Anti-Osteoarthritic Effects of Prunella Vulgaris and Gentiana Lutea In Vitro and In Vivo

Osteoarthritis (OA) is the progressive destruction of articular cartilage with severe symptoms, including pain and stiffness. We investigated the anti-osteoarthritic effects of Prunella vulgaris (PV) and Gentiana lutea (GL) extract in primary cultured chondrocytes RAW 264.7 cells in vitro and destabilization of the medial meniscus (DMM)-induced OA mice in vivo. Primary chondrocytes were induced with IL-1β, and RAW 264.7 cells were treated with LPS and co-incubated with either individual extracts of PV and GL or different ratios of PV and GL mixture. For the OA animal model, the medial meniscus (DMM) was destabilized in 9-week-old male C57BL/6 mice. Treatment of individual PV and GL and combination of PV and GL extracts inhibited the mRNA expression level of COX2 in chondrocytes and RAW 264.7 cells. The optimized inhibitory effect was attained with a PV and GL combination at an 8:2 ratio (PG) without cytotoxic effects. PG extracts prevented the expression of catabolic factors (COX2, Mmp3, Mmp9, and Mmp13) and inflammatory mediator levels (PGE2 and collagenase). In addition, PG decreased subchondral sclerosis and increased BMD in the subchondral region of DMM-induced OA mice with protection of articular cartilage destruction by inhibiting inflammatory processes. This study suggests that PG may be an alternative medicinal herb for treatment of OA.


Introduction
Knee 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 GOLD TM 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).

Water-Soluble Tetrazolium Salt (WST) Assay
The cells were seeded in 96-well plates (1 × 10 5 cells) at 37 • C in 5% CO 2 . 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).

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-3 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 −∆∆Ct method.

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.

Histology
The 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].

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).

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 oneway 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).

PV and GL Mixtures Inhibit COX2 mRNA Expression
Inflammation in mouse primary cultured chondrocytes is predominantly initiated by increased expression levels of IL-1β, and the inflammatory responses in RAW 264.7, macrophage have been induced by the administration of LPS [33,34]. First, we confirmed the effects of single extracts of PV and GL on inflammation in primary chondrocytes and RAW 264.7 cells and compared the synergistic effects of the combination of PV and GL extracts at three different ratios (6:4, 7:3, and 8:2). High amounts of PV were combined in the mixture extract because PV is a well-known traditional herbal medicine used for the treatment of inflammation [35]. Primary chondrocytes were induced with IL-1β (1 ng/mL), and RAW 264.7 cells were treated with LPS (1 µg/mL) and co-incubated with either individual extracts of PV and GL or different ratios of PV and GL mixture (100 and 150 µg/mL). Since cyclooxygenase-2 (COX2) is a reliable inflammatory biomarker that is detectable during the inflammatory response [36], we examined the anti-inflammatory effects of PV and GL extracts by evaluating the mRNA expression level of COX2 in chondrocytes and RAW 264.7 cells using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). As expected, individual PV and GL extracts inhibited COX2 mRNA expression in both IL-1β-induced chondrocytes and LPS-induced RAW 264.7 cells (Supplementary Figure S3A,B). Additionally, combined PG extracts at ratios of 6:4, 7:3, and 8:2 significantly decreased the expression level of COX2 in primary chondrocytes and RAW 264.7, respectively (Supplementary Figure S4C,D). Comparison of COX2 expression between the single extracts and PV and GL mixture at 150 µg/mL showed that the inhibitory effect of the optimal extract was a PV and GL combination at an 8:2 ratio ( Figure 1A,B) without cytotoxic effects at 50, 100, and 150 µg/mL ( Figure 1C,D). Consequently, we used a PV and GL mixture at an 8:2 ratio (PG) for subsequent examination.

PV and GL Mixtures Inhibit COX2 mRNA Expression
Inflammation in mouse primary cultured chondrocytes is predominantly initiated by increased expression levels of IL-1β, and the inflammatory responses in RAW 264.7, macrophage have been induced by the administration of LPS [33,34]. First, we confirmed the effects of single extracts of PV and GL on inflammation in primary chondrocytes and RAW 264.7 cells and compared the synergistic effects of the combination of PV and GL extracts at three different ratios (6:4, 7:3, and 8:2). High amounts of PV were combined in the mixture extract because PV is a well-known traditional herbal medicine used for the treatment of inflammation [35]. Primary chondrocytes were induced with IL-1β (1 ng/mL), and RAW 264.7 cells were treated with LPS (1 µ g/mL) and co-incubated with either individual extracts of PV and GL or different ratios of PV and GL mixture (100 and 150 µ g/mL). Since cyclooxygenase-2 (COX2) is a reliable inflammatory biomarker that is detectable during the inflammatory response [36], we examined the anti-inflammatory effects of PV and GL extracts by evaluating the mRNA expression level of COX2 in chondrocytes and RAW 264.7 cells using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). As expected, individual PV and GL extracts inhibited COX2 mRNA expression in both IL-1β-induced chondrocytes and LPS-induced RAW 264.7 cells (Supplementary Figure S3A,B). Additionally, combined PG extracts at ratios of 6:4, 7:3, and 8:2 significantly decreased the expression level of COX2 in primary chondrocytes and RAW 264.7, respectively (Supplementary Figure S4C,D). Comparison of COX2 expression between the single extracts and PV and GL mixture at 150 µ g/mL showed that the inhibitory effect of the optimal extract was a PV and GL combination at an 8:2 ratio ( Figure  1A,B) without cytotoxic effects at 50, 100, and 150 µ g/mL ( Figure 1C,D). Consequently, we used a PV and GL mixture at an 8:2 ratio (PG) for subsequent examination.

