Abstract

   Osteoarthritis (OA) is one of the most prevalent chronic degenerative
   joint diseases affecting adults in their middle or later years. It is
   characterized by symptoms such as joint pain, difficulty in movement,
   disability, and even loss of motion. Moreover, the onset and
   progression of inflammation are directly associated with OA. In this
   research, we evaluated the impact of Flavokawain A (FKA) on
   osteoarthritis. In-vitro effects of FKA on murine chondrocytes have
   been examined using cell counting kit-8 (CCK-8), safranin o staining,
   western blot, immunofluorescence staining, senescence β-galactosidase
   staining, flow cytometry analysis, and mRFP-GFP-LC3 adenovirus
   infection. An in-vivo model of destabilization of the medial meniscus
   (DMM) was employed to investigate FKA’s effect on OA mouse. An analysis
   of bioinformatics was performed on FKA and its potential role in OA. It
   was observed that FKA blocked interleukin (IL)-1β-induced expression of
   inflammatory factors, i.e., cyclooxygenase-2 (COX2) and inducible
   nitric oxide synthase (iNOS) in chondrocytes. In addition, FKA also
   downregulated the catabolic enzyme expression, i.e., aggrecanase-2
   (ADAMTS5) and matrix metalloproteinases (MMPs), and helped in the
   upregulation of the anabolic protein expression, i.e., type II collagen
   (Col2), Aggrecan, and sry-box transcription factor 9 (SOX9). Moreover,
   FKA ameliorated IL-1β-triggered autophagy in chondrocytes, and it was
   observed that the FKA causes anti-inflammatory effects by the
   mitogen-activated protein kinase (MAPK) and
   phosphoinositide-3-kinase/Akt/mammalian target of rapamycin
   (PI3K/AKT/mTOR) signaling pathways inhibition. The results of
   immunohistochemical analysis and microcomputed tomography from the in
   vivo OA mouse model confirmed the therapeutic effect of FKA. Finally,
   we assessed the anti-arthritic impacts of FKA by conducting in vivo and
   in vitro analyses. We concluded that FKA can be employed as a useful
   therapeutic agent for OA therapy, but the findings require needs
   further clinical investigation.

   Keywords: flavokawain A, osteoarthritis, apoptosis, autophagy,
   3-methyladenine, MAPK, PI3K/AKT/mTOR

Introduction

   Osteoarthritis (OA) is one of the most prevalent joint diseases around
   the globe. Around 18% of women and 10% of men over 60 years of age are
   affected by it ([46]Woolf and Pfleger, 2003). The most common clinical
   features of OA are joint pain, swelling, stiffness, and subchondral
   bone sclerosis ([47]Aicher and Rolauffs, 2014). Non-steroidal
   anti-inflammatory drugs (NSAIDs) are the main drug of choice for the
   conservative treatment of OA. But NSAIDs have some significant side
   effects, including gastrointestinal irritation, ulceration and bleeding
   ([48]Katz et al., 2021). The treatment of end-stage OA is knee
   arthroplasty, but the functional results may be poor, and the life of
   the prosthesis is limited ([49]Glyn-Jones et al., 2015). Therefore, the
   focus of OA research has shifted to prevention and early treatment.
   Drug development for joint protection interventions is in progress.
   Cartilage metabolic imbalance and chronic inflammatory responses are
   critical in the progression of OA ([50]Appleton, 2018). Increased
   levels of the proinflammatory cytokine IL-1β, which promotes the
   cartilage matrix catabolic enzyme synthesis, such as ADAMTS5 and MMPs
   ([51]Le Maitre et al., 2007), and suppresses the levels of important
   extracellular matrix (ECM) proteins, including Col2 and Aggrecan
   ([52]Kobayashi et al., 2005), were detected in the synovial fluid of
   individuals suffering from OA ([53]Chen et al., 2020). Therefore, drugs
   that reverse the IL-1β′s effect can help in designing a potential
   therapy for OA.

   In recent years, plant extracts have been recognized for their
   pharmaceutical significance. Flavokawain A (FKA) is a natural chalcone
   extracted from Piper methysticum Forst, which has exhibited
   anti-apoptotic and anti-inflammatory effects ([54]Rajendran et al.,
   2021). The in vitro studies have revealed that FKA inhibits the NFκB
   and MAPK pathways to relieve lipopolysaccharide-induced inflammation of
   Raw 264.7 macrophages ([55]Kwon et al., 2013). Furthermore, the ability
   of FKA to regulate oxidative stress has been published ([56]Hseu et
   al., 2019). However, the role of FKA in OA has not yet been
   investigated. Hence, the objective of this research is to elucidate the
   possible role of FKA in OA treatment.

