Apigenin Inhibits the Progression of Osteoarthritis by Mediating Macrophage Polarization

Objective: The overall purpose of this study was to investigate the mechanism of macrophage polarization on chondrocyte injury in osteoarthritis and the protective effect of apigenin on chondrocytes in osteoarthritis. Method: Primary chondrocytes were isolated from the knee cartilage of three-day-old mice, and cells positive for Alsine blue staining and type II collagen immunocytochemical staining were identified and used in followup experiments. Transwell coculture was performed. Chondrocytes were inoculated in the inferior compartment, and macrophages were inoculated in the upper compartment. The experimental groups were the N group, LPS group, and LPS+ apigenin group. The effect of macrophage polarization on chondrocyte inflammation and the protective effect of apigenin on chondrocytes were verified by the drug administration. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of RNA and protein. Experimental OA was induced by modified Hulth surgery in mice. Modified Hulth surgery was performed on the mouse’s right knee to induce experimental osteoarthritis in mice, with the nonoperative right knee serving as an ipsilateral control. The mice were randomly assigned to three groups (six mice per group): the sham group, the modified Hulth group, and the modified Hulth + apigenin group. Animals were given gavage for four weeks. The protective effect of apigenin on articular cartilage was verified by histological staining and immunohistochemical analysis. Results: Histological staining showed that apigenin had a protective effect on cartilage degeneration induced by modified Hulth surgery. The PCR results showed that apigenin significantly reduced the expression levels of IL-1, IL-6, MMP3, and MMP13 in the articular cartilage of OA mice, and it had a protective effect on articular cartilage. Apigenin reduced the levels of IL-1, IL-6, TNF-α, and IL-12 in macrophages and increased the levels of MG-L1, MG-L2, ARG-1, and IL-10, which can inhibit the M1 polarization of macrophages and promote M2 polarization. In the coculture system, apigenin decreased the protein levels of TRPM7, P-mTOR, BAX, and c-caspase3 in macrophages, while significantly increasing the protein levels of Bcl2. The levels of IL-1, IL-6, MMP13, TNF-α, P38, JNK, and ERK phosphorylation were reduced in chondrocytes. Conclusion: Apigenin alleviates cartilage injury in OA mice induced by modified Hulth. Apigenin inhibits chondrocyte inflammation through the MAPK pathway. Apigenin alleviates macrophage-polarization-induced inflammatory response and chondrocyte apoptosis in the macrophage–chondrocyte coculture system through the TRPM7-mTOR pathway.


Introduction
Osteoarthritis (OA)-a progressive and degenerative disease, and the most common form of arthritis-is a leading cause of musculoskeletal pain, disability, and socioeconomic loss worldwide [1]. The pathogenesis of OA is characterized by extracellular matrix (ECM) damage and chondrocyte death [2]. Chondrocytes preserve the maintenance of articular cartilage by regulating articular cartilage structure and function, as well as ECM turnover [3].

Protective Effect of Apigenin on Articular Cartilage in Modified Hulth Surgically Induced OA Mice
To investigate the protective effect of apigenin on OA, we first established a surgically induced knee OA mouse model. H&E, Safranin-O Fast green, and toluidine blue staining were employed for the histological evaluation ( Figure 1). We observed that, compared with normal mice, OA mice presented serious degradation of the cartilage, obvious proteoglycan loss, and deep cartilage erosion. In contrast, the mice in the modified Hulth + apigenin group exhibited a smoother cartilage surface and reduced loss of proteoglycan. These results demonstrate that apigenin exerts protective effects against modified-Hulth-induced cartilage degeneration. In addition, TUNEL assay results showed more apoptotic cells in the cartilage of OA mice, while cell apoptosis was decreased in the cartilage of mice in the modified Hulth + apigenin group ( Figure 2). + apigenin group exhibited a smoother cartilage surface and reduced loss of proteoglyc These results demonstrate that apigenin exerts protective effects against modified-Hu induced cartilage degeneration. In addition, TUNEL assay results showed more apopto cells in the cartilage of OA mice, while cell apoptosis was decreased in the cartilage mice in the modified Hulth + apigenin group ( Figure 2).  and OA + API groups (magnification, ×40, ×100). Compared with the N group, the OA group exhibited serious degradation of the cartilage, obvious proteoglycan loss, and deep cartilage erosion. In contrast, the OA + API group exhibited a smoother cartilage surface and reduced the loss of proteoglycan.

