Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1β-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.


Introduction
Osteoarthritis (OA) is the most prevalent type of degenerative joint disorder, characterized by cartilage degradation, osteophyte formation, and synovial inflammation [1][2][3].Articular cartilage primarily consists of the extracellular matrix (ECM) and chondrocytes.In chondrocytes, the activation of biochemical pathways leads to OA-causing mechanisms, which disrupt the production of cartilage-specific ECM components [4,5].Activation of the biochemical pathway activation involves pro-inflammatory cytokine production, inflammation, and ECM degradation by catabolic matrix-degrading enzymes, such as matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS) [6][7][8].
Upon the activation of catabolic enzymes, proteoglycans and collagen in the articular cartilage are degraded [7].
According to the Chinese Pharmacopoeia (Chinese-Pharmacopoeia-Commission, 2020), Mume Fructus (MF, commonly known as Omae), a traditional drug and health food, is processed from the Prunus mume (PM) fruit (commonly known as Measil) by fumigating it at a low temperature (40˚C) until it turns black.MF has been traditionally used to treat cough, dysentery, asthenia, consumptive thirst, vomiting, and abdominal pain.Recently, it has also been reported to exhibit antibacterial, antioxidant, antitussive, antiallergic, antiviral, antitumour, and anti-inflammatory effects [20][21][22][23][24][25][26].Furthermore, MF is the main herbal ingredient in 'Jaseng Woongayoungsin-hwan', which has been clinically used to treat herniated discs and lower limb pain.Woongayoungsin-hwan is an extract purified from a mixture of oriental herbs (Atractylodis rhizoma alba, Crataegus pinnatifida, Pueraria lobata Ohwi, Cyperus rotundus L., Chaenomeles sinensis Koehne, Panax ginseng C.A. Meyer, Alpiniae officinarum rhizoma, Cinnamomum zeylanicum Breyne, Triticum aestivum L., Zingiber officinale Roscoe, Citrus unshiu Markovich, Amomum villosum Lour., Piper longum L., Lemmaphyllum microphyllum, Syzygium aromaticum, Inula helenium, Pterocarpus indicus, Mentha arvensis var.piperascens, Arisaema amurense, and Mume Fructus) [27].For patients with a herniated intervertebral disc, herbal medicine treatment with Woongayoungsin-hwan has been proven effective in reducing low back pain, showing an improvement with a quick return to the activities of daily life activities and routine [27].For patients with muscular atrophy in the lower limbs caused by a condition of peripheral neuropathy, Korean medicine treatment with Woongayoungsin-hwan has exhibited a significant therapeutic effect with a gradual increase in the duration of self-walking exercise time [28].Disc pain and osteoarthritis have a similar etiopathogenesis, since proteoglycans, predominantly aggrecan, in the ECM of the nucleus pulposus and articular cartilage are lost in both cases [29].However, the effects of MF on articular cartilage chondrocytes and OA progression remain unclear.
Considering the anti-inflammatory effect of MF and analgesic effect of Woongayoungsinhwan on spinal disc problems, we hypothesized that MF is also effective against osteoarthritis.Therefore, in this study, we aimed to investigate the effects of MF extracts on IL-1β-induced OA progression in rat chondrocytes and to elucidate the potential underlying mechanisms.

Preparation of MF and PM extracts
MF and PM were purchased from Green M. P. Pharm.Co., Ltd.(Gyeonggi-do, South Korea).PM was purchased as a dried powder.The aqueous extract of MF was prepared with distilled water, boiled using a reflux apparatus maintained at 88˚C for 6 h, cooled to 20-22˚C, and filtered using filter paper.The filtrate was cooled to −20˚C and lyophilized using a freeze dryer

Nitric oxide (NO) assay
The RAW 264.7 cells were seeded at a density of 1.5 × 10 4 cells/well in 96-well plates, incubated at 37˚C under 5% CO 2 for 16 h, and treated with different MF concentrations.After 1 h of treatment, the cells were stimulated with 1 μg/mL LPS for 24 h.
The NO assay reagent was prepared prior to experiments.For preparing reagent A, 1 g of sulfanilamide (Sigma-Aldrich) and 85% phosphoric acid (Junsei Honsha Co., Ltd., Tokyo, Japan) were dissolved in 50 mL distilled water.For preparing reagent B, 1 g of N-(1-naphthyl) ethylene diamine (Sigma-Aldrich) was dissolved in 50 mL distilled water.Next, 50 μL of the culture medium and an equal volume of NO assay reagent were added to a 96-well microplate in the order A and B. The absorbance was measured at 560 nm using a microplate reader (Bio-Tek).The amount of nitrite in the medium was calculated using the sodium nitrite standard curve.