Materials and methods

Ethics approval

   For the purpose of minimizing animal suffering, the research was
   conducted as per the guiding principles and protocols established by
   the Animal Care and Use Committee for Teaching and Research at the
   Tongji Medical College of Huazhong University of Science and
   Technology.

Chemicals and materials

   FKA (T3S0737, purity: 99.41%) and 3-Methyladenine (3 MA) were acquired
   from TOPSCIENCE (Shanghai, China), and the mouse IL-1β cytokine was
   obtained from the R&D system (501-RL-010, United States). Safranin O
   solution were provided by Solarbio (Beijing, China). The primary
   antibodies for iNOS, COX2, Col2, Aggrecan, Sox9, and immunofluorescence
   secondary antibodies were acquired from Abcam (Shanghai, China). Atg12,
   Beclin, LC3I/II, P21, P16, Bax, Bcl2, GAPDH, and antibodies for all
   pathways were purchased from CST (Beverly, MA, United States).
   Corresponding primary antibodies for MMP3/13 and Nrf2/HO-1/NQO1 were
   supplied by the Proteintech Group (Wuhan, Hubei, China). ADAMTS5
   primary antibody, secondary antibody, phosphate-buffered saline (PBS),
   trypsin, collagenase type II, the CCK8 assay kit, bovine serum albumin
   (BSA), and protein extraction kit were ordered and acquired from Boster
   Biological Technology (Wuhan, Hubei, China). RFP-GFP-LC3-adenovirus was
   purchased from Hanbio (Shanghai, China). The Senescence β-galactosidase
   staining kit and the Annexin V-FITC apoptosis detection kit were
   supplied by Beyotime (Shanghai, China).

Isolation and culture of chondrocytes

   In our study, five-day-old male C57BL/6J mice were sacrificed to obtain
   chondrocytes. The cartilage was obtained from around the knee joint
   region of the mice and was cut into granules after removing the
   surrounding synovial tissue. The granules were digested with trypsin
   for half an hour. Later, 0.2% type II collagenase was added for
   digestion for a period of 6 h. Subsequently, the cell suspension was
   concentrated by the process of centrifugation at 300g for a period of
   5 min to obtain primary chondrocytes. Moreover, the incubation of
   primary chondrocytes was performed at a temperature of 37°C, with 5%
   CO[2] in DMEM/F12 (1:1) medium containing penicillin/streptomycin
   solution (1%) and FBS (10%). The cells of the third passage were
   utilized for subsequent experiments.

Cell viability assay

   For the measurement of the viability of cells, the CCK8 assay was
   employed. Mouse chondrocytes were seeded at a density of 1×10^4
   cells/well in a 96-well plate containing the required culture medium.
   Following cell adhesion, 0.1 ml of medium with or without IL-1β
   (5 ng/ml) was added at various concentrations of FKA (0, 5, 10, 20, and
   40 μM) for a period of 24 h. Subsequently, an addition of 10 μl of CCK
   8 solution was carried out in all the wells, and incubation was
   performed for a period of 1 h. Thereafter, we employed a microplate
   reader (Bio-Rad, Richmond, CA, United States) to measure the viability
   of cells by measuring the absorbance at 450 nm.

Safranin O staining

   Seeding of chondrocytes was performed in 24-well plates. When
   chondrocytes reached 80% confluency, FKA with a concentration of 40 μM
   was administered alone or in combination with IL-1β (5 ng/ml). Cells
   were incubated for 24 h and washed with PBS. Cells were then fixed with
   4% paraformaldehyde, and after discarding the cell fixative, they were
   stained with 0.5% Safranin O reagent for 2 h. The dye was finally
   removed and washed once again with distilled water.

Western blot

   The protein extraction was performed by first washing the mouse
   chondrocytes seeded in a 6-well plate using PBS thrice, followed by the
   addition of 100 μl RIPA lysate buffer, 1 μl phosphatase inhibitor, and
   protease inhibitor to each well. Additionally, the protein
   concentration was measured by employing the Bicinchoninic Acid Assay.
   Moreover, 25 μg of protein samples were added to each well in the
   SDS-PAGE gel (8.0–12.5%) for electrophoresis. The electroblotting
   method was employed for the separation of protein bands and
   transferring onto a polyvinylidene membrane, and blocking of the
   membrane was performed at room temperature using BSA (5%) for a period
   of 1 h, and overnight incubation of the target band was carried out in
   the corresponding primary antibody at a temperature of 4°C. Washing of
   the membrane was conducted in TBST three times, and a second incubation
   was performed in the corresponding secondary antibody for a period of
   1 h, then rinsed thrice with TBST again. The Image Lab software
   (Bio-Rad) was used to obtain clear western blotting bands.

