Kaempferia parviflora Extract Stabilizes Cartilage Homeostasis via TIMP-1–Associated Matrix Modulation in Monosodium Iodoacetate–Induced Rat Osteoarthritis

TL;DR

Kaempferia parviflora extract helps protect cartilage in osteoarthritis by reducing inflammation and stabilizing matrix balance.

Contribution

The study demonstrates KPE's novel role in stabilizing cartilage via TIMP-1 modulation in an OA rat model.

Findings

KPE improved weight-bearing and preserved cartilage structure in OA rats.

KPE reduced serum CTX-II levels, indicating less collagen degradation.

KPE restored TIMP-1 expression and modulated systemic inflammation.

Abstract

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by extracellular matrix (ECM) breakdown, inflammation, and pain-associated functional impairment. Current pharmacological treatments primarily provide symptomatic relief without preventing cartilage degeneration. Kaempferia parviflora extract (KPE), rich in polymethoxyflavonoids, has been reported to have anti-inflammatory properties; however, its in vivo effects on cartilage homeostasis in OA remain incompletely defined. Methods: A monosodium iodoacetate (MIA)–induced rat model of knee OA was used to evaluate the therapeutic effects of KPE. Following OA induction, rats received oral KPE at low, medium, or high doses for 19 days. Pain-associated functional impairment was assessed by static weight-bearing analysis. Cartilage integrity was evaluated histologically, serum inflammatory and cartilage degradation biomarkers were quantified, and expression of matrix-degrading enzymes and their endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), was analyzed in articular cartilage. Results: MIA injection induced marked joint dysfunction, including an approximately 50% reduction in weight bearing on the affected limb. While KPE did not significantly reduce acute knee swelling, all KPE doses significantly improved weight-bearing imbalance compared with MIA controls. Histological analysis demonstrated preservation of cartilage structure and proteoglycan content in KPE-treated groups. Serum CTX-II levels were significantly reduced across all KPE doses, indicating attenuation of collagen degradation. Systemic inflammatory markers showed differential modulation: significant reductions in serum CRP and COX-2 at medium and high doses, while PGE2 showed a consistent downward trend that did not reach statistical significance. In articular cartilage, KPE treatment restored TIMP-1 expression, whereas modulation of individual MMPs was modest and variable. Conclusions: KPE alleviates OA-associated functional impairment and cartilage degeneration in an experimental OA model. The therapeutic effects are associated with reinforcement of TIMP-1–mediated matrix homeostasis and modulation of inflammatory pathways, supporting the potential of KPE as a natural adjunct candidate for OA management.

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1. Introduction

Osteoarthritis (OA) is the most prevalent degenerative joint disorder, characterized by the progressive breakdown of articular cartilage, subchondral bone remodeling, synovial inflammation, and consequent joint pain and stiffness [1,2,3]. The pathogenesis of OA is multifactorial, involving mechanical stress, inflammation, oxidative stress, and imbalances in cartilage matrix synthesis and degradation [4].

At the molecular level, OA is characterized by an imbalance between anabolic and catabolic activities within the joint, leading to the progressive degradation of articular cartilage. A central feature of this process is the upregulation of inflammatory mediators, including interleukins (such as IL-1β and IL-6), prostaglandins (particularly prostaglandin E2), and matrix metalloproteinases (MMPs), which together contribute to the breakdown of cartilage extracellular matrix (ECM) [5,6,7]. These mediators promote cartilage erosion by stimulating chondrocytes and synovial cells to produce catabolic enzymes and pro-inflammatory cytokines, thereby amplifying inflammatory and degradative cascades. Among the MMPs, MMP1, MMP3, and MMP13 are particularly implicated in the degradation of type II collagen and aggrecan, two key structural components of cartilage [7,8,9]. Excessive activation of these catabolic pathways disrupts tissue homeostasis and accelerates OA progression. Conversely, tissue inhibitor of metalloproteinase-1 (TIMP-1) plays an essential role in maintaining ECM stability by counterbalancing MMP activity, and restoration of this regulatory balance is increasingly recognized as a critical determinant of cartilage homeostasis.