PG Decreases Inflammation in Mouse Primary Chondrocytes and RAW 264.7 Cells
To investigate the effect of PG extract on inflammation in RAW 264.7 cells and primary chondrocytes, catabolic and inflammatory-associated factors, such as COX2, Mmp3, and Mmp13, were measured by qRT-PCR. Treatment with IL-1β in chondrocytes

PG Decreases Inflammation in Mouse Primary Chondrocytes and RAW 264.7 Cells
To investigate the effect of PG extract on inflammation in RAW 264.7 cells and primary chondrocytes, catabolic and inflammatory-associated factors, such as COX2, Mmp3, and Mmp13, were measured by qRT-PCR. Treatment with IL-1β in chondrocytes and LPS in RAW 264.7 cells increased the mRNA expression levels of COX2, Mmp3, Mmp9, and Mmp13. However, the PG extract significantly reduced the mRNA expression of COX2, Mmp3, Mmp9, and Mmp13 in both RAW 264.7 cells and primary chondrocytes (Figure 2A,B). Furthermore, Western blot analysis showed that protein levels of NF-κB and COX2 were decreased, and NO production was significantly decreased in RAW 264.7 after the PG extract treatment (Figure 3). These results suggest that the PG extract decreased inflammation in mouse primary chondrocytes and RAW 264.7 cells.

PG Decreases Inflammation in Mouse Primary Chondrocytes and RAW 264.7 Cells
To investigate the effect of PG extract on inflammation in RAW 264.7 cells and primary chondrocytes, catabolic and inflammatory-associated factors, such as COX2, Mmp3, and Mmp13, were measured by qRT-PCR. Treatment with IL-1β in chondrocytes and LPS in RAW 264.7 cells increased the mRNA expression levels of COX2, Mmp3, Mmp9, and Mmp13. However, the PG extract significantly reduced the mRNA expression of COX2, Mmp3, Mmp9, and Mmp13 in both RAW 264.7 cells and primary chondrocytes (Figure 2A,B). Furthermore, Western blot analysis showed that protein levels of NF-κB and COX2 were decreased, and NO production was significantly decreased in RAW 264.7 after the PG extract treatment (Figure 3). These results suggest that the PG extract decreased inflammation in mouse primary chondrocytes and RAW 264.7 cells.

PG Inhibits Secretion of Prostaglandin E2 (PGE2) and Collagenase in IL-1β-Induced Primary Chondrocyte Inflammation
To further confirm the anti-inflammatory effect of PG leaf extract on destructive

PG Inhibits Secretion of Prostaglandin E2 (PGE2) and Collagenase in IL-1β-Induced Primary Chondrocyte Inflammation
To further confirm the anti-inflammatory effect of PG extract on destructive mediators, the levels of secreted PGE2 and collagenase were investigated in IL-1β-induced primary chondrocytes. The administration of IL-1β and co-treatment with PG extract (100 and 150 µg/mL)induced inflammation of primary cultured chondrocytes. To measure the release of PGE2 and collagenase, the culture supernatant was concentrated using Vivaspin ® , and the levels of destructive mediators were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. IL-1β-induced primary chondrocytes exhibited increased PGE2 and collagenase levels. However, treatment with the PG extract significantly suppressed the secretion of PGE2 and collagenase in IL-1β-induced chondrocytes. (Figure 4A,B). These results indicate that PG treatment inhibited the secretion of PGE2 and collagenase by downregulating the COX2, Mmp3, Mmp9, and Mmp13 expressions.
Western blot analysis. β-actin was used as a loading control. (B) NO production was examined using nitrate/nitrite assay. * p < 0.05 vs. Mock, CONT; non-treated group.

PG Inhibits Secretion of Prostaglandin E2 (PGE2) and Collagenase in IL-1β-Induced Primary Chondrocyte Inflammation
To further confirm the anti-inflammatory effect of PG leaf extract on destructive mediators, the levels of secreted PGE2 and collagenase were investigated in IL-1β-induced primary chondrocytes. The administration of IL-1β and co-treatment with PG extract (100 and 150 µ g/mL)induced inflammation of primary cultured chondrocytes. To measure the release of PGE2 and collagenase, the culture supernatant was concentrated using Vivaspin ® , and the levels of destructive mediators were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. IL-1β-induced primary chondrocytes exhibited increased PGE2 and collagenase levels. However, treatment with the PG extract significantly suppressed the secretion of PGE2 and collagenase in IL-1β-induced chondrocytes. (Figure 4A,B). These results indicate that PG treatment inhibited the secretion of PGE2 and collagenase by downregulating the COX2, Mmp3, Mmp9, and Mmp13 expressions.

