1. IntroductionKnee osteoarthritis (OA), also referred to as degenerative joint disease of the knee, is characterized by pain, stiffness, and changes in knee joint constructs, such as the synovium, ligament, and articular cartilage [1]. Approximately 37% people over 60 years of age have knee OA worldwide, and it is a global issue in an aging society [2]. Various risk factors for OA, such as heavy work or sports activities, injury, frequent kneeling, being overweight, and genetics, can stimulate mechanical stress on the knee joint, resulting in degeneration of the articular cartilage [3].Joint instability caused by excessive physical kneeling promotes the formation of subchondral sclerosis and periarticular tissue abnormalities [4]. Damaged joint cells, such as chondrocytes, synovial fibroblasts, monocytes, and macrophages, induce the secretion of inflammatory cytokines such as interleukin-1-beta (IL-1β) [5]. In addition, IL-1β induced by external stress upregulates nuclear factor kappa B (NF-κB) and various mitogen-activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK), jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (p38MAPK), which can accelerate the inflammatory response [5]. Consistently, increased MAPKs have been observed in OA patients, and activated MAPKs are known to promote matrix metalloproteinase (Mmp) synthesis, which destroys articular cartilage [6].Both IL-1β and NF-κB enhance the synthesis of OA-associated catabolic factors, such as cyclooxygenase-2 (COX2), matrix metalloproteinase 3 (Mmp3), matrix metalloproteinase 13 (Mmp13), nitric oxide (NO), and prostaglandin E2 (PGE2), by initiating the SRY-box transcription factor (Sox9) and the Src/PI3K/AKT pathway and inflammatory cytokines, including interferon gamma (IFN-γ), IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in chondrocytes [7,8,9]. These processes damage articular cartilage and accelerate OA [10]. A study revealed that OA treatment relieves inflammatory responses in OA pathogenesis [11]. For example, non-steroidal anti-inflammatory drugs (NSAIDs), such as cyclooxygenase inhibitors (dexamethasone, meloxicam, and aspirin), relieve pain and inhibit PGE2 secretion by ameliorating inflammatory responses in chondrocytes [12]. However, the long-term use of these drugs is limited because of their renal, cardiac, gastrointestinal, and hepatological side effects, leading to an increased risk of NSAIDs use [13].Herbal plants have been widely used as alternative treatments because of their beneficial effects, such as relieving pain and decreasing inflammatory responses [14,15]. In addition, herbal plants have fewer side effects than chemical medications, supporting the increased demand for herbal plants [16]. Phytochemical studies have demonstrated that some single compounds derived from herbal plants, such as curcumin (Curcuma longa) and resveratrol (Polygonum cuspidatum), have been successfully researched for their anti-osteoarthritic effects, and the demand for the identification of new herbal plants for the prevention and treatment of OA has increased [17,18,19]. In addition, combined therapy with two or more plant extracts has shown synergistic effects on specific diseases, such as cardiovascular disease, cancer, and inflammation, to minimize the dosage of each extract [20,21,22].Prunella vulgaris (PV) and Gentiana lutea (GL) are traditional medicines in east Asia. PV belongs to the Lamiaceae family, which is rich in polyphenols and promotes α-glucosidase inhibitory and antioxidant effects for treating type 2 diabetes [23,24]. A study reported that PV exerted anti-inflammatory properties in the RAW 264.7 cell inflammatory response by regulating PGE2 and NO secretion, which plays a central role in OA development [25]. GL, known as bitter root, yellow gentian, and bitterwort, belongs to the genus Gentiana in the family Gentianaceae [26]. Bioactive compounds of gentian promote anti-atherosclerotic [27], antioxidant [28], anti-obesity [29] and anti-inflammatory effects [27,28]. One of the main components isolated from GL showed protective effects against IL-1β-induced articular chondrocyte inflammation by reducing COX2, Mmp, and PGE2 expressions [30]. Although the anti-inflammatory effects of PV and GL on OA have been reported, the combined treatment of PV and GL on OA in vitro and in vivo has not been reported.

This study investigated the protective effects of PV and GL extracts on OA on catabolic factors and destructive mediators in primary cultured chondrocytes and RAW 264.7 cells in vitro. Subsequently, the inhibitory effects of PV and GL extracts on subchondral sclerosis, histological analysis, and inflammatory cytokines in a medial meniscus (DMM)-induced OA mouse model were examined in vivo.