Despite the high global prevalence and socioeconomic burden of OA, current therapeutic strategies remain largely palliative, focusing on symptom relief rather than halting or reversing disease progression [10]. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to manage pain and inflammation. However, their long-term use is associated with gastrointestinal, cardiovascular, and renal adverse effects, limiting their applicability in chronic conditions such as OA [11]. These limitations underscore the need for safer therapeutic strategies that not only alleviate symptoms but also preserve cartilage structure and ECM homeostasis. Accordingly, increasing attention has been directed toward natural compounds and herbal extracts with anti-inflammatory, antioxidant, and chondroprotective properties as potential disease-modifying approaches for OA [10,12].

The rhizomes of Kaempferia parviflora are rich in polymethoxyflavonoids (PMFs) [13], with 5,7-dimethoxyflavone (DMF) and 5,7,4′-trimethoxyflavone (TMF) identified as major bioactive components that are readily absorbed following oral administration [13]. Previous studies have suggested that Kaempferia parviflora and its extracts may alleviate knee OA in rats, suppress inflammatory responses in rheumatoid arthritis–related cell models, and exert chondroprotective effects in vitro and in vivo [14]. However, in vivo evidence defining whether Kaempferia parviflora extract (KPE) directly regulates cartilage ECM homeostasis, particularly through endogenous matrix-protective mechanisms such as TIMP-1, remains limited.

Our previous study using a human chondrocyte cell line (CHON-1) demonstrated that KPE attenuated inflammation and ECM degradation in an IL-1β-induced OA-like environment [15,16]. While these findings indicated suppression of inflammatory mediators and MMP expression, it remained unclear whether KPE could modulate the MMP/TIMP regulatory axis and preserve cartilage structure in vivo. This gap in knowledge limits the mechanistic interpretation and translational relevance of KPE in OA.

Therefore, the present study was designed to address this gap by evaluating the effects of KPE in a monosodium iodoacetate (MIA)-induced rat model of knee OA. By integrating functional assessment, biochemical analysis, and histological evaluation, we investigated whether KPE could alleviate pain-associated functional impairment, suppress systemic inflammation, and restore TIMP-1–associated ECM regulatory balance in vivo.

In this preclinical model, we demonstrate that KPE improves joint function, attenuates cartilage degradation, and reduces inflammatory responses following OA induction. Importantly, these protective effects are associated with restoration of ECM homeostasis, in part through upregulation of TIMP-1, supporting the potential of KPE as a natural therapeutic candidate for OA.

2. Results

2.1. Validation of DMF, TMF, and Tectochrysin as KPE Markers

Quantitative profiling of principal polymethoxyflavones in Kaempferia parviflora extract (KPE) was performed to evaluate chemical consistency and establish reference markers for standardization. The targeted compounds—5,7-dimethoxyflavone (DMF), 5,7,4′-trimethoxyflavone (TMF), and tectochrysin—were chosen as index constituents based on their reported bioactivity and high abundance in KP rhizomes. HPLC analysis showed that KPE contained 34.75 mg/g DMF, 16.35 mg/g TMF, and 4.00 mg/g tectochrysin. Under the established chromatographic conditions, the retention times of the reference standards were 13.360 min (DMF), 28.430 min (TMF), and 41.290 min (tectochrysin) (Figure 1). Analysis of the KPE sample under identical conditions yielded retention times of 13.370 min (DMF), 28.435 min (TMF), and 41.283 min (tectochrysin). The retention-time deviations between standards and KPE were minimal for all three compounds. The quantitative contents and retention-time data for these index compounds are summarized in Table 1.

2.2. Effects of KPE on Knee Swelling and Pain-Related Functional Impairment in MIA-Induced OA Rats

To verify successful induction of OA, rats were injected intra-articularly with MIA, which is known to induce acute joint inflammation and pain-associated functional impairment. As expected, MIA injection led to marked alterations in knee morphology and weight-bearing behavior, confirming establishment of the OA model.

