteins are inducible (HSP90α) and constitutive isoforms (HSP90β) (52), while HSP90 chaperones that reside in the endoplasmic reticulum (GRP94/GP96) and mitochondria (TRAP1) are also present in humans (53).

HSP27 is a small heat shock protein that plays a crucial role in cellular protection and homeostasis (54). During fetal development and early childhood, HSP27 is highly expressed in the chondrocytes of the endplate cartilage, particularly in zones associated with chondrocyte maturation, suggesting a role in supporting metabolic and morphologic changes during tissue growth and differentiation (55). The cellular functions of HSP27 in OA are outlined below:

The Hsp40 protein family, also known as DNAJ proteins, is an essential molecular chaperones that regulate protein folding and quality control within cells, particularly in the ER (60). Among them, DNAJB12 plays a key role by guiding Hsp70 chaperones to the ER surface and participating in the degradation of misfolded proteins through ER-associated autophagy (ERAA), helping maintain intracellular protein homeostasis (61). In OA, dysfunction in protein quality control and autophagy pathways is increasingly recognized as contributing to disease progression. DNAJB12 is identified as a potential risk factor for knee OA. Thus, targeting Hsp40 family members, such as DNAJB12, could offer new therapeutic strategies for OA by restoring proper protein handling and reducing ER stress, thus potentially slowing or altering disease progression (62). HSP40 family members remain largely understudied in OA, and their precise roles in disease pathogenesis require further experimental investigation.

HSP60 is a highly conserved molecular chaperone involved in protein folding and cellular stress responses (63). Hsp60, a mitochondrial chaperone protein, is crucial in maintaining mitochondrial function and protecting cells from oxidative stress-induced damage (64).

HSP70 is a molecular chaperone that maintains cellular protein homeostasis by assisting in proper protein folding, preventing aggregation, and facilitating protein repair or degradation (74). HSPs, particularly HSP70 (and its inducible form HSP72), play a pivotal role in the pathogenesis and potential management of OA (Figure 4) (75).

GRP78 (Glucose-Regulated Protein 78), or BiP as a member of the HSP70 family, is an essential ER chaperone protein that helps maintain protein-folding homeostasis by binding to misfolded or unfolded proteins and facilitating their proper folding or degradation (93). In osteoarthritic cartilage, GRP78 is upregulated as part of the protective ER stress response, primarily via the IRE1-XBP1 pathway. It is key in enhancing the cell's capacity to manage ER stress. However, although GRP78 expression increases in OA cartilage, its expression remains localized (mainly to the upper middle zones). It does not increase significantly with cartilage degeneration, showing only a weak correlation with OA severity. In contrast, markers of the apoptotic ER stress response, such as CHOP, pPERK, and Ub, strongly correlate with cartilage damage and chondrocyte apoptosis. This imbalance, where the protective GRP78-mediated response is insufficient or diminished, while the pro-apoptotic signaling intensifies, suggests that a reduced protective response alongside an enhanced apoptotic response contributes to ER stress, induced chondrocyte apoptosis, and ECM degradation in OA cartilage (94). In OA in humans, Grp78 and Bag-1 are upregulated in chondrocytes, indicating the presence of ERS, which is likely triggered by the heightened metabolic activity required for excessive synthesis of ECM proteins, including collagen VI. Grp78, a molecular chaperone and marker of the UPR, is elevated in response to this ER stress, reflecting disrupted protein folding and processing. Bag-1, which has anti-apoptotic and cell survival functions, is upregulated throughout all cartilage zones in advanced OA, possibly as a compensatory mechanism to counteract apoptosis and promote cell survival and proliferation. Simultaneously, collagen VI is significantly upregulated and redistributed from the pericellular matrix to the interstitial space, suggesting impaired matrix organization and cell-matrix interaction (95). In knee OA, the synovial fluid contains microparticles (MPs) that present the intracellular stress proteins vimentin and GRP78 on their outer membrane, confirming that these vesicles can externalize putative autoantigens even in a largely "non-autoimmune" arthropathy. However, the proportion of vimentin⁺ and GRP78⁺ MPs in OA fluid is markedly lower than in rheumatoid arthritis. When OA-derived MPs were added to primary fibroblast-like synoviocytes cultured from OA tissue, they elicited only a modest secretory response: BAFF (B-cell activating factor) rose reliably, and smaller increases were seen in CXCL6, CCL8, and CCL15. At the same time, classical pro-inflammatory mediators such as TNF-α, IL-8, CXCL2, and CXCL5 were either unchanged or minimally induced. This subdued profile contrasts with the broad, high-level cytokine/chemokine burst triggered by rheumatoid MPs, indicating that although OA MPs can engage synoviocytes via surface vimentin and GRP78, the downstream activation is limited, reflecting both their lower abundance and the comparatively restrained inflammatory environment of osteoarthritic joints (96).