Immunofluorescence

   Laser confocal culture dish (34.8 mm diameter) was utilized to seed the
   chondrocyte cells at a density of 1 × 10^4 cells/well. 5 ng/ml of IL-1β
   was added for treating the cells for 24 h in the absence or presence of
   40 µM FKA. Furthermore, PBS was utilized for washing (three times);
   cell fixation was carried out using 4% paraformaldehyde for a period of
   15 min; cell permeabilization was performed by employing 0.5% Triton
   X-100 for a period of 15 min, and 5% BSA was employed for blocking.
   Overnight incubation of the chondrocytes was done with primary antibody
   against MMP13 (18165-1-AP, 1:50) and Aggrecan (ab36861, 1:100) at 4°C.
   After that, they were rinsed using PBS three times, and incubation was
   performed again with fluorescent secondary antibodies (ab150083, 1:200)
   for a period of 60 min. In addition, 0.5 μg/ml DAPI was utilized for
   the nuclei staining. The cell imaging was carried out with the aid of a
   confocal microscopy (Leica, United States).

Senescence β-galactosidase staining

   The senescent β-galactosidase staining was utilized to check the degree
   of cellular senescence. The washing of cells was conducted using PBS,
   and fixation was carried out for 15 min. After that, the staining
   solution was added for overnight incubation at 37°C in the absence of
   carbon dioxide. A microscope was employed for the observation and
   recording of the number of blue spots in the field of view.

Flow cytometry analysis

   Chondrocytes were collected using 0.1% trypsin following the
   instructions provided by the manufacturer; PBS was used for washing
   (twice), and cell resuspension was performed in a binding buffer.
   Apoptotic cells were assessed by flow cytometry (Beckman Coulter, Inc.
   United States) after performing staining using propidium iodide and
   Annexin V ([57]Sun et al., 2021).

Detection of autophagic flux

   RFP-GFP-LC3-adenovirus was employed for the detection of autophagic
   flux. When the cells reached about 50% confluency, the cells were
   infected with adenovirus vectors for a day. Then use IL-1β (5 ng/ml)
   pretreated cells in the presence or absence of FKA (40 μM) for 24 h.
   Due to acid sensitivity, green fluorescence is extinguished after
   autolysosome formation, and only red fluorescence can be detected
   ([58]Pei et al., 2021). The number of autophagosomes and autolysosomes
   was then observed by confocal microscopy (Leica, United States).

Bioinformatics analysis of FKA

   Through the PharmMapper and miRWalk2.0 databases, the FKA and
   OA-related pathways were identified, respectively. The prediction of
   FKA-target genes was performed based on the PharmMapper database, which
   was subsequently imported into the Database for Annotation,
   Visualization, and Integrated Discovery (DAVID) to carry out the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.
   Moreover, in order to obtain FKA-related OA pathways, the incorporation
   of the KEGG pathways was conducted with OA-related pathways acquired
   using the miRWalk 2.0 database. Additionally, the Venn diagram
   (Venny2.1, [59]http://bioinfogp.cnb.csic.es/tools/venny/index.html) was
   utilized for the visualization of various intersected pathways related
   to FKA. Furthermore, Cytoscape 3.8.2 was employed for the screening of
   hub genes based on the degree, betweenness, and closeness centrality of
   FKA-target genes. The Pathway Builder Tool 2.0
   ([60]https://www.proteinlounge.com) was employed to conduct KEGG
   pathway enrichment analysis, and the leading KEGG pathways were
   selected from the crossover pathways. Finally, the proximity of
   FKA-target genes in the network were illustrated by constructing a
   circos plot with the aid of the circlize R package, which also included
   the location of the genes on the chromosome and their connections.