At seven days post-MIA injection, control animals exhibited pronounced edema in the right knee joint. Quantitative analysis revealed that the thickness of the MIA-injected knee was 1.19 ± 0.09-fold greater than that of the contralateral left knee, indicating localized swelling associated with acute inflammation (Table 2). All MIA-injected groups, including those treated with KPE at low (L), medium (M), or high (H) doses, also developed knee edema. The right-to-left knee thickness ratios were 1.17 ± 0.04, 1.17 ± 0.05, and 1.13 ± 0.03 in the L, M, and H KPE groups, respectively. Although the high-dose KPE group showed a modest numerical reduction in knee thickness ratio, this difference did not reach statistical significance, indicating that KPE did not significantly attenuate acute MIA-induced knee swelling at this early time point.

In contrast, MIA injection resulted in a substantial imbalance in weight-bearing distribution between the left and right hind limbs, with approximately 50% reduction in load bearing on the injured limb, reflecting pain-related functional impairment (Figure 2A). Consistent with this observation, the MIA control group showed a significant decrease in weight bearing on the affected leg. Notably, KPE treatment at all tested doses significantly improved weight-bearing balance, indicating alleviation of OA-associated pain and functional deficits (Figure 2B).

KPE treatment did not significantly reduce knee edema at day 7 after MIA injection, but it did significantly improve weight-bearing imbalance compared with the MIA control group.

2.3. Histological Preservation of Cartilage by KPE in a MIA-Induced Rat OA Model

Histological examination of knee joint sections using hematoxylin and eosin (H&E) and Safranin O staining revealed pronounced cartilage damage in the MIA-treated control group (Figure 3A,B). In these animals, the articular cartilage surface appeared irregular and disrupted, accompanied by a marked reduction in Safranin O staining intensity, particularly in the superficial zone, indicating loss of proteoglycan-rich matrix. In contrast, KPE-treated groups (L, M, and H) exhibited improved preservation of cartilage morphology compared with the MIA control. The articular surfaces appeared relatively smoother, and Safranin O staining was more clearly retained across cartilage layers, suggesting attenuation of matrix loss (Figure 3A,B). These histological differences were consistently observed across KPE-treated groups, although the degree of preservation varied among individual samples. Given that formal quantitative histological scoring was not applied, the present observations are reported as qualitative morphological comparisons rather than numerical assessments.

2.4. KPE Modulates Serum Biomarkers of Cartilage Degradation in an MIA-Induced Rat OA Model

To evaluate the effects of KPE on cartilage degradation, serum levels of glycosaminoglycan (GAG), CTX-II, aggrecan, and osteocalcin were measured following MIA injection. The high-dose KPE (H) group showed a numerical decrease in serum GAG levels compared with the MIA group; however, this difference did not reach statistical significance (Figure 4A). In contrast, serum CTX-II levels were significantly reduced in all KPE-treated groups (L, M, and H) compared with the MIA control, indicating attenuation of collagen type II degradation across the tested dose range (Figure 4B; Supplementary Table S3). While the magnitude of reduction differed among doses, each KPE-treated group showed a statistically significant decrease relative to the MIA group. Furthermore, KPE treatment at all doses significantly attenuated MIA-induced increases in serum aggrecan and osteocalcin levels (Figure 4C,D). Both markers were significantly reduced in the KPE-treated groups compared with the MIA-only group, reflecting suppression of cartilage matrix breakdown and altered bone–cartilage metabolic activity.