HSP90 is a molecular chaperone that stabilizes and assists in properly folding a wide range of client proteins, many of which are involved in cellular stress responses, inflammation, and apoptosis (123). In OA, HSP90 acts as a damage-associated molecular pattern (DAMP), meaning stressed or necroptotic chondrocytes release it and trigger inflammatory responses in the joint environment (124).

Synthetic OA drugs primarily include non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen and celecoxib, and corticosteroids such as hydrocortisone and dexamethasone (136). These drugs work by inhibiting key inflammatory pathways, particularly the cyclooxygenase (COX) and NF-κB signaling pathways, which produce pro-inflammatory mediators like TNF-α and IL-1β (137). Synthetic drugs reduce inflammation, pain, and swelling associated with OA by blocking these pathways (138). However, while effectively alleviating symptoms, they do not halt or reverse cartilage degradation. Long-term use of synthetic drugs may also lead to serious side effects such as gastrointestinal issues, increased infection risk, mood changes, or bone loss, which limits their use for chronic treatment (139). High-molecular-weight hyaluronic acid (HMW-HA), typically ranging from 1000 to 2000 kDa, is a therapeutic agent commonly used in OA treatment for its viscoelastic and anti-inflammatory properties (140). In the context of OA, where synovial inflammation and macrophage dysregulation contribute to disease progression, HMW-HA significantly suppresses IL-1β-induced inflammatory responses in synoviocytes (141). It achieves this by downregulating the expression of GRP78, a key marker of ERS, inhibiting activation of the NF-κB pathway, a central mediator of proinflammatory signaling. This suppression decreases proinflammatory cytokines (such as IL-6 and PGE2) and shifts macrophage polarization from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype (142, 143). The effect is enhanced when HMW-HA treatment is combined with GRP78 knockdown, highlighting the GRP78-NF-κB axis as a crucial target of HMW-HA's anti-inflammatory action in OA (144). VA692 is a novel, selective COX-2 inhibitor derived from the 1,5-diarylpyrrole scaffold with dual anti-inflammatory and nitric oxide-releasing properties. It has demonstrated strong anti-inflammatory, antioxidant, anti-nociceptive, and chondroprotective effects in both IL-1β-stimulated human chondrocyte cell lines (T/C-28a2) and primary OA chondrocytes. Compared to celecoxib, VA692 more effectively modulates inflammation by suppressing COX-2, IL-1β, IL-6, IL-8 expression, and PGE2 release, reducing oxidative stress and apoptosis. Notably, proteomic analysis revealed that VA692 downregulates key heat shock proteins, specifically HSP90α, HSP90β, and HSP70 (HSP7C), which are typically elevated in OA and contribute to cartilage degeneration by promoting MMP expression, NO synthesis, and COX-2 production. By inhibiting these HSPs, VA692 may restore cellular homeostasis and reduce catabolic responses in chondrocytes, suggesting its potential role in modulating HSP-mediated stress responses and preserving cartilage integrity in OA (145). In addition, ITZ-1 is a novel small molecule identified as a client-selective inhibitor of Hsp90 that exhibits chondroprotective effects in OA. It exerts its action primarily by binding to the C-terminal domain of Hsp90, unlike most known Hsp90 inhibitors that target the N-terminal ATP-binding site. By binding to Hsp90, ITZ-1 selectively induces degradation of the client protein Raf-1, suppressing IL–1β–induced ERK activation, inhibiting MMP-13 production, a key enzyme responsible for cartilage degradation. Moreover, ITZ-1 promotes dissociation of the HSF1/Hsp90 complex, thereby activating HSF1 and significantly upregulating cytoprotective Hsp70 without causing widespread degradation of other Hsp90 client proteins, minimizing cytotoxicity (146). Likewise, MG132 is a reversible proteasome inhibitor that exerts chondroprotective effects in OA by inducing Hsp70, which plays key roles in cell survival and stress response (78). In OA, excessive chondrocyte apoptosis contributes to cartilage degradation (147). MG132, when administered intra-articularly in a rat model of OA, significantly upregulates Hsp70 expression in chondrocytes without causing chondrotoxicity (148). Hsp70 overexpression mitigates chondrocyte apoptosis by acting as a molecular chaperone, preserving mitochondrial integrity, inhibiting caspase activation, and suppressing inflammatory signaling pathways such as NF-κB (149). This dual anti-apoptotic and anti-inflammatory action helps maintain cartilage integrity and reduces synovial inflammation, suggesting that MG132 could be a promising disease-modifying agent for OA by enhancing chondrocyte resilience to stress and cytokine-mediated damage (148). Lastly, 4-Phenylbutyric acid (4-PBA), a chemical chaperone and ER stress inhibitor, protects cartilage in OA by modulating HSP-mediated stress responses. It downregulates GRP78, CHOP, pro-inflammatory cytokines (TNF-α, IL-1β), and pro-apoptotic markers (Bax, Caspase-3), while upregulating Bcl-2, thereby reducing ER stress, inflammation, and apoptosis (150).