2. Materials and Methods 2.1. Extraction and Confirmation of Standards Compounds from PV and GL ExtractPV extract powder, GL extract powder, and the combination (PG extract) were provided by NINE B Co., Ltd. (Daejeon, Republic of Korea). PV and GL were extracted with water and 30% ethanol, respectively. The extracts were sequentially processed using filtration, evaporated, and spray-dried with dextrin. The combined product, PG extract, was prepared by mixing PV and GL extract powder at a weight ratio (w/w) of 8:2. PV, GL, and PG extraction was performed by subjecting it to gradient water with a sequential 0.1% trifluoroacetic acid-acetonitrile solvent system (5. 20, 25, 40, 5, and 5% acetonitrile) using high-performance liquid chromatography (HPLC; Agilent Technologies, Santa Clara, CA, USA) with a Hypersil GOLDTM column (Thermo Fisher Scientific, Waltham, MA, USA). The HPLC conditions were as follows: column temperature, 25 °C; detection wavelength, 280 nm, and flow rate, 0.6 mL/min. The standard compounds of rosmarinic acid for PV and Gentiopicroside for GL are shown in Supplementary Figure S1A,B, and the standard compounds of gentiopicroside (1) and rosmarinic acid (2) were compared with the combination of PV and GL with the chemical structures of gentiopicroside and rosmarinic acid (Supplementary Figure S2A–D). 2.2. Cell Culture and Primary Chondrocyte IsolationRAW264.7 cells were obtained from the Korean Cell Line Bank (KCLB No. 46609) and maintained in cultured medium (high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA)). Primary chondrocytes from the knee articular cartilage of 5 day-old mice were isolated using a culture medium containing 1% collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) and 0.5% trypsin EDTA for 2 h at 37 °C, as previously described [31]. Digestive solutions were filtered using a Falcon® 40 μm cell strainer (Corning, Riverfront Plaza Corning, NY, USA), and chondrocytes were obtained by centrifugation at 1200 rpm for 2 min. To induce an inflammatory response, chondrocytes were treated with IL-1β (1 ng/mL; Genscript, Piscataway, NJ, USA) for 2 d and RAW264.7 cells were stimulated with lipopolysaccharide (LPS, 1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 d. 2.3. Water-Soluble Tetrazolium Salt (WST) Assay

The cells were seeded in 96-well plates (1 × 105 cells) at 37 °C in 5% CO2. After 3 d, the cells were incubated with three different concentrations of the PV and GL (PG) extract (50, 100, and 150 µg/mL) for 2 d. Cytotoxicity was measured using the D-Plus™ CCK cell viability assay kit (Dongin Biotech, Seoul, Korea) at an absorbance of 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

2.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Quantitative Reverse-Transcription PCR (qRT-PCR)

Cells were treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to isolate total RNA, according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 100 ng of RNA using a RevertAid™ H Minus First Strand cDNA synthesis kit (Fermentas, Hanover, NH, USA). Reverse transcriptase PCR (RT-PCR) was performed using HiPi Plus 5X PCR MasterMix (ELPIS Biotech, Daejeon, Republic of Korea), and quantitative RT-PCR was performed using the SYBR Green I qPCR kit (TaKaRa, Shiga, Japan). The specific primers used for this study were as follows: forward 5′-TCA CTG CCA CCC AGA-3′ and reverse 5′-TGT AGG CCA TGA GGT CCA C-3′ for mouse Gapdh, forward 5′-GGT CTG GTG CCT GGT CTG ATG AT-3′ and reverse 5′-GTC CTT TCA AGG AGA ATG GTG C-3ʹ for mouse COX2, forward 5′-CTG TGT GTG GTT GTG TGC TCA TCC TAC-3′ and reverse 5′-GGC AAA TCC GGT GTA TAA TTC ACA ATC-3′ for mouse Mmp3, forward 5′-CTT CGA CAC TGA CAA GAA GTG G-3′ and reverse 5′-GGC ACG CTG GAA TGA TCT AAG-3′ for mouse Mmp9, forward 5′-CTT CTT CTT GTT GAG CTG GAC TC-3′ and reverse 5′-CTG TGG AGG TCA CTG TAG ACT-3′ for mouse Mmp13. The mRNA expression levels were normalized to Gapdh expression, and the relative expression levels of the genes were calculated using the 2−ΔΔCt method.

