Arctigenin prevents the progression of osteoarthritis by targeting PI3K/Akt/NF‐κB axis: In vitro and in vivo studies

Abstract Osteoarthritis (OA), which is principally featured by progressive joint metabolic imbalance and subsequent degeneration of articular cartilage, is a common chronic joint disease. Arctigenin (ATG), a dietary phyto‐oestrogen, has been described to have potent anti‐inflammatory effects. Nevertheless, its protective effects on OA have not been clearly established. The target of our following study is to evaluate the protective effects of ATG on IL‐1β–induced human OA chondrocytes and mouse OA model. Our results revealed that the ATG pre‐treatment effectively decreases the level of pro‐inflammatory mediators, such as prostaglandin E2 (PGE2), nitrous oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), IL‐6 and tumour necrosis factor alpha (TNF‐α) in IL‐1β–induced human chondrocytes. In addition, ATG protects against the degradation of extracellular matrix (ECM) under the stimulation of IL‐1β and the possible mechanism might be connected with the inactivation of phosphatidylinositol‐3‐kinase (PI3K)/Akt/nuclear factor‐kappa B (NF‐κB) axis. Furthermore, a powerful binding capacity between ATG and PI3K was also uncovered in our molecular docking research. Meanwhile, ATG may act as a protector on the mouse OA model. Collectively, all these findings suggest that ATG could be utilized as a promising therapeutic agent for the treatment of OA.


| INTRODUC TI ON
Osteoarthritis (OA) is the leading cause of disability worldwide, and in the United States, the number of patients is more than 22.7 million. Most patients have been previously described to experience arthritis-attributable activity restrictions without any valid treatments except total joint replacement in late stage. [1][2][3] Moreover, the incidence and scale of OA is predicted to have been continuously increasing in the following years owing to the ripening of the population, ascending obesity rates and a large amount of traumatic joint injuries. [4][5][6] Its pathological changes are commonly characterized by the progressive cartilage loss, synovial inflammation, subchondral bone remodelling and osteophyte formation. 7 As a further comment, accumulating evidence has shown that inflammatory mediators, particularly interleukin-1 beta (IL-1β), play a significant role in OA progress via promoting the secretion of many pro-inflammatory factors or catabolic enzymes. 8 Meanwhile, some researchers have found that the level of IL-1β is dramatically elevated in the synovial liquid, the articular cavity and the serum of OA individuals. [9][10][11] Likewise, the secretion of cartilage catabolic enzymes could be up-modulated by the stimulation of IL-1β in vitro. Among them, metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) function as the main destroyers of extracellular matrix (ECM). 12 Therefore, strategies aiming at the inhibition of IL-1β itself or IL-1β-induced inflammatory responses hold promise in slowing down the progression of OA.
Prior studies have noted the importance of NF-κB on pathogenesis and progression of OA. 13,14 When under normal situation, NF-κB is situated in the cytoplasm as an inactive form, connected with IκBα, an inhibitory subunit. 14,15 When stimulated by IL-1β, IκBα is phosphorylated by IκB kinases (IKKs) and subsequently degraded in the cytoplasm. Afterwards, NF-κB p65 molecule is separated with IκBα and experiences a translocation from the cytoplasm to the nucleus, which further modulated the translation of related downstream devastative gene, such as iNOS, COX-2, MMPs and ADAMTs. 12,[15][16][17] PGE2, activated by COX-2, as an inflammation-related factors during the progress of OA can highly promote the expression of ADAMTS5 and MMPs, which attribute to the of ECM in cartilage tissue. 18 Moreover, NO, as an inflammatory mediator, belongs to the nitric oxide synthase (NOS) family of enzymes, with which MMP production and many other pro-inflammatory factors are up-regulated. 19 Type II collagen and aggrecan are the major structural constituents of the ECM, 12,20 while MMP13, as a vital part of collagenase family, occupies an extremely significant position aiming type II collagen disintegration, and ADAMTS5, as a key member of secreted zinc proteases, shows dramatically destructive effects of aggrecan synthesis. 21,22 Moreover, a specific molecular repressor in the NF-κB p65 nuclear translocation has been verified to delay the process of OA in the mouse OA models. 23 On the other hand, the PI3K/Akt signalling pathway has been expounded to be involved in the adjustment of NF-κB abnormal activation. [24][25][26][27] Briefly, the phosphorylation of the p85 regulatory subunit of PI3K serves activation of PI3K signalling and triggering AKT phosphorylation, which then binds multiple downstream target proteins such as mTOR, NF-kB, GSK-3beta. Among them, to conclude, NF-kB is the most important hinge in inflammatory diseases. The inhibition of PI3K/Akt/NF-κB axis is expected to be a strategy with high efficiency for attenuating the development of OA.
Although there are already drugs for OA in the market, such as sodium hyaluronate, NASIDs, glucosamine, chondroitin sulphate, most of them were proved to relive the pain of joint, but whether it could truly prevent the progression of OA is still debatable and many side effects were also reported. [28][29][30][31][32][33] Thus, our present study is trying to find a safer agent-derived natural plant, which could truly attenuate OA with little side effects. Arctigenin (ATG), a phenylpropanoid dibenzylbutyrolactone lignan, is pulled from the seed of Arctium lappa L (A lappa), which is commonly known as greater burdock, a kind of edibles worldwide. 34 More and more researchers have subscribed to the view that ATG possesses immeasurable pharmacological value, including antioxidant, neuronal protection, antiviral and anti-inflammatory effects. [35][36][37][38] The anti-inflammatory effect has been confirmed on LPS-induced inflammation models in RAW264.7 cells or human U937 macrophage cells by means of restraining NF-κB, JAK-STAT and MAPK pathway. [39][40][41][42] Additionally, a recent study has exposited that ATG remits LPS-induced acute lung injury in rats. 43 To the most important, the ATG was found to exhibit the ability to protect against rheumatoid arthritis by target Akt/NF-κB. 44 All of these may lead to the conclusion that ATG might own a potential therapeutic effect on the developmental process of OA and there is an imperative to figure out the underlying mechanisms.