Nanoparticles have emerged as promising carriers for OA therapy due to their ability to enhance intra-articular drug retention, improve tissue targeting, and provide controlled or stimuli-responsive drug release. By encapsulating anti-inflammatory agents, antioxidants, growth factors, or nucleic acids, nanoparticles can protect therapeutic molecules from enzymatic degradation and deliver them directly to chondrocytes, synoviocytes, or subchondral bone. However, their clinical translation remains limited by pharmacokinetic and biocompatibility hurdles. Rapid synovial and lymphatic clearance, poor penetration through the dense avascular cartilage, and degradation in the inflamed joint environment all shorten their residence time and efficacy (151 –153). Light-inducible hybrid nanomedicine is a multifunctional nanosystem that releases therapeutic agents upon near-infrared (NIR) stimulation. In OA treatment, gold nanocages (AuNCs) loaded with diacerein (DIA) and NGF-targeted siRNA (siNGF) are coated with a cationic polymer (PBAE) for siRNA delivery and a thermo-responsive lipid shell (lauric/stearic acid) for light-triggered release. After intra-articular injection, NIR-induced photothermal heating of AuNCs melts the lipid layer, enabling on-demand release of DIA and siNGF. This dual therapy reduces ROS and NGF expression, alleviates inflammation and pain, and induces HSP70 expression, which protects chondrocytes by stabilizing proteins, preventing apoptosis, and preserving cartilage matrix (151). A multifunctional nanozyme (MPMP) is designed to mimic antioxidases (SOD, CAT) and hyaluronan synthase (HAS). Its MoS₂ core, coated with Mg²⁺-doped polydopamine and zwitterionic polymers, scavenges ROS/RNS and generates oxygen, alleviating oxidative stress in osteoarthritic joints. Concurrently, photothermal Mg²⁺ release activates HAS-like activity and upregulates HSP70, which enhances antioxidant defenses, suppresses pro-inflammatory pathways (NF-κB, IL-17), and promotes mesenchymal stem cell chondrogenesis and HA synthesis. This dual action protects cartilage and stimulates regeneration, restoring joint homeostasis and providing a promising OA therapy (154).

Extracellular vesicles (EVs) are nano-sized, lipid bilayer-enclosed particles secreted by cells that carry bioactive molecules such as proteins, RNA, and lipids, enabling intercellular communication (155, 156). In the context of OA, EVs, particularly those derived from cartilage-derived progenitor cells (CPCs) and bone marrow mesenchymal stromal cells (BM-MSCs), can mediate tissue repair by promoting chondrocyte proliferation, migration, and anabolic gene expression (157, 158). One key mechanism by which EVs may contribute to OA treatment is HSPs, especially HSP70, which are commonly enriched in EVs and act as molecular chaperones that assist in protein folding, protect cells from stress, and modulate immune responses. While BM-MSC EVs tend to contain higher levels of HSP70, CPC EVs, despite having lower HSP70, still enhance meniscal cell viability and repair, possibly by offering a more balanced, less stress-inducing signaling environment (159).