2.5. Western Blot Analysis

Cells were lysed with radioimmunoprecipitation buffer (RIPA; BIOSESANG, Seongnam, Republic of Korea), containing phenylmethylsulfonyl fluoride PMSF (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Total protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with primary antibodies specific for COX2 (ab39012; Abcam, Waltham, MA, USA), NF-κB (#8242; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (#3033S; Cell Signaling Technology, Danvers, MA, USA), β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), and appropriate secondary antibodies (horseradish peroxidase [HRP]-conjugated goat anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA), and HRP-conjugated goat anti-rabbit IgG (Bethyl Laboratories, Montgomery, TX, USA)). Immunolabeled proteins were detected using West-Q Pico Dura ECL Solution (GenDEPOT, Katy, TX, USA).

2.6. Destabilization of Medial Meniscus (DMM) Mouse Model and Micro-CT Analysis

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Ajou University (2020-0036), and experiments were performed according to the guidelines of the committee. To induce osteoarthritis (OA) in a mouse model, the medial meniscus (DMM) was destabilized in 9-week-old male C57BL/6 mice. Mice were anesthetized with tiletamine/zolazepam (Zoletil; Vibrac Laboratories, Carros, France), and the medial meniscus of the left knee joint was dissected. After surgery, mice were fed food pellets containing three different concentrations of PG extract (50, 100, and 200 mg/kg/d) for 8 weeks. At the end of the experiment, the mice were sacrificed, plasma samples were stored at −80 °C, and the left knee joint was fixed with 4% paraformaldehyde (BIOSESANG) at 4 °C until analysis. For micro-CT analysis, articular knee joints were scanned using high-energy spiral scan micro-CT (Skyscan 1173; Bruker, Billerica, MA, USA) and representative CT images were reconstructed using CTvox software (version 3.2, Bruker, Billerica, MA, USA). The bone mineral density of the region of interest was measured using Skyscan 1173 software.

2.7. HistologyThe formalin-fixed knee joint was decalcified using 0.5 M ethylenediaminetetraacetic acid (EDTA, pH 8.0) for 2 weeks, embedded in paraffin blocks, and sectioned (6 µm) using a rotary microtome (Leica Biosystems, Wetzler, Germany). Slide sections were subjected to safranin O staining to analyze the damaged regions of the articular cartilage. To determine the inflammatory responses of the articular cartilage, immunohistochemistry (IHC) was performed using specific primary antibodies, including COX2 (ab39012), Mmp3 (ab52915), and Mmp13 (ab39012) obtained from Abcam (Waltham, MA, USA). All stained slides were scanned using an Axio Scan 7 slide scanner (Carl Zeiss, Oberkochen, Germany). Cartilage destruction scoring was assessed using the Osteoarthritis Research Society International (OARSI) scoring to evaluate the degree of cartilage destruction [32]. 2.8. NO and Plasma Cytokine Analysis

NO levels in the cellular supernatants were examined using a Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Plasma levels of inflammatory cytokines (IFN-γ, IL-1-β, IL-6, and TNF-α) were analyzed using a mouse cytokine/chemokine magnetic bead panel immunology multiplex assay kit (MCYTOMAG-70k; Millipore, Billerica, MA, USA) and a MAGPIX® multiplex analyzer (Luminex, Austin, TX, USA).

2.9. Statistical Analysis

All data in the bar graphs were expressed using GraphPad Prism 9.2.0 software (GraphPad Software, San Diego, CA, USA). Statistical significance was evaluated by one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) post hoc test, using professional Statistical Package software (SPSS 25.0 for Windows, SPSS Inc., Chicago, IL, USA).