2.5. KPE Modulates MMP/TIMP-1 Expression in Knee Cartilage in the MIA-Induced Rat OA Model

To evaluate the effects of KPE on cartilage matrix–related proteins, Western blot analysis was performed to assess the expression of MMP1, MMP3, MMP13, and TIMP-1 in knee cartilage following MIA induction. As shown in Figure 5, Supplementary Figure S2, and Supplementary Table S4, the protein levels of MMP1, MMP3, and MMP13 were increased in the MIA group compared with the normal control. KPE treatment resulted in a significant reduction in MMP3 expression in all treatment groups compared with the MIA control (Figure 5B). In contrast, although MMP1 expression showed a decreasing trend following KPE administration, these changes did not reach statistical significance across the tested doses (Figure 5A). MMP13 expression exhibited only modest changes among groups and did not show statistically significant modulation by KPE treatment (Figure 5C). Notably, TIMP-1 protein expression was significantly increased in the medium- and high-dose KPE groups compared with the MIA control, with levels approaching those observed in the normal control group (Figure 5D).

2.6. KPE Attenuates Systemic Inflammatory Markers in MIA-Induced Rat OA

To assess the systemic inflammatory status in the MIA-induced OA model, serum levels of CRP, COX-2, and PGE_2_ were measured (Figure 6). MIA injection increased serum CRP levels compared with the normal control. KPE treatment was associated with a reduction in CRP levels across treatment groups, with progressively lower mean values observed in the medium- and high-dose groups relative to the MIA control (Figure 6A). For COX-2, serum levels were significantly reduced in the medium- and high-dose KPE groups compared with the MIA control, whereas the low-dose group showed a modest, non-significant decrease (Figure 6B). Serum PGE_2_ levels were markedly elevated following MIA injection. KPE treatment was associated with a downward trend in PGE_2_ levels across all doses; however, these reductions did not reach statistical significance under the current experimental conditions (Figure 6C). Taken together, these findings indicate that KPE administration modulates systemic inflammatory markers in the MIA-induced OA model, with differential sensitivity among individual inflammatory mediators.

3. Discussion

OA is a degenerative joint disorder characterized by an imbalance between anabolic and catabolic processes in articular cartilage, leading to progressive ECM breakdown and joint dysfunction. Current therapies remain predominantly symptomatic and do not halt or reverse disease progression, underscoring the unmet need for disease-modifying strategies [16,17,18]. Although NSAIDs effectively alleviate pain and inflammation, longitudinal data from the Osteoarthritis Initiative indicate no measurable benefit on synovitis or cartilage thickness, together with well-recognized gastrointestinal and cardiovascular risks associated with long-term use [18,19,20,21,22,23].

In the present study, we evaluated KPE in a MIA-induced rat model of knee OA and observed consistent therapeutic effects across functional, biochemical, and histological endpoints. Across the tested dose range, KPE improved weight-bearing capacity, favorably modulated systemic and cartilage-related biomarkers, and preserved cartilage architecture without detectable adverse effects on body weight or general health, supporting a favorable short-term tolerability profile. Quantitative compositional analysis confirmed that KPE contains substantial levels of polymethoxyflavonoids, including DMF and TMF, which have been previously reported to exert anti-inflammatory and cartilage-protective activities [24,25,26]. These data provide a chemical and biological linkage between extract composition and in vivo efficacy, although the present study was conducted using a single production lot and therefore does not address batch-to-batch variability.

As a phytopharmaceutical approach, KPE faces translational challenges common to botanical extracts, including bioavailability, standardization, and incomplete mechanistic characterization [27,28]. Compared with earlier studies that focused on isolated inflammatory or catabolic markers, the present work provides an integrated assessment by concurrently evaluating multiple matrix-degrading enzymes (MMP1, MMP3, and MMP13), the endogenous inhibitor TIMP-1, systemic inflammatory mediators (CRP and PGE_2_), and histological cartilage integrity within a unified experimental framework [8,29,30]. However, plasma exposure and pharmacokinetic profiles of individual KPE constituents were not directly assessed, and exposure–response relationships therefore remain to be established.