Monoclonal antibody 9B8 is a novel therapeutic antibody targeting the HSP90α, developed by immunizing BALB/c mice with T3A-A3 cells (160). In OA, HSP90 is critical in stabilizing hypoxia-inducible factor 1-alpha (HIF-1α), a key regulator of glycolytic metabolism in hypoxic chondrocytes (161). By binding to and inhibiting HSP90α, 9B8 disrupts the proper folding and stabilization of HIF-1α, thereby suppressing downstream genes involved in glycolysis (such as PFKP and ENO2) and inflammation (e.g., TNF-α and IL-6). This results in decreased chondrocyte catabolism, improved extracellular matrix synthesis (COL2A1, ACAN), and reduced inflammatory damage. In both in vitro chondrocyte models and in vivo rat ACLT-induced OA models, 9B8 was shown to attenuate cartilage degradation and subchondral bone destruction, highlighting its potential as a disease-modifying treatment for OA via inhibition of HSP90-mediated glycolytic and inflammatory pathways (160).

Photosensitizers are light-activated compounds that absorb near-infrared (NIR) radiation and convert it into heat, enabling localized thermal stimulation of deep tissues like articular cartilage (162, 163). In OA therapy, intra-articular photosensitizers like indocyanine green (ICG) can be activated by NIR light to safely raise joint temperature to ~40°C. This mild hyperthermia induces HSP70 in chondrocytes, which prevents apoptosis and promotes anabolic matrix components (ACAN, SOX9). Thus, photothermal stimulation provides a minimally invasive strategy to protect cartilage and slow OA progression via HSP70-mediated mechanisms (164). Hyperthermia is being investigated as a non-invasive OA treatment that targets cartilage degeneration by inducing Hsp70. As a cytoprotective protein, Hsp70 inhibits chondrocyte apoptosis, promotes proteoglycan and type II collagen synthesis, and supports cartilage metabolism. Deep-heating methods, such as microwaves, can upregulate Hsp70 and improve cartilage integrity in OA models, though efficacy depends on heat intensity, duration, and frequency, which remain to be optimized. Combining hyperthermia with pharmacological Hsp70 inducers (e.g., glutamine, curcumin, GGA, MG132) may further enhance chondroprotection, offering a cost-effective, minimally invasive strategy to slow OA progression by supporting chondrocyte survival and matrix repair (165). Microwave (MW)-induced thermotherapy is a promising non-invasive approach for OA, as it elevates intra-articular temperature in a dose-dependent manner, with 40 W identified as optimal for stimulating cartilage metabolism without damage. MW heating markedly increased HSP70 expression, particularly at 8–24 h, which correlated with enhanced proteoglycan (PG) and type II collagen (Col II) expression. Quercetin, an HSP70 inhibitor, suppressed PG induction, confirming HSP70's role in matrix regulation. Interestingly, Col II upregulation was HSP70-independent, suggesting the involvement of other stress proteins such as HSP42 (165). Combined MW irradiation and intra-articular glutamine (Gln) synergistically upregulate HSP70 in articular cartilage, enhancing chondrocyte protection and matrix metabolism in a rat OA model. MW at 40 W for 20 min raised joint temperature to ~38.3°C, sufficient to induce HSP70 without tissue damage. While Gln alone had no effect, its combination with MW markedly increased HSP70 and aggrecan expression, reflecting enhanced anabolic activity. Inhibition of HSP70 by quercetin or siRNA reduced aggrecan and abolished the protective effects. Histology further confirmed preserved cartilage and reduced OA severity in the MW + Gln group compared to controls (166). Likewise, laser irradiation applies focused light energy to tissues, modulating cellular responses. In OA, it upregulates HSPs, particularly HSP70, which stabilize proteins, protect chondrocytes from apoptosis, and reduce inflammation. Increased HSP70 expression in articular cartilage has been shown to alleviate joint inflammation, limit cartilage degradation, and improve OA symptoms (167).