RNA isolation and quantitative reverse transcription PCR (qRT-PCR)
Primary chondrocytes (5 × 10 5 cells/well) were seeded in a 6-well culture plates.After incubation for 2 days, the cells were treated with various doses of MF or PM and 10 ng/mL IL-1β at 37˚C for 30 h.RAW 264.7 cells (1 × 10 6 cells/well) were seeded in 6-well plates for 16 h, incubated with various concentrations of MF, and treated with LPS (1 μg/mL).Total RNA was isolated from chondrocytes using TRIzol™ reagent (Life Technologies, Carlsbad, CA, USA).The RNA samples were reverse-transcribed to complementary DNA (cDNA) using an RT-Kit™ (Biofact, Daejeon, Korea) following the manufacturer's instructions.qRT-PCR analysis was performed using SYBR 1 Green Master mix (Bio-Rad, Hercules, CA, USA) and an iCycler iQ™ real-time PCR detection system (Bio-Rad).Results were normalized using β-actin as an internal control.The primer sequences are listed in Table 1.

Western blotting and immunoprecipitation
Primary chondrocytes (5 × 10 5 /well) were seeded into a 6-well culture plates.After incubation for 2 days, the cells were treated with different MF concentrations and 10 ng/mL IL-1β at 37˚C for 30 h.Chondrocytes were lysed using radioimmunoprecipitation assay lysis buffer (Biosesang, Sungnam, Korea) containing protease and phosphatase inhibitors.The primary antibodies used are listed in Table 2. Anti-β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (Santa Cruz Biotechnologies) were used as the loading controls.The protein bands were visualized using an Amersham Imager 600 imaging system (GE Healthcare Life Sciences, Uppsala, Sweden) and an enhanced chemiluminescence (ECL) system (Bio-Rad, Hercules, CA, USA).The protein levels were quantified using ImageJ (NIH, Bethesda, Maryland, USA).

Immunofluorescence assay
Primary chondrocytes were seeded onto glass coverslips in a 24-well culture plates.After incubation at 37˚C for 2 days, the cells were treated with MF at different concentrations and 10 ng/ mL IL-1β at 37˚C for 30 h.Subsequently, the cells were fixed with 4% paraformaldehyde and incubated overnight with the primary antibodies (Table 3) at 4˚C overnight.After washing with phosphate-buffered saline (PBS), the chondrocytes were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Jackson Immuno-Research Labs, West Grove, PA, USA) for 2 h at 20-25˚C.The nuclei were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1 μg/mL in PBS) for 10 min.Images of the cells were obtained using a confocal microscope (Eclipse C2 Plus; Nikon, Konan, Minato-ku, Japan).The fluorescence intensity was analyzed using ImageJ (NIH, Bethesda, Maryland, USA).

MF and PM preparation for high-performance liquid chromatography (HPLC)
Powdered dried samples (1 g) were mixed with methanol:water (7:3; v/v, 10 mL) and extracted via sonication at 24-26˚C for 60 min.The extract was filtered using a 0.45 μm syringe filter, and the crude extract obtained was analyzed directly using high-performance liquid chromatography with diode-array detection (HPLC-DAD).The PM and MF filtrates were subjected to HPLC for quality control.Neochlorogenic acid, chlorogenic acid, and 4-CQA (catalogue #BP0083, #BP0345, and #BP0411, respectively; Chengdu Biopurify Phytochemicals Ltd., Chengdu, China) were used as standard materials.HPLC was performed using an 1260 Infin-ity™ HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump, DAD, auto-sampler, thermostatically controlled column compartment, and a Capcell pak 1 AQ C18 column (250 mm × 4.6 mm, 5 μm).The column temperature was set to 40˚C.The mobile phase consisting of 1% acetic acid (solvent A), and acetonitrile (Solvent B) was used for isocratic elution; the latter was conducted with 90-75% solvent A for 1-25 min.The flow rate and injection volume were 0.7 mL/min and 10 μL, respectively.Absorbance was monitored at 328 nm using DAD spectrophotometer.Compound structures were drawn using Chemdraw Ultra 7.0 (BioByte Corp., Claremont, CA, USA).

Quantification and statistical analysis
Data were analyzed using Prism 8 software (GraphPad).All data are expressed as the mean ± standard deviation (SD) values from at least three independent experiments.Statistical analyses were performed using a one-way analysis of variance (ANOVA) to determine the differences between groups.Dunnett's multiple comparison test was performed to determine significant differences between the groups.Statistical significance was set at P < 0.05.

Effect of Mume Fructus (MF) and Prunus mume (PM) on IL-1β-induced OA signaling in rat articular chondrocytes
MF is processed from PM fruits via fumigation.Therefore, the effects of PM and MF were compared to confirm the effect of PM on the primary chondrocytes in rats.The cytotoxic Table 1.Oligonucleotides used for quantitative reverse transcription-PCR analysis.