Apigenin Inhibits Macrophage M1 Polarization in a Macrophage-Chondrocyte Cocu System
CD86 is a surface marker for M1 macrophages. As shown in Figure 4, the fluorescence intensity (MFI) of CD86 increased significantly in the LPS-treated g the macrophage-chondrocyte coculture system. Apigenin was then demonstrated nificantly reduce the levels of CD86 protein expression in the macrophage-chon coculture system. The qRT-PCR result also proved this ( Figure 5A-D). In the macro chondrocyte coculture system, the levels of M1 macrophage polarization markers,

Apigenin Promotes Macrophage M2 Polarization in a Macrophage-Chondrocyte Coculture System
To further explore the effects of apigenin on M2 polarization, RAW264.7 cells were  results demonstrate that apigenin promoted M2 macrophage polarization in the macrophage-chondrocyte coculture system.

Apigenin Inhibits Chondrocyte Inflammation through the MAPK Pathway
In the macrophage-chondrocyte coculture system, macrophage polarization induced by LPS can cause the inflammation of chondrocytes in the lower chamber of the Transwell plate. As shown in Figure 8, the mRNA levels of IL-1 (8E, p < 0.01), IL-6 (8F, p < 0.01), MMP13 (8G, p < 0.01), and TNFα (8H, p < 0.05) of chondrocytes in the lower chamber of the Transwell plate were significantly increased during macrophage polarization by LPS.

Apigenin Inhibits the Apoptosis of Chondrocytes Induced by Macrophage Polarization
In our study, LPS (100 ng/mL) regulated the polarization of macrophages but could not directly induce the apoptosis of primary chondrocytes that were not cocultured with macrophages. However, in the macrophage-chondrocyte coculture system, chondrocyte cell apoptosis was observed in the lower chamber of the Transwell plate ( Figure 9). In the previous result, we found that apigenin could inhibit chondrocyte apoptosis in OA mice.   The Western blot and flow cytometry analyses verified the results of an antiapoptotic effect of apigenin on OA mouse cartilage. As shown in Figure 9, protein levels of Bax ( Figure 9A, p < 0.01) and cleaved-caspase3 ( Figure 9C, p < 0.01) were significantly increased during M1 polarization by LPS in the macrophage-chondrocyte coculture system, while the protein level of Bcl-2 ( Figure 9B, p < 0.01) was significantly decreased. Apigenin decreased the protein levels of Bax ( Figure 9A, p < 0.01) and cleaved-caspase3 ( Figure 9C, p < 0.01) and increased the protein level of Bcl-2 ( Figure 9B, p < 0.05) significantly. In the two-color flow cytometry scatter plot, the Annexin V-FITC is represented on the X axis and the PI is represented on the Y axis. Living cells were double negative (Annexin V-FITC−/PI−), early apoptotic cells were Annexin V-FITCpositive (Annexin V-FITC+/PI−), late apoptotic cells were Annexin V-FITC and PI positive (Annexin V-FITC+/PI+). We can judge apoptosis by the percentage of cell population in the scatter plot. Compared with the N group, the apoptosis of chondrocytes increased after IL-1β induction, and apigenin reduced the apoptosis of chondrocytes.