Stem cell therapy in OA is an emerging regenerative treatment aimed at addressing the root cause of the disease, articular cartilage degeneration, rather than merely alleviating symptoms. Among the various stem cell types, mesenchymal stem cells (MSCs), particularly those derived from bone marrow (BMSCs) and adipose tissue (ASCs), are most commonly used due to their abundance, immunomodulatory properties, and ability to differentiate into chondrocytes (168). Articular cartilage stem cells (ACSCs) are a specialized population of cartilage-resident progenitor cells possessing self-renewal and multipotent differentiation capabilities, particularly into chondrocytes (169). These cells contribute to cartilage repair by homing to injury sites, modulating the microenvironment, and promoting matrix regeneration (170). In OA, ACSCs exert a therapeutic effect by attenuating ERS in chondrocytes, a key driver of apoptosis in cartilage degeneration (7). Mechanistically, ACSCs influence the PERK–eIF2α–ATF4 pathway, a significant branch of the UPR, activated during ERS. By modulating this pathway, ACSCs enhance the expression of GRP78 and other cytoprotective factors (e.g., TMEM119, BMP6), thereby promoting protein homeostasis, reducing apoptosis (as evidenced by improved Bcl-2/Bax ratios and decreased caspase-3 activation), and supporting chondrocyte survival. Although ACSCs upregulate PERK expression, their overall effect appears to balance protective stress responses with inhibition of terminal apoptosis, making them promising candidates for OA therapy targeting HSP- and PERK-mediated mechanisms (171). Pelleted bone marrow-derived mesenchymal stem cells (hBMMSCs) form aggregates that create a protective microenvironment, enhancing survival under stress compared to dispersed suspensions. In OA, these pellets show greater metabolic stability and lower expression of apoptotic and inflammatory genes. HSP70 mediates their cytoprotective effects by stabilizing proteins, suppressing apoptosis, and promoting resilience under mild heat stress. This response supports hBMMSC survival and function in the inflamed joint, enhancing their regenerative capacity and therapeutic potential for cartilage repair (172). MSCs are multipotent stromal cells capable of differentiating into various cell types, including chondrocytes, which are essential for cartilage repair (173). In the context of OA, MSCs offer promising therapeutic potential due to their ability to regenerate damaged articular cartilage and modulate inflammation (174). However, their in vitro chondrogenic differentiation is slow and often incomplete. Heat shock protein 70, a molecular chaperone induced by mild thermal stress, has emerged as a key enhancer. Periodic heat shock (e.g., 41°C for 1 hour weekly) has been shown to upregulate HSP70 in MSCs, which in turn promotes earlier and more robust chondrogenic differentiation by enhancing the synthesis of cartilage-specific matrix proteins such as type II collagen and aggrecan. Additionally, HSP70 helps prevent chondrocyte apoptosis and may modulate the transition toward hypertrophic differentiation, thereby offering a non-invasive strategy to improve MSC-mediated cartilage regeneration in OA treatment (175).

Ultrasound (US) therapy is a non-invasive physical treatment modality that uses high-frequency sound waves to penetrate tissues and exert therapeutic effects, particularly in musculoskeletal disorders like knee osteoarthritis (KOA) (176). In the context of OA, US therapy helps alleviate joint symptoms by reducing synovial inflammation, inhibiting chondrocyte apoptosis, and slowing cartilage degeneration (177). US treatment significantly alters the rabbit KOA model's synovial fluid (SF) proteome, particularly upregulating heat shock proteins such as HSP90A. HSPs, especially HSP90, play a protective role in OA by stabilizing key signaling molecules, blocking apoptosis (e.g., via Apaf-1 inhibition), and enhancing anti-inflammatory pathways such as PI3K-Akt and NF-κB. By increasing HSP expression in SF, US therapy contributes to cellular homeostasis, synovial protection, and potentially cartilage repair, thereby offering a molecular mechanism for its clinical benefits in managing KOA (178).

Pelotherapy is a therapeutic practice that uses peloids, natural muds, or clay-like materials for medicinal purposes. These substances are often derived from mineral-rich waters and contain various organic and inorganic compounds, microorganisms, and minerals (179). Chaperokines are members of the molecular HSP families which, when released or exposed extracellularly (e.g., via exosomes, on the cell surface or in free form), function as cytokine- or alarmin-like signaling molecules. They thereby bridge proteostasis (folding, stabilization, degradation) and immune/stress signaling by interacting with extracellular receptors (e.g., TLRs, CD91/LRP1) and modulating innate and adaptive responses (180). Pelotherapy is applied externally (by brush and in mud baths at 38–42°C) for consecutive days. The thermal and chemical stimuli trigger a heat-shock response: intracellular Hsp72 synthesis is promoted while pathological elevations of extracellular Hsp72 (eHsp72), which otherwise act as "chaperokines" driving pro-inflammatory cytokine release, are normalized. By lowering systemic eHsp72, pelotherapy dampens the Hsp-cytokine positive feedback loop and rebalances the neuroendocrine-immune network with a heat-induced boost in cortisol. This anti-inflammatory and chondroprotective mechanism underlies the clinical improvements in pain, stiffness, and function observed in knee OA patients (181).