IL-1β
Sense Antisense effects of PM on rat chondrocytes were evaluated using a CCK-8 assay.The viability of PM-or IL-1β-treated chondrocytes remained unaffected at concentrations ranging from 0 to 100 μg/ mL (Fig 1A).Compared with MF, PM did not affect IL-1β-induced OA signaling in the primary chondrocytes of rats (Fig 1B and 1C).These results were confirmed using immunofluorescence staining (Fig 2).MF has a more significant therapeutic effect on chondrocytes than PM.

HPLC analysis identified the chemical composition of Mume Fructus (MF) and Prunus mume (PM) aqueous extracts
To determine the differences in the chemical composition of MF and PM, which could be linked to their diverse effects on rat primary chondrocytes, phytochemical screening and analysis were performed using HPLC (Table 4).HPLC chromatograms showed peaks of neochlorogenic acid (  22.62% whereas that in the PM extract was 5.42% (Table 5), indicating a marked difference between the two peak areas.These results suggest that the 4-CQA component in MF inhibits OA signaling in the primary chondrocytes of rats.These results suggest that the 4-CQA component in MF inhibits OA signaling in the primary chondrocytes of rats.

Mume Fructus (MF) treatment suppresses IL-1β-induced OA signaling in rat articular chondrocytes
The CCK-8 assay was used to confirm the cytotoxic effects of MF on rat chondrocytes.The viability of MF-treated rat chondrocytes remained unaffected at MF concentrations ranging from 0 to 100 μg/mL, and the survival rate was dose-dependent in IL-1β-treated rat chondrocytes (Fig 1A).IL-1β is known to trigger the expression of various extracellular proteolytic enzymes in chondrocytes, such as MMP3, MMP13, and ADAMTS5 [13,14].To elucidate the effect of MF extract on ECM synthesis, we examined IL-1β-induced ECM degradation in rat articular

Mume Fructus (MF) treatment inhibits LPS-induced inflammation in macrophages
The CCK-8 assay was used to examine the cytotoxic effects of MF on macrophages.The viability of MF-or LPS-treated RAW 264.7 cells was not affected at concentrations of MF ranging from 0 to 100 μg/mL (Fig 6A ).LPS can activate macrophages to produce various pro-inflammatory cytokines, such as TNF-α and IL-6, and other inflammatory mediators, including NO [31,32].To assess the effect of MF on NO production in RAW 264.

Mume Fructus (MF) treatment inhibits IL-1β-induced inflammatory signaling in rat chondrocytes
Previous studies have reported that IL-1β-induced MAPK and NF-kB activation promotes MMP overproduction, thereby causing cartilage degradation [12,17].To confirm the mechanism underlying the effects of MF on rat chondrocytes, the MAPK signaling pathways were assessed using western blot analysis.The p-ERK, p-JNK, and p-p38 levels were significantly increased in IL-1β-stimulated cells and were decreased in MF-treated cells (Fig 7A