| Primary human chondrocyte culture
Afterwards, chondrocytes were seeded in a 25-cm 2 task at a density of 2 × 105 cells/mL in complete DMEM/F12 medium (with 10% FBS and 1% antibiotic) in a 5% CO 2 atmosphere at 37°C. To avoid the phenotype loss of chondrocytes, cells at least in two passages were employed for subsequent experiments.

| Cell viability
The toxicity of ATG towards human OA chondrocytes was determined via the CCK-8 kits obeying the protocols of the manufacturer.

| Nitrous oxide measurement and ELISA
The activity of NO in culture medium was appraised via the Griess reagent as previous descriptions did. 47 According to the manufacturer's introductions, the PGE2, TNF-α, IL-6, collagen II, aggrecan, MMP13 and ADAMTS-5 level in culture medium were assessed using the ELISA kit (R&D Systems). The experiment was performed five times.

| Western blotting
The total protein was gained from cells using the RIPA lysis buffer with 1% phenylmethanesulphonyl fluoride (PMSF) on the ice bath for 10 minutes, and subsequent centrifugation (15 338 g) was at 4°C for 20 minutes. Later, the concentrations of protein were detected by virtue of the BCA protein assay kit. The protein (30 mg) of the respective group was separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, followed by transferring to a polyvinylidene difluoride (PVDF) membrane (Millipore).
After being blocked with 5% BSA for 2 hours, the obtained membranes were then incubated with the primary antibodies: Lamin B

| Real-time PCR
The total RNA from the human chondrocytes was gained through TRIzol reagent (Invitrogen), and the procedures obey the manufacturer's guidelines. The first-strand complementary DNA (cDNA) was synthesized by dint of 1000 ng of total RNA and the QuantiTect Reverse Transcription Kit. As for the quantitative real-time PCR (qPCR), the reactions were acted in a 10 μL system (diluted cDNA  48 The NCBI Primer-Blast tool (https ://www.ncbi.nlm.nih.gov/tools/ primer-blast/ ) was applied to design and form the necessary primers, and the sequences are shown in Table 1.

| Immunofluorescence
The cells were planted in a 25-mm glass in 6-well plate and incubated with serum-starved medium overnight. Subsequently, the cells were solely treated with IL-1β (10 ng/mL) or co-treated with ATG (50 μmol/L) and IL-1β (10 ng/mL) for 24 hours. However, the incubation time of the immunofluorescence staining of p65 with

Gene
Forward primer Reverse primer

| Molecular modelling
The structure of ATG and PI3K, downloaded from Chemicalbook

| Mice OA models
Forty-five eight-week-old C57BL/6 male wild-type mice were ac- The animals were killed by cervical dislocation at 8 weeks after surgery. The whole knee joints were collected for further evaluation.

| Histological assessment
The harvested joint samples were treated as follows: fixation in 4% paraformaldehyde for 1 day and subsequent decalcification in 10% EDTA solution at 4°C for 2 months. Afterwards, the samples were dried using graded ethanol, vitrification by dimethylbenzene and implanting into paraffin slabs. To assess the cartilage tissue degradation, sagittal sequential segments (7 μm thick) of full joints were attained with per joint, which owns 10 slides (70 μm thick) to select.

| Statistical analysis
All tests were achieved independently fivefold. Outcomes were expressed as means ± SD The variances of two or more groups were analysed using (ANOVA) statistical test. Thereafter, Tukey's test analysed the disparities of the treatment and control groups using SPSS version 20.0. Statistical significance was present for values of P < .05.

| Arctigenin did not show toxicity on chondrocyte at the dose of 0-50 μmol/L
The chemical structure of ATG is presented in Figure 1A.