4. DiscussionOsteoarthritis (OA) is a common degenerative disorder, among which knee arthritis is the most common global disease [37]. Although many pharmaceutical drugs have been used for osteoarthritis treatment [38], their long-term side effects have been reported in older, frail, and comorbid patients [39]. This study describes the potential effected of anti-inflammatory herbal plants, PV and GL, in vitro and in vivo.In OA patients, chondrocytes are stimulated by external stress, leading to the upregulation of various catabolic molecules such as IL-1, COX2, and MMPs [40]. COX2 expression is upregulated by inflammatory cytokines, promoting increased production of MMP3 and MMP13, which induces proteoglycan and collagen degradation with apoptosis of chondrocytes [41]. In addition, increased inflammatory responses in osteoarthritis upregulate the release of destructive mediators such as NO, prostaglandin, and collagenase [42]. Prostaglandin, synthesized by the cyclooxygenase (COX) enzyme, is a derivative lipid molecule that regulates homeostasis and inflammation in the body [43]. It has been reported that osteoarthritic patients present elevated levels of PGE2 and collagenase in articular cartilage, promoting the degradation of collagen in cartilage [44,45]. Hence, NO, PGE2, and collagenase play important roles in regulating knee remodeling during inflammation. Studies have reported that both PV and GL promote anti-inflammatory activities [27,46]. In this study, the PG combination showed synergistic effects on inflammation in primary chondrocytes and RAW 264.7 cells without cytotoxic effects by reducing catabolic factors. In addition, treatment with the PG extract decreased inflammation-mediated secretion of PGE2, collagenase, and NO production. The results showed that upregulation of NF-κB, COX2, and MMPs induced by external stress inhibited using PG treatment prevented the secretion of inflammatory factors. This study suggests that the PG extract has protective effects against inflammation in primary chondrocytes and RAW 264.7 cells.The knee joint comprises several compartments, including articular cartilage, the meniscus, and ligaments [47]. Articular cartilage is a white connective tissue that provides a smooth, lubricated surface for the articulation to protect the ends of bones where they come together to form joints [48]. The meniscus of the knee joint is a crescent-shaped wedge of fibrocartilage located between the femoral condyle and tibial plateau, which provides increased stability to femorotibial articulation, distributes axial load, absorbs mechanical stress, and provides lubrication to the knee joint [49]. Various types of osteoarthritic mouse models, including surgical, chemical, and spontaneous methods, have been reported, and the DMM model has been widely used to evaluate degenerative OA [50]. Patients with OA typically exhibit high levels of BMD in physically damaged knees [51] and an osteosclerotic phenotype in the subchondral region of the tibial plateau [52]. In addition, a sclerotic phenotype of the OA model was observed in DMM-induced mice [53]. Similarly, our results showed that the OA mouse model induced by DMM showed subchondral osteophyte formation with increased BMD. However, micro-CT images of the left knee joint showed that treatment with PG extract inhibited the DMM-induced BMD increase in the tibial plateau. These results suggest that PG prevents subchondral sclerosis in a mouse model of DMM-induced OA.High levels of inflammatory cytokines, including IFN-γ, IL-1β, IL-6, and TNF-α, have been detected in OA patients, promoting the activation of MMPs and catabolic factor synthesis [54]. These processes are present in damaged knees with a reduction in type 2 collagen and proteoglycan expression and destruction of articular cartilage [55]. As histological analysis of destructive knees is important for degenerative arthritis studies [56], cartilage destruction and inflammatory responses were measured by detecting proteoglycan content using safranin O staining and immunohistochemistry of catabolic factors (COX2, Mmp3, and Mmp13) in the articular cartilage. Our results showed that the PG extract reduced articular cartilage destruction and decreased the inflammatory cytokine levels of IFN-γ, IL-1β, IL-6, and TNF-α and catabolic factors in DMM-induced OA mice. These results suggest that PG treatment inhibits DMM-induced destruction and inflammation with osteophyte formation in the subchondral cartilage region by reducing inflammatory responses and cytokine production (Figure 8). Previous studies have reported that phytopharmaceutical compounds identified from natural products could exert beneficial effects on patients with OA [57,58]. However, a limitation of our study is that it is unclear whether PG presents clinical efficacy in the treatment of patients with knee osteoarthritis. In addition, this study investigated only male DMM mice; however, it is uncertain whether the anti-osteoarthritic effects of PG are sex specific. Our in vitro and in vivo results showed beneficial effects in OA experimental models, and it is necessary to study the application of PG extract to humans in future studies. Consequently, PG may be a potential candidate for the inhibition and prevention of osteoarthritis.