PG Prevents Subchondral Sclerosis in the DMM-Induced OA Mouse Model
Based on the in vitro results, the anti-inflammatory effects of PG extract were evaluated in an osteoarthritic mouse model by surgical DMM in vivo. DMM-induced mice were fed either food pellets or pellets containing different concentrations of PG extract (50, 100, and 200 mg/kg/d) for eight weeks. The left knee joint was removed and scanned on the last administration day using micro-computed tomography (micro-CT). Micro-CT images showed that DMM mice exhibited subchondral sclerosis and increased BMD in the subchondral region. However, treatment with the PG extract reduced DMM-induced sclerosis ( Figure 5A, red) and increased BMD ( Figure 5B). These results suggest that PG prevents tibial subchondral osteophyte formation in a mouse model of DMM-induced OA.
(50, 100, and 200 mg/kg/d) for eight weeks. The left knee joint was removed and scanned on the last administration day using micro-computed tomography (micro-CT). Micro-CT images showed that DMM mice exhibited subchondral sclerosis and increased BMD in the subchondral region. However, treatment with the PG extract reduced DMM-induced sclerosis ( Figure 5A, red) and increased BMD ( Figure 5B). These results suggest that PG prevents tibial subchondral osteophyte formation in a mouse model of DMM-induced OA.

PG Reduces Cartilage Destruction in DMM-Induced OA Mouse Model
Histological analysis of articular cartilage destruction was performed to confirm the protective effects of PG extract on cartilage destruction in the DMM-induced OA model. By safranin O staining, DMM mice presented a loss of cartilage in the knee joint compared with the sham-operated group. In addition, immunohistochemical analysis showed that the DMM group exhibited upregulated expression of catabolic factors such as COX2, Mmp3, and Mmp13 in the articular cartilage. However, PG extract treatment significantly decreased cartilage destruction ( Figure 6A, left) with reduced expression levels of catabolic factors (COX2, Mmp3, and Mmp13) compared to the DMM-induced OA group ( Figure 6A, right). Administration of PG extract decreased DMM-induced vertical clefts and erosions extending to the calcified layer of the articular surface using the OARSI grade and quantified by the relative expression of COX2, Mmp3, and Mmp13 ( Figure 6B). the DMM group exhibited upregulated expression of catabolic factors such as COX2, Mmp3, and Mmp13 in the articular cartilage. However, PG extract treatment significantly decreased cartilage destruction ( Figure 6A, left) with reduced expression levels of catabolic factors (COX2, Mmp3, and Mmp13) compared to the DMM-induced OA group ( Figure 6A, right). Administration of PG leaf extract decreased DMM-induced vertical clefts and erosions extending to the calcified layer of the articular surface using the OARSI grade and quantified by the relative expression of COX2, Mmp3, and Mmp13 ( Figure 6B).

PG Decreases Inflammatory Cytokines in the DMM-Induced OA Mouse Model
To examine the effects of PG on inflammatory cytokines, the secretion of IFN-γ, IL-1β, IL-6, and TNF-α was analyzed in a mouse model of DMM-induced OA. As expected, DMM mice exhibited elevated plasma levels of inflammatory cytokines IFN-γ, IL-1β, IL-6, and TNF-α. However, the PG extract significantly inhibited the plasma levels of OA-induced cytokines (Figure 7). Taken together, these results suggest that PG treatment prevents subchondral osteophyte formation by inhibiting catabolic factors and inflammatory cytokines in DMM-induced OA mice.
1β, IL-6, and TNF-α was analyzed in a mouse model of DMM-induced OA. As expected, DMM mice exhibited elevated plasma levels of inflammatory cytokines IFN-γ, IL-1β, IL-6, and TNF-α. However, the PG extract significantly inhibited the plasma levels of OAinduced cytokines (Figure 7). Taken together, these results suggest that PG treatment prevents subchondral osteophyte formation by inhibiting catabolic factors and inflammatory cytokines in DMM-induced OA mice.

Discussion
Osteoarthritis (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 antiinflammatory 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

Discussion
Osteoarthritis (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.

Conclusions
In summary, this study investigated the effects of PG extracts in vitro and in vivo. The PG extract decreased the expression of catabolic factors and inflammatory mediator levels in primary chondrocytes and RAW 264.7 cells. Administration of the PG extract in a DMM-induced OA mouse model protected articular cartilage destruction by inhibiting inflammatory processes. These results suggest that the PG extract can be used as an

Conclusions
In summary, this study investigated the effects of PG extracts in vitro and in vivo. The PG extract decreased the expression of catabolic factors and inflammatory mediator levels in primary chondrocytes and RAW 264.7 cells. Administration of the PG extract in a DMM-induced OA mouse model protected articular cartilage destruction by inhibiting inflammatory processes. These results suggest that the PG extract can be used as an alternative herbal medicine to treat patients with OA.