| Arctigenin suppressed the IL-1β and increased PGE2, IL-6, NO, iNOS, TNF-α and COX-2 levels in chondrocytes
The Not surprisingly, however, the adverse effects were reversed by the pre-treatment with ATG in a dose-dependent manner, and the 50 μmol/L of ATG group decreases these levels to 1.5-5.1-fold of control ( Figure 2B,F). Moreover, application of ATG dramatically exhibited an inhibitory effect on the endogenous NO and PGE2 increases in a dose-dependent manner (decrease to around twofold of control), which were up-regulated by around sevenfold of control after IL-1β stimulation ( Figure 2E). The results in this chapter indicate that ATG prominently inhibited inflammatory factor levels in human OA chondrocytes with IL-1β stimulation.

| Arctigenin enhanced ECM synthesis and reduced ECM degradation induced by IL-1β in chondrocytes
Next

| Arctigenin attenuated IL-1β-induced NF-κB activation in chondrocytes
To assess the role ATG played in the NF-κB signalling pathway, we firstly separated the nuclear and cytosol proteins and then utilized Western blotting to examine the protein levels of IκBα in cytoplasm and p65 in nuclei, respectively. As is shown in Figure 4A,B, IL-1β markedly promoted the degradation of IκBα (decrease to 36% of control) in the cytoplasm and more dissociated p65 was phosphorylated (up-regulated to 2.58-fold of control) and be freed to translocate from cytoplasm to nucleus. However, ATG pre-treatment highly inhibited these changes. The IκBα level was recovered to 89% of control, and the p65 level in nucleus was down to 1.13-fold of control in 50 μmol/L of the ATG group. Furthermore, we carried out experiments to measure the nuclear translocation of NF-κB p65 via the immunofluorescence staining of p65 ( Figure 4C). This experiment contained three groups: (a) control group, (b) IL-1β group and (c) IL-1β + ATG (50 μmol/L) group. Making a comparison between experimental groups and control group, the result shows that the p65-active proteins were transferred from the cytoplasm to the nucleus followed by IL-1β stimulation. However, ATG pre-treatment sharply decreases this motion. In brief, the findings from this study provide a new understanding that the pre-treatment of ATG highly inhibits IL-1β-induced NF-κB activation.

| Arctigenin inhibited the PI3K/Akt signalling pathway induced by IL-1β in human OA chondrocytes
The association of upstream axis of NF-κB with ATG was firstly examined by performing docking analysis of ATG with the antagonist binding site of PI3K. 55 According to all returned molecular models, ATG was found to exert wonderful interaction with PI3K ( Figure 5A).

| Arctigenin ameliorated OA development in a mouse DMM model
The therapeutic action of ATG in vivo was also confirmed in DMM model. As illustrated in the image of Safranin O/Fast Green staining, results indicated that the cartilage erosion, hypocellularity and a high proteoglycan loss were clearly presented in the DMM group compared with the sham one ( Figure 6A). Nevertheless, oral administration of ATG (30 mg/kg) could mitigate these detrimental effects. The OARSI scores were also applied for quantitative analysis ( Figure 6B). Altogether, these results indicate that ATG ameliorates OA development in vivo. among which NF-κB is considered as the most significant one. 26 Hence, PI3K/Akt/NF-κB axis can be considered as a potential therapeutic target for treating OA. 26 As the docking analysis shows, we found that ATG strongly binds to the inhibitory pocket of PI3K.

| D ISCUSS I ON
Accordingly, the IL-1β-induced PI3K/Akt phosphorylation was inhibited by ATG intervention, which was in agreement with the previous  39 Thus, the inhibitory effects of ATG on NF-kB activation were followed by inhibition of PI3K/AKT. Although many other signalling molecules might also participate in ATG-induced protective effects in OA, we did not exclude or verify all potential factors one by one in the present study. However, it is our directions in future's works.
To further investigate the protective effect of ATG in vivo, we launched an animal model of OA in the present work. DMM is a highly authentic and efficient method to make an animal OA model for the in vivo analysis. Equally for OA patients, the DMM mice featured with a damaged cartilage surface, hypocellularity and a high proteoglycan loss. However, all the histological changes were attenuated by the pre-treatment of ATG. In addition, compared with the DMM group, the ATG pre-treatment group had lower OARSI scores.
These results above were in accordance with the in vitro work and suggested that ATG was safe and effective in vivo.
In conclusion, this is the first study that demonstrates ATG is a potent natural agent for OA protection via inhibiting the PI3K/Akt/ NF-κB axis and the latent molecule mechanism is displayed specifically in Figure 7. Although no significant improvements were noted in comparison with ATG-treated group with the healthy control at present data, ATG treatment could attenuate the progression of OA in pathological condition both in vitro and in vivo, indicating its potential as a prophylactic treatment option of OA.

CO N FLI C T S O F I NTE R E S T S
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of the study are available from the corresponding author upon request.