DMM OA model

   Eighteen specific pathogen-free C57BL/6J male mice were acquired from
   the Experimental Animal Center of Tongji Hospital. Mouse DMM OA model
   was established as previously described ([61]Lu et al., 2021). The
   mouse were anesthetized by intraperitoneal injection of pentobarbital
   (100 μl/10g body weight). Right knee surgery of all the mice was
   carried out and randomly classified into 3 groups. FKA group mice (n =
   6) underwent DMM surgery and were intra-articularly injected with
   50 mg/kg FKA weekly. Sham-operated group mice (n = 6) underwent sham
   surgery (capsulotomy and suturing) and were injected with saline (equal
   volume). OA group mice (n = 6) underwent DMM surgery and were injected
   intra-articularly with saline (equal volume). After Eight weeks of
   surgery, all mice were sacrificed with overdose anesthetics, and sample
   collection and fixation were performed using 4% paraformaldehyde for
   24 h.

Histological evaluation

   Decalcification of the fixed samples was performed for 30 days using
   the EDTA-Decalcifying-fluid, and 5-μm-thick fragments of the right knee
   joint were cut. In addition, hematoxylin and eosin (H&E),
   safranin-o-fast green as well as toluidine blue were used for the
   staining of these fragments ([62]Pan et al., 2022). The expression of
   Aggrecan and Col2 in mouse cartilage was assessed by
   immunohistochemistry. The assessment of the changes in mouse articular
   cartilage was carried out as per the Osteoarthritis Research Society
   International (OARSI) scoring system ([63]Glasson et al., 2010).
   Scoring was done by two independent investigators who have no knowledge
   of the grouping.

Micro-computed tomography

   We used a small animal micro-CT (Scanco Medical AG, Bassersdorf,
   Switzerland) for the purpose of scanning the right knee joint of the
   mice. After obtaining the image, the internal software of the microCT
   system was used for 3D reconstruction. Statistical analysis was then
   performed using the trabecular numbers (Tb.N), trabecular separation
   (Tb.Sp), and bone volume/tissue volume (BV/TV) of the samples.

Statistical analysis

   Independent repetition of each experiment was conducted at least
   thrice, and analysis of data was carried out by employing the GraphPad
   prism V.8 software (GraphPad Inc., La Jolla, CA, United States).
   Additionally, all the data were represented by mean +standard deviation
   (SD), and relevant analyses were conducted using one-way analysis of
   variance (ANOVA) followed by Turkey’s post-hoc test. Statistical
   significance was determined by p ≤ 0.05.

Results

Effect of FKA on cell viability

   We used the CCK 8 kit to determine the impact of various concentrations
   of FKA on the viability of chondrocytes. The molecular structure of FKA
   can be seen in [64]Figure 1A. The illustrations in [65]Figures 1C,D
   reveal that the presence or absence of IL-1β (5 ng/ml) and different
   concentrations of FKA had no effect on cell viability. Based on current
   and previous findings ([66]Kwon et al., 2013), we used 20 μM and 40 μM
   of FKA for subsequent experiments. In addition, Safranin O staining
   showed that the morphology of chondrocytes did not change after FKA
   intervention ([67]Figure 1B).

FIGURE 1.

   [68]FIGURE 1
   [69]Open in a new tab

   Effects of FKA on chondrocyte viability (A) Chemical Structure of FKA.
   (B) Safranin O staining of chondrocyte cells after treatment with IL-1β
   (5 ng/ml) and FKA for a period of 24 h (scale bar 100 μm). Viability of
   chondrocyte cells after 24 h intervention with various concentrations
   of FKA in the absence (C) or presence of IL-1β (D) tested using the
   CCK8 kit. The values are presented as means ± SD (n = 3).

FKA inhibits chondrocyte inflammation and catabolism induced by IL-1β

   The western blot was utilized to assess the impact of FKA on cartilage
   inflammation and metabolism. [70]Figure 2(A–D) illustrates that IL-1β
   elevated the expression levels of chondrocyte inflammatory cytokines
   (COX2 and iNOS), MMPs (MMP3/13), and ADAMTS5, whereas FKA lowered their
   expression levels in a concentration-dependent manner. Additionally,
   FKA also reversed the impacts of IL-1β on the anabolism of
   chondrocytes. The expression of Col2, Aggrecan, and Sox9 in
   chondrocytes showed a significant downward trend under the influence of
   IL-1β, but the effect was reversed under the effect of FKA ([71]Figures
   2E,F). The results of immunofluorescence were consistent with the
   western blot data ([72]Figures 2G–I).

FIGURE 2.