Mild electrical stimulation (MES) is a noninvasive physical therapy that uses low-intensity direct electrical current to stimulate biological responses in tissues (182). In the OA context, MES has been shown to increase HSP70 levels, a molecular chaperone that helps protect cells from stress and promotes tissue repair. MES does not enhance HSP70 production by boosting gene expression but by inhibiting its degradation via the ubiquitin-proteasome pathway, leading to protein accumulation. When combined with heat stimulation (HS), which induces HSP70 transcription, MES and HS synergistically elevate HSP70 levels more effectively than either treatment alone. This combined treatment enhances cartilage matrix metabolism and upregulates protective proteins like proteoglycans, contributing to chondroprotection and potentially slowing OA progression (183).

Natural products play a crucial role in osteoarthritis therapy by providing multi-targeted anti-inflammatory, antioxidant, and chondroprotective effects with fewer side effects than conventional drugs (184). Tunicamycin (TM) is a natural antibiotic that disrupts protein maturation by inhibiting N-linked glycosylation, thereby inducing ERS (185). In OA, TM has been used to model ER stress in chondrocytes, the sole cell type in cartilage responsible for maintaining ECM homeostasis. TM-induced ER stress activates the UPR, initially promoting protective autophagy via the upregulation of GRP78. This chaperone senses misfolded proteins and regulates UPR activation. GRP78-mediated autophagy, marked by increased expression of LC3B and Beclin-1 and autophagosome formation, helps protect chondrocytes from apoptosis. However, persistent or excessive ER stress overwhelms this protective mechanism, suppresses autophagy, and increases cell death. Knockdown of GRP78 significantly reduced autophagy-related protein expression and autophagosome formation, confirming its central role in mediating autophagy during ER stress (186). Melatonin, a natural pineal gland hormone, exerts potent antioxidant, anti-inflammatory, and mitochondrial-protective effects (187). Melatonin alleviates disease progression in OA by inhibiting ferroptosis, an iron-dependent cell death induced by lipid peroxidation, in chondrocytes (188). A key mechanism involves its regulation of NOX4, a ROS-generating enzyme that is upregulated in OA and promotes ferroptosis (189). On one hand, melatonin suppresses NOX4 expression, especially in mitochondria, thus reducing oxidative stress, preserving mitochondrial function, and enhancing energy production (190). Importantly, NOX4 was found to bind directly to GRP78, inhibiting the enzyme GPX4. By inhibiting NOX4, melatonin helps maintain GRP78 function, stabilizes GPX4, and ultimately prevents chondrocyte ferroptosis (189). Conversely, melatonin stabilizes GRP78, easing ER stress, limiting CHOP-driven apoptosis, and reducing misfolded protein buildup in chondrocytes. This stabilization supports chondrocyte survival, reduces cartilage degradation, and ultimately slows OA progression (191). Icariin is a bioactive flavonol glycoside derived from traditional Chinese herbal medicine, widely recognized for its anti-inflammatory, immunomodulatory, cartilage-regenerative, and analgesic effects in OA treatment (192). Icariin targets HSPs, especially HSP90AA1 and HSPA1A, key regulators of protein folding, stress response, and inflammation. By binding HSP90AA1, it influences client proteins controlling chondrocyte survival and inflammatory signaling (MAPK, PI3K/Akt), thereby reducing inflammation, preventing apoptosis, and supporting cartilage homeostasis (193). Icariin dose-dependently inhibits OA–FLS proliferation and migration without cytotoxicity up to 10 μM. It downregulates IL-1β, MMP14, and GRP78 at mRNA and protein levels, thereby reducing inflammation, suppressing ECM degradation, limiting ER stress–mediated apoptosis, and alleviating synovial hyperplasia (194). Similarly, Diacerein, an anti-inflammatory anthraquinone used in OA, acts mainly by inhibiting IL-1β and downstream mediators like NF-κB, thus limiting inflammation and cartilage degradation (195). Diacerein exerts its protective effects in OA through modulation of HSPs, particularly HSP70, which are molecular chaperones that protect cells from stress-induced damage (196). HSP70 can inhibit key inflammatory pathways, suppress chondrocyte apoptosis, and reduce oxidative stress, contributing to cartilage preservation (197). Thus, Diacerein's therapeutic benefits in OA involve cytokine suppression and enhancement of cellular stress defense mechanisms via HSP regulation (196). Chrysin (CHR) is a natural flavonoid derived from plants such as Scutellaria baicalensis and Mucuna pruriens, known for its anti-inflammatory, antioxidative, and antiapoptotic properties (198) (199). In KOA, CHR has demonstrated therapeutic potential by targeting synovitis and synovial fibrosis, key pathological features of KOA. CHR achieves these effects by modulating the PERK/TXNIP/NLRP3 signaling axis, which is correlated with ERS activity and inflammasome activation in synovial fibroblasts (SFs). Notably, CHR downregulates GRP78, thereby inhibiting downstream activation of PERK and its pro-fibrotic mediators, including CHOP, TXNIP, and NLRP3. This cascade ultimately reduces the production of fibrotic proteins like COL1A1, PLOD2, and TIMP1, and inflammatory cytokines such as IL-6, TNF-α, and IL-1β, alleviating joint inflammation, fibrosis, and pain (199). Taraxasterol (TAX), a pentacyclic triterpene from Taraxacum officinale (dandelion), has long been used in Chinese medicine for its anti-inflammatory properties (200). In OA, TAX reduces joint inflammation and damage by inhibiting TNF-α, IL-6, IL-1β, and NF-κB signaling. It also upregulates miR-140 and miR-146a, which suppress overexpressed targets in OA, including HSPA4L, ST5, and ERBB4, thereby limiting inflammatory responses (201).