Discussion
OA is a common joint disease with a steadily increasing prevalence that leads to chronic pain and impaired joint function and poses a global social burden [33,34].OA is a form of degenerative arthritis that affects the joints of the knees, hips, shoulders, hands, and feet and induces articular cartilage destruction and thinning [1].Non-surgical modalities for the treatment of OA include medications such as hyaluronic acid, nonsteroidal anti-inflammatory drugs, and acetaminophen, while surgical treatment options include total knee arthroplasty, osteotomy, and unicompartmental knee arthroplasty [35].However, complications, such as blood pressure problems and infections, have been reported following some of these treatments [36].Therefore, plant extracts with joint protective effects and few side effects have been prioritized in the search for an alternative treatment for OA [37][38][39].One candidate is MF, a traditional herbal product with anti-inflammatory, antioxidant, and abdominal pain-reducing effects [21,26]; however, its effect on OA remains unknown.PM, a herbal medicinal plant commonly used in traditional Korean medicine and folk remedies, is the fruit of the Chinese plum (Prunus mume Sieb.et Zucc.).It has been reported to exhibit antimicrobial activity [40], and is effective against gastric secretion in rats [41] and diabetes [42].PM has different names and uses depending on the time of harvest and processing method, and it is generally classified as follows: Cheongmae is a green fruit with hard pulp and a strong sour and astringent taste; Cheongmae in steamed and dried state is called Geummae; Cheongmae pickled in brine and sun-dried is called Baekmae; Cheongmae with its pericarp removed and blackened to charcoal is called Omae (MF); and the yellow fruit with the ripe, fragrant smell is called Hwangmae [43].MF is processed by removing the pericarp and pits of near-mature Cheongmae picked from mid-June to early July, dried, and steam baked to black in a straw fire.MF is widely used as a medicinal herb in traditional Korean and Chinese medicine [44].Previous studies on the effects of MF have reported its antioxidant activity [45], antimicrobial effects against bacteria causing food poisoning/gastroenteritis [46], antitumor effects [47], and hypoglycemic effects [48].However, few studies have specifically investigated the potential of using MF in the treatment of OA.PM is used to treat various symptoms, including fatigue, cough, headaches, constipation, food poisoning, and stomach disorders [49].Pharmacologically, PM has been studied as an antidiabetic [50], hepatoprotective [51], antitumour [52], anti-inflammatory [53,54], antibacterial [55], and anticancer agent [50].We predicted that PM may also have an anti-inflammatory effect, thereby exerting an inhibitory effect on cartilage destruction due to an inflammatory response.However, PM, the raw material of MF, did not affect OA signaling in the chondrocytes.Assuming that the differential responses could be ascribed to different  chemical compositions, we conducted phytochemical analysis of MF and PM via HPLC.Our results revealed the presence of three main compounds, with the 4-CQA content differing between MF and PM.4-CQA is a phenolic acid and unique isomer of chlorogenic acid [56].Its pharmacological effects include improvement in the blood glucose levels, iron content, and lipid peroxide accumulation via activation of the system XC/glutathione peroxidase 4/factorerythroid factor 2 system in patients with diabetes [57].Additionally, 4-CQA attenuates LPSinduced inflammatory responses and oxidative stress and ameliorates myocardial hypertrophy through a hypoxia-inducible factor 1 subunit alpha-related pathway [57,58].The anti-inflammatory effects of 4-CQA may be attributed to its ability to inhibit OA through MF-mediated inflammatory signaling regulation.IL-1β induces the expression of several inflammatory mediators, such as IL-6, TNF-α, leukaemia inhibitory factor IL-6 family cytokine, prostaglandin E2, NO, COX-2, and inducible NO synthase, and various catabolic processes; it further contributes to synovial inflammation that degrades the cartilage.This process of IL-1β-induced cartilage destruction activates all three MAPKs (ERK, p38, and JNK) and the NF-κB signal cascade pathway [19].These intracellular signaling pathways induce OA owing to their participation in ECM degradation homeostasis, resulting in increased MMP3, MMP13, and ADAMTS5 levels and reduced COL2A1 levels [59,60].
The pathophysiology of OA is characterized by the breakdown of the ECM of articular cartilage by proteinases, whose expression is upregulated by inflammatory stimuli [61].These inflammatory responses occur in chondrocytes and synovial macrophages [62].Among the pathways involved in inflammatory responses, MAPKs and NF-κB are well known to play key roles [63].Therefore, we examined and confirmed the anti-inflammatory effects of MF through gene expression of TNF-α, IL-6, and IL-1β in RAW264.7 cells and also in chondrocytes.
Our results verified the effects of MF treatment in inhibiting MMP3/13 and increasing COL2A1 expression, which were combined to demonstrate the efficacy of MF treatment in OA signaling inhibition (Fig 8).Additionally, MF treatment reduced TNF-α, IL-6, and IL-1β levels in a dose-dependent manner in macrophages and chondrocytes.These OA signaling inhibitions and anti-inflammatory effects were induced by MF, which regulated all three MAPKs and NF-kB signaling.Moreover, MF treatment reduced the levels of COX-2, which is another inflammatory mediator.
Notably, the increase in the COL2A1 expression in the MF treatment group was considerably higher than that in the control group.Collagen is the most abundant protein in mammals [64].In humans, different types of collagens are present in the connective tissues such as discs, cartilage, tendons, and ligaments of diverse organs such as bones and joints [65].COL2A1, which comprises discs and cartilages, is degraded by MMP3 and MMP13 [15].A recent study indicated that the degradation of COL2A1 and a decrease in its level might initiate and promote OA [15]; moreover, the study suggested that collagen degradation played a central role in the pathogenesis of OA.Therefore, the upregulation of COL2A1 expression induced by MF in cartilage may be an OA treatment strategy.Additionally, MF could induce COL2A1 expression and inhibit MMP3 expression in discs during the treatment of herniated discs using Jaseng Woongayoungsin-hwan; however, further research is warranted in this regard.
Our study has a limitation.As we focused on the efficacy of MF extract in chondrocytes, we were able to analyze the marker components of MF but not to the extent of component-specific effects.Further studies are needed to determine the specific effects of 4-CQA on chondrocytes and OA pathogenesis to determine the specific effects of 4-CQA on chondrocytes and OA pathogenesis.

Conclusions
The current study revealed that MF treatment reduces OA pathogenesis by inhibiting inflammation via MAPK-NF-κB signaling and induces COL2A1 expression.Thus, MF could be a potential therapeutic agent for OA, promoting ECM regeneration and inhibiting its degradation.