   [73]FIGURE 2
   [74]Open in a new tab

   Protective effects of FKA against IL-1β-induced inflammation and ECM
   destruction in mouse chondrocytes. Chondrocyte cell treatment with
   5 ng/ml of IL-1β only or with FKA (20 and 40 μM) for a period of 24 h.
   Western blot analysis of inflammatory cytokines (COX2 and iNOS),
   catabolic (ADAMTS5, MMP3, and MMP13), and anabolic markers (Col2,
   Aggrecan, and Sox9) (A,C and E). Quantitative analysis of protein
   expression (B,D and F). (G and H) 5 ng/ml of IL-1β was added for
   treating the cells for 24 h in the absence or presence of 40 µM FKA.
   Confocal microscopy showing the expression of MMP13 and Aggrecan in
   immunofluorescence experiments (scale bar 10 μm). (I) Relative
   quantification of fluorescence intensity. The values are presented as
   means ± SD (n = 3). #p < 0.05 vs the control group; *p < 0.05 and **p <
   0.01 vs the IL-1β group.

FKA regulates the expression of senescence-related genes in chondrocytes

   The chondrocyte cells during OA exhibited elevated levels of various
   senescence markers, for instance, β-galactosidase, as well as the
   increased expression of senescence-related genes ([75]McCulloch et al.,
   2017). [76]Figures 3A,B show that the levels of expression of P16 and
   P21 proteins were elevated in chondrocytes under the influence of
   IL-1β, whereas FKA suppressed their expression. Additionally, the
   senescence β-galactosidase staining kit was employed to test the
   β-galactosidase activity in different groups. Under elevated IL-1β
   levels, the activity of β-galactosidase was increased (more blue
   spots), and FKA decreased its activity ([77]Figures 3C,D).

FIGURE 3.

   [78]FIGURE 3
   [79]Open in a new tab

   The regulation of FKA on the expression of senescence-related genes in
   chondrocytes (A) Western blot analysis revealing P16 and P21 expression
   in chondrocytes. (B) Quantitative analysis of protein expression (C).
   β-Galactosidase used for the staining of chondrocyte cells (scale bar
   100 μm) (D) Quantitative analysis of β-galactosidase expression. The
   values are presented as means ± SD (n = 3). #p < 0.05 vs control group;
   *p < 0.05 and **p < 0.01 vs IL-1β group.

FKA suppresses IL-1β-induced apoptosis in mouse chondrocytes

   Chondrocyte apoptosis has a close correlation with the severity of
   cartilage damage in OA. In the current study, we treated chondrocytes
   with FKA in the IL-1β presence or absence for a period of 24 h.
   [80]Figures 4A,B indicate that treatment with IL-1β markedly
   upregulated Bax and downregulated Bcl2 expression in chondrocytes.
   However, FKA alleviated these trends. In order to determine the
   apoptosis status of chondrocytes, we employed the Annexin V-FITC/PI
   Apoptosis Detection kit. [81]Figures 4C,D illustrate that FKA treatment
   led to a remarkable decrease in IL-1β-induced apoptosis of
   chondrocytes.

FIGURE 4.

   [82]FIGURE 4
   [83]Open in a new tab

   Impacts of FKA on IL-1β-induced apoptosis in mouse chondrocytes (A)
   Western blot findings revealing Bax and Bcl2 expression in
   chondrocytes. (B) Quantitative analysis of protein expression. (C) The
   apoptosis rate of chondrocyte cells was detected by flow cytometry. (D)
   The apoptosis rate of chondrocyte cells in all groups were detected
   with Annexin V-FITC/PI staining and flow cytometry. The values are
   presented as means ± SD (n = 3). #p < 0.05 vs control group; *p < 0.05
   and **p < 0.01 vs IL-1β group.

FKA regulates IL-1β-induced suppression of the expression of
autophagy-related genes in chondrocytes

   Numerous pieces of evidence indicate that a crucial role is played by
   autophagy in the progression of OA ([84]Musumeci et al., 2015; [85]Liu
   et al., 2021; [86]Yan et al., 2022). Therefore, we analyzed the impacts
   of FKA on autophagy in IL-1β-treated mouse chondrocyte cells.
   [87]Figures 5A,B reveal that IL-1β treatment remarkably suppressed the
   ATG12, LC3Ⅱ/LC3, and Beclin, expression levels in mouse chondrocytes.
   However, the administration of 40 μM FKA significantly reversed these
   changes. Moreover, RFP-GFP-LC3-adenovirus was employed to further
   verify the impact of FKA on autophagy. We observed that the number of
   autophagolysosomes and autophagosomes decreased in chondrocyte cells
   treated with IL-1β, and FKA pretreatment reversed this trend as
   illustrated in [88]Figure 5E. Furthermore, the autophagy inhibitor,
   3MA, attenuated the promoting effect of FKA on IL-1β-induced
   chondrocyte autophagy ([89]Figures 5C,D).