Daphnoretin, a natural dicoumarin from plants like Leguminosae and Glycosmis pentaphylla, is known for anti-tumor and antiviral effects. In OA, it promotes chondrocyte survival and inhibits apoptosis by attenuating ER stress and inactivating the NLRP3 inflammasome. Through downregulating ER stress markers (GRP78, CHOP, ATF6, Caspase-12), daphnoretin reduces inflammation, limits apoptosis, and helps preserve joint integrity (202).

Tetramethylpyrazine (TMP), ligustrazine, is a nitrogen-containing compound derived from the Chinese herb Ligusticum wallichii (203). Clinically known for vascular and anti-inflammatory effects, TMP has emerged as a potential OA therapy. It protects chondrocytes by reducing ER stress, primarily through downregulating GRP78 and CHOP, thereby limiting apoptosis. TMP also suppresses cytokines and matrix-degrading enzymes (MMPs, ADAMTS), preserving ECM integrity. Thus, it may alleviate OA by modulating HSP-related ER stress, reducing inflammation, and protecting cartilage (204). Sirtuins (SIRT1–7) play protective roles in osteoarthritis by regulating chondrocyte energy metabolism, oxidative stress, inflammation, autophagy, senescence, and circadian rhythm, thereby maintaining cartilage homeostasis and slowing OA progression (205, 206). Echinacoside (ECH) is a natural phenylethanoid glycoside predominantly found in Cistanche species, widely recognized for its antioxidant, anti-inflammatory, and anti-aging properties (207). ECH protects chondrocytes by targeting oxidative stress and ER stress. It upregulates Sirt1, which alleviates ER stress via the PERK–eIF2α–ATF4–CHOP pathway. By downregulating GRP78 and CHOP, ECH prevents apoptosis and preserves ECM integrity by increasing Collagen II and Aggrecan while reducing MMP13 and ADAMTS5. These effects disappear with Sirt1 silencing or ER stress induction, confirming that ECH mitigates OA mainly through Sirt1-mediated inhibition of HSP-related ER stress (208).

Celastrol, a bioactive compound from Tripterygium wilfordii, shows anti-inflammatory, antioxidant, and anti-apoptotic effects in OA. It protects chondrocytes by inhibiting ER stress via the Atf6/Chop pathway, downregulating markers (Bip, Atf6, Chop, Xbp-1) and reducing apoptotic proteins such as caspase-3 and caspase-9 (209).