Discussion
OA is a degenerative disease of articular cartilage with high incidence, affecting more than 500 million people worldwide (~7% of the global population), and is induced by various factors in the elderly (>65 years of age) [20]. OA not only affects the articular cartilage but also involves pathological changes across all the joint tissues, including subchondral bone, ligaments, capsules, synovium, and periarticular muscles, as a result of a combination of risk factors [21]. Multiple studies have documented that low-grade synovial inflammation (synovitis) contributes to cartilage degeneration and pain progression in OA, although the etiology of OA is complicated [20,22,23]. During the process of OA, macrophages can become activated M1 macrophages that can release inflammatory mediators and MMPs [23].
Recent studies have shown that a variety of flavonoids can act on osteoarthritis and delay its progression. Licochalcone A (Lico A) inhibits the NLRP1 inflammatorome through the nuclear factor erythropoie-3 associated factor 2 (Nrf2)-heme oxygenase-2 (HO-1)-nuclear factor κ-B (NF-κB) axis. Nrf2 small interfering RNA (siRNA) can reverse the antipyroptosis effect of Lico A in mouse OA chondrocytes and has a potential therapeutic effect on OA [24]. Quercetin inhibits ER stress by activating the sirtuin1-adenosine monophosphate activated protein kinase (SIRT1-AMPK) signaling pathway. Protective effects of quercetin were also observed in rat models of OA [25]. Luteolin decreased the IL-1β-induced production of NO, PGE2, TNF-α, MMP-2, MMP-8, and MMP-9 and the expression of COX-2, iNOS, MMP-1, MMP-3, and MMP-13, and it reversed the IL-1β-induced degradation of collagen II [26]. Tangeretin can eliminate OA progression by inhibiting inflammation and ECM degradation in chondrocytes and animal models through the Nrf2-NF-κB and MAPK-NF-κB pathways [27]. Icariin has a strong inhibitory effect on proinflammatory signaling, such as NF-κB and MAPK, and can also upregulate anti-inflammatory signaling, such as GR and Nrf2 [28]. Fisetin is available for orthopedic biologic therapy as an adjunct to orthopedic surgery to improve clinical outcomes [29].
In this study, apigenin protected chondrocytes by repressing M1 macrophage polarization and promoting M2 macrophage polarization. Transient receptor potentioid melatonin seven (TRPM7) is a nonselective Ca 2+ conducting ion channel that plays an important role in lipopolysaccharide (LPS)-induced activation of mouse macrophages [30]. mTOR plays an important role in the regulation of the polarization of macrophages. Previous studies have confirmed that the activation of mTORC1 can inhibit autophagy and promote the apoptosis of articular chondrocytes during OA development [31]. Apigenin can attenuate inflammatory responses in a macrophage-chondrocyte coculture system induced by macrophage polarization through the TRPM7-mTOR pathway. In this study, we demonstrated the anti-inflammatory and antiapoptotic effects of apigenin in a macrophage-chondrocyte coculture system. Mitogen-activated protein kinases (MAPKs) are crucial regulators of cellular pathology and physiology, playing a crucial role in chondrogenic differentiation, including JNK, ERK, and p38 [32]. We found that apigenin inhibited chondrocyte apoptosis and inflammation through the MAPK pathway. Apigenin is a well-known nutraceutical compound that has been shown to have effects on diabetes, amnesia and Alzheimer's disease, depression, insomnia, cancer, and female diseases [12,33]. It has also been demonstrated that apigenin modulates bone formation by regulating AnxA6 and TNAP [34]. We demonstrated herein that the chondroprotective effect of apigenin is mainly ascribed to its regulatory effects on macrophage polarization. There is increasing evidence that synovial inflammation plays a major role in the progression of osteoarthritis. Therefore, alleviating synovial inflammation may be an effective method of treating patients with osteoarthritis [35,36].

Cell Culture of Chondrocytes and Macrophages
The isolation and identification of primary chondrocytes by Alcian blue staining and immunocytochemical staining of type II collagen from mice were performed as previously described [37]. In brief, the primary chondrocytes used for experiments were prepared from the knee joint cartilage of newborn mice. After digestion by trypsinase and collagenase II, sequentially, the cells were maintained in DMEM/F12 1:1 + 10% fetal bovine serum. The cells identified as positive for Alcian blue staining and immunocytochemical staining of type II collagen were used in subsequent experiments. RAW264.7 was purchased from the Shanghai Institute of Cell Biology (Shanghai, China). High-glucose DMEM containing 10% FBS was used for RAW264.7 cell culturing. The cells were incubated in a 37 • C incubator with 5% CO 2 [38].
Transwell coculture systems were performed. Chondrocytes were seeded onto 12-well culture plates at 1 × 10 6 cells per 1 mL. After overnight seeding, the cells were washed 3 times with PBS, and RAW cells were directly added to the chondrocytes. The control group received serum-free medium only. After apigenin treatment, the supernatant and cells were collected separately for further analyses [39]. M1 macrophages were polarized by the addition of LPS (100 ng/mL) and IFN-γ (50 ng/mL) for 24 h, and M2 macrophages were obtained by the addition of IL-4 (20 ng/mL) for 24 h [40]. The cells of the drugtreated group were treated with apigenin (10 µM). Then, the polarization transitions were evaluated via q-PCR, Western blot, and immunohistochemical staining.