Histological analysis demonstrated that MIA induced pronounced cartilage surface disruption and proteoglycan loss, as evidenced by reduced Safranin O staining. KPE treatment preserved cartilage structure and matrix staining intensity, with more evident protection observed at the medium and high doses. These morphological findings are consistent with the biochemical modulation of cartilage degradation markers and support a chondroprotective effect of KPE in vivo. Synovial inflammation was not systematically assessed using standardized histopathological scoring; therefore, quantitative conclusions regarding synovitis cannot be drawn from the present dataset.

Biochemical analyses demonstrated that serum markers associated with cartilage degradation, including CTX-II, aggrecan, and osteocalcin, were reduced following KPE administration. CTX-II levels were significantly decreased in all KPE-treated groups compared with the MIA control, indicating that the cartilage-protective effect of KPE is observed across the tested dose range. Although the magnitude of reduction differed among doses, a strictly linear dose–response relationship was not evident. Such variability is consistent with the pharmacological characteristics of phytochemical-rich extracts, which exert biological effects through multiple convergent signaling pathways rather than a single dose-limiting target [31,32].

At the molecular level, OA induction was associated with increased expression of MMP family members and reduced TIMP-1 expression, consistent with a catabolic shift in matrix turnover. KPE treatment significantly suppressed MMP-3 expression across doses, whereas modulation of MMP-1 and MMP-13 was more limited. These differential responses likely reflect the distinct regulatory hierarchies of individual MMPs, with MMP-3 acting as an upstream stromelysin sensitive to inflammatory modulation, and MMP-13 representing a terminal collagenase less responsive to short-term intervention [33,34]. Importantly, KPE robustly restored TIMP-1 expression, suggesting reinforcement of endogenous matrix-protective mechanisms rather than uniform inhibition of all catabolic enzymes.

With respect to inflammatory mediators, KPE treatment was associated with a significant reduction in serum COX-2 levels at the medium and high doses, whereas PGE_2_ levels exhibited a downward trend without reaching statistical significance. This dissociation suggests that KPE may preferentially modulate inflammatory signaling at the level of enzyme expression or activity rather than uniformly suppressing downstream prostaglandin production [24,25]. Similar context-dependent regulation of COX-2 and prostaglandin output has been reported in OA-related settings, reflecting complex post-translational and substrate-dependent control mechanisms [26,35].

Several limitations of the present study should be acknowledged. Sample sizes were modest and may have limited statistical power for selected endpoints. Formal quantitative histological scoring, such as OARSI grading, was not applied, potentially reducing cross-study comparability. Safety assessment was restricted to short-term observations based on clinical monitoring and gross organ weights. Serum biochemical toxicity markers or organ histopathology were also not evaluated. Therefore, conclusions regarding long-term systemic safety cannot be drawn. Nevertheless, the concordant patterns observed across functional, biochemical, and histological endpoints support the robustness of the overall findings and justify further confirmatory and mechanistic investigations.

Future studies should incorporate larger and sex-balanced cohorts, standardized quantitative histological scoring, and comprehensive pharmacokinetic and toxicological analyses to define exposure–response relationships and long-term safety. Elucidation of upstream pathways governing TIMP-1 regulation will further clarify the molecular basis of KPE-mediated cartilage protection and support its translational development.

4. Materials and Methods

4.1. Manufacturing of Kaempferia parviflora Extracts

Rhizomes of black ginger (Kaempferia parviflora) harvested in Thailand were air-dried, finely pulverized, and subjected to enzymatic hydrolysis under controlled conditions. The hydrolysate was then extracted with ethanol, and the extract was sequentially filtered, vacuum-concentrated, and sterilized before spray-drying to yield a stable powder. The final Kaempferia parviflora extract (KPE) was manufactured and supplied by Hamsoa Pharm Co., Ltd. (Iksan, Republic of Korea).