Quercetin is a polyhydroxy flavonoid widely present in plants and traditional Chinese herbs, known for its diverse biological activities, including antioxidant, anti-inflammatory, and anti-arthritic effects (210). Quercetin targets key proteins in OA, notably HSPA2 and HSP90AA1, which regulate stress responses and protein homeostasis. By modulating these HSPs, it affects nitric oxide production, estradiol response, and mitochondrial ATP synthesis, processes linked to OA progression. Network pharmacology and docking studies confirmed HSPA2 as a direct target, indicating that quercetin's protective effects may stem from regulating HSP-mediated stress responses and inflammation, thereby preserving cartilage integrity (211).

Curcumin is a naturally occurring polyphenolic compound with strong antioxidant, anti-inflammatory, and antiapoptotic properties, traditionally used in herbal medicine and as a dietary spice (212). Curcumin protects chondrocytes from oxidative stress–induced apoptosis in OA by upregulating SIRT1, which suppresses the PERK–eIF2α–ATF4–CHOP axis of the UPR. By lowering ER stress markers (CHOP, GRP78, ATF4) and related HSPs, it limits apoptosis, preserves cartilage integrity, and slows OA progression (213).

Baicalin, a flavonoid from Scutellaria baicalensis roots, is noted for its, cytoprotective, antioxidant, and anti-inflammatory effects (214). Baicalin demonstrated protective effects on human chondrocytes, the sole cellular component of articular cartilage, by reducing oxidative stress and preventing apoptosis (215). Its mechanism involves attenuating endoplasmic reticulum stress, contributing to chondrocyte death in OA progression. Specifically, baicalin downregulates ER stress markers such as BiP and CHOP, which are upregulated during cellular stress and promote apoptosis. By suppressing these markers, baicalin helps maintain cellular homeostasis and cartilage integrity, suggesting that modulation of HSP-related ER stress pathways is a central mechanism in its anti-OA activity (216).

Taurine is a naturally occurring, abundant free amino acid in humans, recognized for its roles in calcium modulation, osmoregulation, membrane stabilization, and especially for its antioxidant and anti-apoptotic properties (217). Taurine alleviates ER stress in OA by downregulating HSP-related markers (GRP78, CHOP, Caspase-12) in H₂O₂-stimulated chondrocytes. This suppression reduces apoptosis and restores Collagen II production, thereby preserving chondrocyte viability and cartilage function (218).

Harpagide (HPG), an iridoid glycoside from Harpagophytum procumbens, shows strong anti-inflammatory and chondroprotective effects. It suppresses TNF-α–induced inflammation by downregulating IL-1β, IL-6, COX-2, and MMP-13, while restoring COL2A1 and ACAN. HPG also alleviates ER stress by inhibiting GRP78 and p-IRE1α and restoring p-AMPK, suggesting protection via the ER stress/AMPK axis. Through these mechanisms, HPG preserves chondrocyte viability, reduces cartilage degeneration, and improves OA outcomes in vitro and in vivo (219).

Bushen Zhuangjin decoction (BZD) is a traditional Chinese natural formula composed of ten medicinal herbs, historically used to nourish the liver and kidneys while strengthening tendons and bones (220). BZD has shown therapeutic potential in OA by inhibiting chondrocyte apoptosis, a key factor in cartilage degradation (221). BZD protects chondrocytes from tunicamycin-induced apoptosis by modulating ER stress pathways. It downregulates Bip, Atf4, Chop, Bax, caspase-3, and caspase-9 while upregulating Xbp1 and Bcl-2, thereby limiting ER stress–mediated apoptosis and preserving chondrocyte viability, highlighting its potential as a disease-modifying OA therapy (222).

Similarly, Duhuo Jisheng decoction (DHJSD) is a traditional Chinese herbal medicine composed of multiple botanicals, known for its effects in alleviating OA symptoms by reducing pain, joint stiffness, and improving mobility (223). In OA, chondrocyte apoptosis contributes to cartilage degeneration, often driven by ER stress (6). DHJSD inhibits ER stress–induced apoptosis in chondrocytes, particularly in tunicamycin models, by downregulating miR-34a. This reduces ER stress markers (Bip, Atf4, Chop) and apoptotic proteins (Bax, caspase-9, caspase-3) while upregulating protective factors Xbp1, Xbp1s, and Bcl-2 (224).