Western Blot Analysis
Protein expression was measured via Western blot analysis. In brief, cells were washed with PBS and lysed using a RIPA buffer with 1% phenylmethanesulfonyl fluoride (PMSF); then, the protein concentration of cell lysates was quantified using the BCA method (Beyotime, Shanghai, China). Subsequently, 20 µg amounts of protein were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 10% gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. After transfer, membranes were blocked with 5% bovine serum album in Tris-buffered saline at room temperature before incubation overnight with primary antibodies in a blocking buffer at 4 • C. The excess primary antibody was removed by washing with Tris-buffered Saline and 0.1% Tween-20 (TBST) three times for 5 min, and the membranes were subsequently incubated with secondary antibodies at room temperature for 1 h. After rinsing with TBST, the band density was quantified using ImageJ software.

qRT-PCR
Macrophages were inoculated in the upper compartment and chondrocytes were inoculated in the lower compartment of the Transwell plate. The cells were cultured for another 48 h after administration before they were harvested. The methods and parameters of the qPCR assay were employed as previously described. Total RNA was extracted from collected cells at the end of the treatment period using Trizol reagent (cat#: 9109; Invitrogen, Carlsbad, CA, USA) and was reverse transcribed into cDNA using the Prime-Script™ RT Reagent Kit (cat#: RR037A; TAKARA BIO INC, Shiga, Japan) according to the manufacturer's instructions. The primer sequences used for qPCR are listed in Table 1. The cycling conditions were 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 1 min. The relative graphs and statistical analyses of transcript quantities were calculated using the 2 −∆∆Ct method with GAPDH as the endogenous reference gene amplified from the samples. The q-PCR analysis was carried out 3 times.   R:TCC AAC CCG AGG AAC TTC TG  MMP13  F:CAG TGC TGC GGT TCA CTT TG  R:TCA TCA TAA CTC CAC ACG TGG TT

Immunofluorescent Staining
Macrophages were stained for immunofluorescence on coverslips according to the standard protocol. Following removal of the supernatant, cells were fixed in a fixation/permeabilization buffer for 30 min at 4 • C. Next, the cells were incubated with primary antibodies against CD86 or CD206 overnight at 4 • C. After that, the cells were rinsed three times in PBS, then incubated with FITC-conjugated secondary antibodies. The coverslips of the three groups were counterstained with DAPI, washed three times in PBS, and imaged under a fluorescent microscope [41].

Flow Cytometry
The apoptotic degree of chondrocytes in each group was measured by flow cytometry using the Annexin V-FITC/PI apoptosis detection kit according to the manufacturer's instructions (cat#: A211-01; Vazyme, Nanjing, China). Flow cytometry analysis was performed using the NovoExpress (Agilent Technologies, Inc., Nanjing, China).

OA Protocol
The animal experimental operations were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University (202209S020). Modified Hulth surgery was performed in mice to induce experimental OA. C57BL/6J mice (8 weeks of age) were used for this study. In the treatment study, modified Hulth surgery in the right knee joints was performed in mice to induce experimental OA according to previous studies. The nonoperated right knees were used as ipsilateral controls. OA was surgically induced by modified Hulth (the surgical modified Hulth model of osteoarthritis in the 129/SvEv mouse). Mice were divided into 3 groups (6 mice per group) by random assignment: sham, modified Hulth, and modified Hulth + apigenin. Animals were gavaged daily for 4 weeks. Then, the mice were sacrificed to evaluate OA severity and joint inflammation.

Histological and Immunohistochemical Analyses
The femoral cartilage tissue from mice was fixed in 10% formalin, decalcified, and embedded in paraffin. Five-micrometer serial sections were obtained and stained with hematoxylin and eosin (H&E), Safranin-O Fast green, and toluidine blue. In order to detect the apoptosis of chondrocytes, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed using a TUNEL assay kit according to the manufacturer's instructions (Beyotime, Shanghai, China). Staining was visualized by fluorescence microscopy.

Statistical Analyses
Statistical analyses were performed using SPSS software, and results are presented as the mean ± SD. Graphs were drawn using GraphPad Prism (version 6.0 for Windows). Every experiment was repeated at least three times. A p-value of 0.05 or less was considered statistically significant.

Conclusions
Apigenin could ameliorate modified Hulth-induced cartilage damage in OA mice. Apigenin attenuated inflammatory responses and alleviated chondrocyte apoptosis induced by macrophage polarization in a macrophage-chondrocyte coculture system through the TRPM7-mTOR pathway. These results suggest a potential therapeutic treatment strategy involving apigenin for OA.

Institutional Review Board Statement:
The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Institutional Animal Care and Use Committee of Xuzhou Medical University (202209S020).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.