4.2. Determination of Index Compounds by HPLC

Dimethoxyflavone (DMF), trimethoxyflavone (TMF), and tectochrysin were chosen as index compounds for KPE based on their abundance and stability. Quantification was carried out using an HPLC system (Agilent 1260 Infinity; Agilent Technologies, Santa Clara, CA, USA) equipped with an Eclipse Plus C18 column (4.6 × 250 mm, 5 µm; Agilent Technologies). The mobile phase consisted of methanol and water containing 0.5% acetic acid and was applied under isocratic conditions at 65% methanol for 60 min at a flow rate of 1.0 mL/min. Standard and sample solutions (5 µL each) were injected and monitored by UV detection at 280 nm (Supplementary Table S1). Reference standards for tectochrysin (Cat. No. 83915), 5,7-dimethoxyflavone (DMF; Cat. No. 84211), and 4′,5,7-trimethoxyflavone (TMF; Cat. No. 85780) were purchased from PhytoLab (Vestenbergsgreuth, Germany) (Table 2 and Table S2). A 50 ppm stock solution of each reference standard was prepared in methanol and used for calibration. KPE samples were dissolved in methanol at 20 g/L, filtered through a 0.45 µm syringe filter, and analyzed under identical chromatographic conditions. All experiments were performed using a single production lot of KPE prepared from one large-scale extraction batch under standardized manufacturing conditions to ensure batch consistency.

4.3. Sprague Dawley Rats

Seven-week-old male Sprague–Dawley rats (Hanabio Corp., Pyeongtaek, Republic of Korea) were housed individually in separate cages in pathogen-free barrier facilities under standard conditions (22 ± 2 °C, 50 ± 10% humidity, 12-h light/dark cycle) with free access to food and water. After a 7-day acclimatization period, the animals were randomly assigned to experimental groups and used to induce experimental OA. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of SouthEast Medi-chem Institute Corp., Busan, Republic of Korea (approval number: SEMI-24-005).

4.4. Experimental Design and Treatment Regimen

Animals were divided into five experimental groups (n = 10 per group): normal control (N), MIA control (C), and three KPE-treated groups receiving low (L), medium (M), or high (H) doses. All groups except the normal control received a single intra-articular injection of monosodium iodoacetate (MIA; 60 mg/mL, prepared concentration; Sigma-Aldrich, St. Louis, MO, USA) into the right knee joint [36]. A fixed injection volume of 50 µL per rat was administered, corresponding to an absolute dose of 3 mg MIA per rat (60 mg/mL × 0.05 mL). Given that rats weighed approximately 250–300 g at the time of OA induction, this dose is equivalent to approximately 10–12 mg/kg, within the commonly used range for inducing OA-like pathology in rats. Seven days after MIA injection, treatments were initiated. The MIA control group received vehicle (water), while the KPE-treated groups received oral KPE once daily for 19 consecutive days at doses of 20.57 mg/kg (L), 25.71 mg/kg (M), or 51.42 mg/kg (H), respectively. The normal control group received no MIA injection and was administered vehicle only. The selected KPE doses were determined based on (i) prior in vivo studies and toxicological evaluations of KPE in rats, which have reported oral administration across a broad dose range (e.g., 5–500 mg/kg/day in a 6-month study and higher-dose subchronic regimens) [37], (ii) published pharmacokinetic evidence demonstrating systemic exposure of major methoxyflavones after oral administration of KPE in rats [13], and (iii) our previous in vitro findings, together with a dose-range design to probe potential efficacy differences while avoiding overt toxicity [15]. Accordingly, the low and medium doses were selected to reflect ranges reported to exert anti-inflammatory and chondroprotective effects in preclinical models. In contrast, the high dose was included to explore upper-range efficacy without exceeding doses previously shown to be well tolerated. A formal a priori power analysis was not performed because reliable variance estimates and expected effect sizes for multiple endpoints were not available at the study planning stage. Therefore, the group size was determined based on commonly used sample sizes in comparable MIA-induced OA rat studies [38] and feasibility considerations.