FIGURE 5.

   [90]FIGURE 5
   [91]Open in a new tab

   FKA regulates IL-1β-induced autophagy-related gene expression in
   chondrocytes (A) Western blot results show Beclin, ATG12, and
   LC3I/LC3II expression in chondrocytes. (B) Quantitative analysis of
   protein expression. (C) Effect of FKA on autophagy in chondrocytes
   after treatment with the autophagy inhibitor 3 MA. (D) Quantitative
   analysis of protein expression. (E) After 5 ng/ml of IL-1β was added
   for treating the cells for 24 h in the absence or presence of 40 µM
   FKA, detection of autophagy in chondrocytes transfected with
   RFP-GFP-LC3-adenovirus was performed (scale bar 25 μm). Autophagosomes
   denoted by the green dots, autophagolysosomes denoted by the red dots.
   The values are presented as means ± SD (n = 3). #p < 0.05 vs control
   group; *p < 0.05 and **p < 0.01 vs IL-1β group.

FKA alters antioxidant gene expression in chondrocytes

   A crucial role is exerted by oxidative stress in the advancement of OA
   ([92]Feng et al., 2019). According to a previous study, FKA alters the
   expression of antioxidant genes in primary splenocytes ([93]Yang et
   al., 2020). Therefore, we assessed whether FKA could alter the
   IL-1β-induced expression of antioxidant genes, including NQO1, Nrf2,
   and HO-1 in chondrocytes. The results in [94]Figures 6A,B illustrate
   that FKA substantially increased the expression of antioxidant genes in
   chondrocytes.

FIGURE 6.

   [95]FIGURE 6
   [96]Open in a new tab

   FKA alters antioxidant gene expression in chondrocytes (A) Western blot
   assay revealing Nrf2, HO-1, and NQO1 expression in chondrocytes. (B)
   Quantitative analysis of protein expression. The values are presented
   as means ± SD (n = 3). #p < 0.05 vs control group; *p < 0.05 and **p <
   0.01 vs IL-1β group.

Results of bioinformatics analysis of FKA

   The identification of potential FKA-target genes was performed using
   the PharmMapper database. The KEGG pathway enrichment analysis revealed
   the enrichment of FKA -target genes in 155 pathways. The four leading
   pathways identified with the most enriched genes were PI3K/Akt,
   proteoglycans in cancer, prostate cancer, and MAPK signaling pathways
   ([97]Figure 7A). In addition, 105 OA-related pathways were identified
   based on the mirWalk2.0 database. We identified 45 common pathways
   related to FKA and OA ([98]Figure 7B). Information on the six KEGG
   pathways with the most enriched genes, including the PI3K/Akt, MAPK
   signaling pathway, proteoglycans in cancer, Chemical carcinogenesis
   -receptor activation, Chemical carcinogenesis -reactive oxygen species,
   and Lipid and atherosclerosis is presented in [99]Figure 7C.
   [100]Figure 7D illustrates the circular diagram revealing the
   FKA-target genes’ chromosomal position and connectivity. Moreover, the
   AKT1 gene also illustrates a superior closeness, degree, and
   betweenness centrality.

FIGURE 7.

   [101]FIGURE 7
   [102]Open in a new tab

   Bioinformatics analysis of FKA pathways and target genes (A) KEGG
   pathway enrichment analysis of FKA target genes. (B) The intersection
   between FKA-related KEGG pathways and OA-related pathways. (C) Top six
   paths in cross paths (KEGG enriched chord diagram). (D) Assessment of
   Centrality and chromosomal investigation of FKA-related central genes.

Effect of FKA on the PI3K/Akt/mTOR and MAPK pathways induced by IL-1β in
mouse chondrocytes

   The PI3K/Akt/mTOR and MAPK pathways exert a significant function in the
   autophagy, inflammation, and progression of OA ([103]Park et al.,
   2018). In this study, treatment of chondrocyte cells was performed with
   IL-1β only or in combination with 20 μM or 40 μM FKA for 30 min, and
   protein expression in chondrocytes was assessed using the western blot
   technique. As shown in ([104]Figure 8), FKA pretreatment partially
   inhibited IL-1β-stimulated phosphorylation of the genes involved in the
   MAPK and PI3K pathways.