4.5. Sample Collection and General Observations

Body weight, food intake, water consumption, and general health status were monitored regularly throughout the experimental period. Animals were fasted for 16 h prior to sacrifice. At termination, rats were euthanized, and blood samples were collected via the abdominal vein for serum analyses. Major organs, including the liver, kidneys, and spleen, were harvested, weighed, and processed for subsequent evaluation. During the experimental period, no significant differences in body weight or food intake were observed among the groups, indicating that KPE administration did not adversely affect general health or appetite. In addition, organ weights and gross morphology of the liver, kidneys, and spleen remained unchanged at study termination, suggesting the absence of overt systemic toxicity or organ-specific adverse effects (Supplementary Figure S1).

4.6. Static Weight-Bearing Test

The static weight–bearing imbalance associated with spontaneous pain in MIA-induced OA was quantified using an incapacitance tester (Model 600, Ugo Basile, Comerio, Italy). Prior to each session, the device was calibrated according to the manufacturer’s instructions. Animals were placed in a plexiglass chamber with each hind paw resting on a separate force plate; after a 5-s acclimation, weight-bearing on each hind limb was measured continuously over the next 5-s sampling period. From these data, both the weight-bearing ratio ([right paw weight] ÷ [left + right paw weight] × 100) and the absolute difference in grams between the two limbs were calculated. Measurements were performed on day 26 post-MIA injection to assess pain progression and the efficacy of KPE treatment.

4.7. Measurement of Serum Biomarkers

Blood was collected via the abdominal vein into serum-separator tubes (SST; Beckman Coulter, Brea, CA, USA), allowed to clot at room temperature for 20 min, and centrifuged at 1650× g for 15 min to obtain serum. Serum levels of aggrecan (AGC), glycosaminoglycan (GAG), osteocalcin (OC), and cross-linked C-telopeptide of type II collagen (CTX-II), TNF-α, COX-2, PGE2, and C-reactive protein (CRP) were measured using commercial ELISA kits: AGC (MBS2022017), GAG (MBS1609791), OC (MBS728975), CTX-II (MBS2533577), TNF-α (MBS2507393), COX-2 (MBS266603), PGE2 (MBS262150), and CRP (MBS2508830) from MyBioSource (San Diego, CA, USA), according to the manufacturers’ instructions.

4.8. Western Blotting

Dissected knee cartilage tissues were homogenized in ice-cold lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, 0.2% SDS, 5 mM NaF) supplemented with protease and phosphatase inhibitor cocktails. After incubation on ice for 30 min, lysates were centrifuged at 12,000× g for 15 min at 4 °C, and the supernatants were collected for protein analysis. Protein concentrations were determined using a standard protein assay, and equal amounts of total protein were loaded for each sample. Equal amounts of protein were mixed with SDS sample buffer, heated at 95 °C for 5 min, and separated on 10–12% SDS–PAGE gels. Proteins were transferred to nitrocellulose membranes using a wet-transfer system (100 V, 1 h, 4 °C). Membranes were blocked with 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies (1:500–1:1000 in TBST containing 5% skim milk): anti-MMP1 (A306151; Abcam, Cambridge, UK), anti-MMP3 (14351; Cell Signaling Technology, Danvers, MA, USA), anti-MMP13 (ab39012; Abcam, Cambridge, UK), anti-TIMP-1 (sc-21734; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA). After washing, membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (1:5000–1:10,000; anti-rabbit IgG-HRP, #7074; anti-mouse IgG-HRP, #7076; Cell Signaling Technology). Protein bands were developed using a femto-level ECL substrate (Donginbiotech, Seoul, Republic of Korea) and detected using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). For densitometric analysis, images were acquired under identical exposure settings, and only non-saturated bands within the linear detection range of the chemiluminescent signal were used for quantification. Band intensities were quantified using ImageJ (version 1.46; NIH) and normalized to β-actin as an internal loading control. Western blot analyses were performed using independent biological replicates, and representative blots shown in the figures reflect consistent results observed across samples within each experimental group.

4.9. Histology

Cartilage destruction was evaluated by histology using hematoxylin and eosin and Safranin-O staining. In brief, knee joints were fixed in 4% PFA, decalcified in 0.5 M EDTA, and embedded in paraffin. The paraffin blocks were sectioned at 5 µm thickness. Serial sections were obtained from the entire joint at 80-μm intervals, and the sections were deparaffinized in xylene, hydrated with graded ethanol, and stained with H&E and Safranin-O. Histological observations were performed under a light microscope at a magnification of X65 (E600, Nikon, Tokyo, Japan). For standardized and reproducible assessment of OA-like cartilage pathology, evaluation was performed using predefined morphological criteria aligned with key OARSI histopathology recommendations for cartilage structure, matrix staining, and cellularity [39,40]. Briefly, articular cartilage degeneration was assessed across serial sections in predefined anatomical regions (medial femoral condyle and medial tibial plateau), focusing on: (i) surface fibrillation/clefting or erosion, (ii) loss of Safranin-O staining intensity reflecting proteoglycan depletion, (iii) chondrocyte disorganization, clustering, or loss, and (iv) overall structural integrity and lesion extent. Two independent investigators blinded to treatment allocation evaluated all sections, and discrepancies were resolved by consensus. Representative images were selected based on these predefined criteria and consistent histological features observed across animals within each group. Although serial sections spanning the entire joint were examined, formal numerical OARSI scoring was not applied because the sections were not prospectively collected at predefined scoring levels required for compartment- and level-matched quantitative scoring. Therefore, standardized qualitative assessment using OARSI-aligned morphological criteria was employed to ensure reproducible comparisons across experimental groups.

4.10. Statistical Analysis

Statistical analyses were performed using StatView (ver. 5.0.1). Data distribution was first assessed for normality using the Shapiro–Wilk test. For datasets that satisfied the assumption of normality, group differences were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test when overall significance was detected. For datasets that did not meet normality assumptions, non-parametric analysis was performed using the Kruskal–Wallis test with appropriate post hoc comparisons. Accordingly, normally distributed data are presented as mean ± standard deviation (SD), whereas non-normally distributed data are presented as median with interquartile range and visualized using box-and-whisker plots. Exact p-values, effect sizes, statistical tests applied, and sample sizes for all analyzed endpoints are comprehensively summarized in Supplementary Table S3. The number of biological replicates (n) for each experiment is indicated in the corresponding figure legends. A two-sided p-value < 0.05 was considered statistically significant.

5. Conclusions

This study demonstrates that KPE exerts therapeutic effects in a MIA–induced rat model of OA. Oral administration of KPE was associated with improved joint function, attenuation of pain-related weight-bearing imbalance, and preservation of cartilage structure, as supported by complementary functional, histological, and biochemical assessments. At the molecular level, KPE did not uniformly suppress all matrix-degrading enzymes but modulated cartilage catabolism selectively. In particular, KPE significantly attenuated MMP-3 expression and restored TIMP-1 levels, suggesting reinforcement of endogenous ECM homeostasis rather than broad inhibition of matrix turnover. Modulation of MMP-1 and MMP-13 was more limited, consistent with their distinct regulatory roles in osteoarthritic cartilage. KPE treatment was also associated with attenuation of systemic inflammatory responses, as reflected by reduced CRP levels and a downward trend in PGE_2_, indicating partial modulation of inflammatory signaling in the OA setting. Across the tested dose range, KPE produced consistent protective effects, although the magnitude of response varied among endpoints, without evidence of a strict linear dose–response relationship. Safety evaluation was limited to short-term observations, including general clinical monitoring and gross organ assessment, under which no overt adverse effects were detected. However, conclusions regarding long-term systemic safety cannot be drawn from the present study. Collectively, these findings suggest that KPE possesses structure-preserving and anti-inflammatory potential in experimental OA through modulation of matrix regulatory balance. Further studies are warranted to evaluate long-term efficacy, pharmacokinetics, and comprehensive safety, and to elucidate the signaling mechanisms underlying TIMP-1–associated matrix regulation, in support of the translational development of KPE as an adjunct strategy for degenerative joint disease management.