FIGURE 8.

   [105]FIGURE 8
   [106]Open in a new tab

   Effect of FKA on the PI3K/Akt/mTOR pathway and MAPK pathway induced by
   IL-1β in mouse chondrocyte cells (A) The expression of proteins of the
   MAPK pathway illustrated by western blot analysis. (B) Quantitative
   analysis of protein expression. (C) Western blot results show the
   expression of proteins of the PI3K/Akt/mTOR pathway. (D) Quantitative
   analysis of protein expression. The values are presented as means ± SD
   (n = 3). #p < 0.05 vs control group; *p < 0.05 and **p < 0.01 vs IL-1β
   group.

Effects of FKA on mouse OA model

   Furthermore, the effects of FKA on OA onset and progression were
   detected in the mouse models. The histological analysis revealed
   remarkably less cartilage damage in the FKA group in comparison to the
   OA model group ([107]Figures 9A,B). Moreover, immunohistochemical
   results revealed that the FKA group showed significantly increased
   expression of Aggrecan and Col2 in comparison to the OA model group
   ([108]Figures 9C–E).

FIGURE 9.

   [109]FIGURE 9
   [110]Open in a new tab

   Impacts of FKA on mouse OA model (A) H&E, safranin, and toluidine blue
   staining of cartilage in the mouse OA model. (B) Osteoarthritis
   Research Society International (OARSI) scoring system (C)
   Immunohistochemistry staining of the expression of Aggrecan and Col2
   were conducted on three groups of cartilage tissues (scale bars, 50 and
   100 μm). (D and E) Quantitative analysis of immunohistochemistry. The
   values are presented as means ± SD (n = 6). #p < 0.05 vs sham group; *p
   < 0.05 vs OA group.

Micro-CT evaluation of the effect of FKA on subchondral bone remodeling in
mouse OA model

   A micro-CT was conducted for the evaluation of the impact of FKA on
   subchondral bone remodeling in the OA models. In [111]Figure 10, we can
   observe that the bone volume fraction (BV/TV) and the trabecular number
   (Tb.N) in the OA model group were evidently increased in comparison to
   the sham group, and the degree of trabecular separation (Tb.Sp) was
   significantly reduced. However, after FKA treatment, these trends were
   significantly reversed. These findings suggest that FKA had a promising
   amelioration impact on subchondral bone remodeling in OA mice.

FIGURE 10.

   [112]FIGURE 10
   [113]Open in a new tab

   Micro-CT evaluation of the impact of FKA on subchondral bone remodeling
   in the mouse OA model. (A) 3D image of mouse OA model generated by
   micro-CT scan. (B) Coronal image views of the right knee from the sham,
   OA, and OA+ FKA group mice. (C) BV/TV, Tb.N, and Tb. Sp of the three
   groups. *p < 0.05 and **p < 0.01 vs sham group (scale bar 1 mm).

Discussion

   As the existing treatments are unsatisfactory, osteoarthritis will
   undoubtedly continue to deteriorate the quality of life of the elderly.
   In recent years, natural plant extracts have attracted a lot of
   attention in the treatment of diseases ([114]Robinson et al., 2016).
   FKA, a chalcone from Piper methysticum Forst extract, has been reported
   to have multiple biological activities. In the research conducted by
   our group, we reported FKA’s therapeutic effects in a mouse model of OA
   and its underlying mechanism of action. Local inflammatory responses
   are crucial in the development of OA as they induce the secretion of
   inflammatory mediators, i.e., TNFα and IL-1β ([115]Dumond et al.,
   2004). For the activation of different inflammatory pathways, for
   instance, the PI3K/Akt and MAPK pathways, IL-1β plays a crucial role.
   Several studies have shown that inhibition of these pathways can
   contribute to the reduction of inflammation and matrix-degrading
   enzymes, and to the improvement of cartilage degeneration in the long
   run ([116]Lianxu et al., 2006; [117]Herrero-Beaumont et al., 2019). We
   observed that the inflammatory factors such as COX2 and iNOS and
   catabolic factors, like ADAMTS5, MMP3, and MMP13 significantly
   increased after IL-1β intervention in chondrocytes, but anabolic
   enzymes, for instance, Col2, Aggrecan, and SOX9 were significantly
   reduced. These trends have been reversed with the addition of FKA in a
   dose dependent manner. Cellular senescence is also an important part of
   OA progression. Senescent cells in joint tissues are thought to be
   responsible for the development of OA ([118]Coryell et al., 2021). Our
   study shows that FKA can substantially suppress the expression of P16
   and P21, senescence cell-related genes, in chondrocytes induced by
   IL-1β. OA is also characterized by enhanced apoptosis in chondrocytes
   ([119]Hwang and Kim, 2015). Therefore, reducing the apoptosis rate of
   chondrocytes is a potential treatment strategy for OA. We found that
   the rate of IL-1β-induced apoptosis can be attenuated by FKA in
   chondrocyte cells. [120]Barranco, (2015) found that the AKT-mTOR
   signaling pathway directly mediates autophagy in chondrocyte cells. The
   onset and progression of OA mediated by excessive production of
   reactive oxygen species (ROS) are countered by autophagy ([121]Charlier
   et al., 2016). Autophagy decreases cellular ROS, resulting in the
   protection of chondrocytes. Therefore, a significant role is played by
   autophagy in the protection of chondrocyte cells from oxidative stress
   ([122]Ansari et al., 2018). Furthermore, the Nrf2/HO-1 signaling
   pathway contributes to the resistance to oxidative stress, and its
   activation can promote the expression of multiple antioxidant factors
   downstream. In our study, it was confirmed that FKA could partially
   reverse IL-1β-induced downregulation of autophagy. The protective
   impact of FKA on IL-1β-induced chondrocyte cells was reduced under the
   influence of 3MA, an autophagy inhibitor. Moreover, FKA also promoted
   the Nrf2/HO-1 signaling pathway expression. Therefore, FKA protects
   against oxidative stress by promoting autophagy and activating
   anti-oxidative pathways. As a result of bioinformatic analysis, we
   identified AKT1 as a potential target of FKA. Previous study confirmed
   that miR-495 induced chondrocyte apoptosis and senescence via directly
   targeting AKT1 and regulating the AKT/mTOR signaling pathway ([123]Zhao
   et al., 2019). It may be possible in future studies to determine
   whether FKA acts as an anti-arthritis agent by targeting AKT1. Also,
   bioinformatic analysis suggested that the PI3K/AKT pathway and MAPK
   pathway could be two potential mechanisms of FKA involved in the
   occurrence and progression of OA. These mechanisms are directly linked
   to the onset and progression of OA ([124]Wu et al., 2021), as well as
   closely related to autophagy. It has been demonstrated in previous
   studies that the PI3K/AKT/mTOR and MAPK pathways inhibition can reduce
   the inflammatory response in OA ([125]Zhou et al., 2019). Our results
   show that FKA can protect chondrocytes by inhibiting the
   phosphorylation of the genes involved in PI3K/AKT/mTOR and MAPK
   pathways induced by IL-1β. In order to validate the therapeutic effects
   of FKA, we performed in vivo experiments. As expected, FKA alleviated
   the OA progression in a mouse OA model.

   In summary, these results emphasize FKA’s potential as a reliable drug
   for treating OA. However, there are some limitations to this study.
   Firstly, detrimental concentrations of FKA on chondrocytes were not
   validated. In addition, the possible effects of FKA on other organs
   (liver and kidney, etc.) were not investigated. In comparison with
   systemic drug delivery, intraarticular delivery offers numerous
   advantages, including the increase in local bioavailability, the
   reduction of adverse events, and the lower cost ([126]Emami et al.,
   2018). In spite of this, intraarticular therapies remain controversial
   in terms of their efficacy. The drug residence time in the joint is
   largely determined by the size of the molecule ([127]Jones et al.,
   2019). The drug residence time of FKA in the articular cavity has not
   been studied in this study, which is another limitation. Moreover,
   ineffectiveness of systemic delivery and low bioavailability of natural
   plant extracts are also factors that have not yet been translated into
   clinical applications. During the past decade, remarkable progress has
   been made in the development of novel drug delivery systems
   (nanoparticles) for encapsulating plant active metabolites. Hence,
   combining FKA and novel nanocarriers may be the key to making it
   applicable in clinical settings, which can be explored in further
   studies ([128]Bharali et al., 2011; [129]Rahman et al., 2020).

Conclusion

   In conclusion, this report is the first to observe that FKA can protect
   chondrocytes by inhibiting the MAPK and PI3K pathways. FKA can promote
   the autophagy of chondrocytes and improve their antioxidant capacity.
   Our findings offer insights into developing novel treatment strategies
   for OA.

Acknowledgments