Decursin alleviates the aggravation of osteoarthritis via inhibiting PI3K-Akt and NF-kB signal pathway

Linjie He a, b, Yinan Pan a, b, Jiapei Yu a, b, Ben Wang a, b, Gaole Dai a, b, Xiaozhou Ying a,*
a Department of Orthopedics Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
b The Second School of Medicine, Wenzhou Medical University, Wenzhou, Zhejiang Province, China

A B S T R A C T

Osteoarthritis (OA) is a common joint disease that takes joint degeneration or aging as its pathological basis, and joint swelling, pain or dysfunction as its main clinical manifestations. Decursin (DE), the major active component isolated from Angelica gigas Nakai, has been demonstrated to possess anti-inflammatory effect in many diseases. But, the specific physiological mechanism of DE on OA is not clear yet. Therefore, the object of this study was to assess the therapeutic effect of DE on OA, and to explore its potential anti-inflammatory mechanisms. In vitro cell experiments, the inflammatory response in chondrocytes is mediated via interleukin-1β (IL-1β), which led to abnormal secretion of pro-inflammatory factors, such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), cyclooXygenase-2 (COX-2), nitric oXide (NO) and inducible nitric oXide synthase (iNOS). These cytokines were all decreased by the preconditioning of DE in a dose-dependent form of 1, 5, and 10 µM. Moreover, DE could restrain IL-1β-mediated inflammatory reaction and the collapse of extracellular matriX (ECM) via reducing the secretion of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and MMPs (matriX metalloproteinases). In short, DE restrained IL-1β-mediated abnormal excitation of PI3K/AKT/NF-κB axis. Furthermore, molecular docking analysis showed that DE has a strong binding affinity with the inhibitory targets of PI3K. In vivo animal studies, DE treatment could helped to improve destruction of articular cartilage and decreased the serum inflammatory factor levels in an operationally induced mouse OA model. To sum up, these data obtained from the experiment indicate that DE has good prospects for the treatment of osteoarthritis.

Keywords: Decursin Osteoarthritis
Inflammatory NF-κB
PI3K
Chondrocyte

1. Introduction

As the most common musculoskeletal disease so far, Osteoarthritis (OA) can cause joint pain, dysfunction and other clinical symptoms, and seriously affect people’s quality of life [1]. With the aging of the world population, the number of patients with osteoarthritis presents a rapid upward trend [2]. And known risk factors for OA include aging, obesity, inflammation, trauma, and genetics [3,4]. Numerous studies have shown that the occurrence and development of OA are closely related to many inflammatory cytokines [5]. Among them, IL-1β is one of the most critical factors in the inflammatory process of Osteoarthritis, which can stimulate the overexpression of associated inflammatory factors and catabolic enzymes such as NO, PGE2, ADAMTS, and MMPs that ultimately lead to the collapse of the ECM [6–8]. Moreover, there are re- ports that abnormal elevation of IL-6, IL-1 and TNF-α have been detected in synovial membrane, the synovial fluid and cartilage of OA patients, confirming their important roles in the pathogenesis of Osteoarthritis [9]. Obviously, blocking IL-1β-mediated inflammation is a promising approach for the treatment of osteoarthritis.
As everyone knows, nuclear factor kappa B (NF-κB) is a dimer transcription factor that coordinates diverse physiological and patho- logical processes, peculiarly the development of inflammation [10]. To date, a number of studies have demonstrated that pro-inflammatory cytokine stimulate the NF-κB signaling pathway via binding to corre- sponding receptor subunits, provoking a signaling cascade that leads to p65 phosphorylation and succeeding nuclear translocation [11–13]. Simultaneously, the omnipresent PI3K/AKT pathway is involved in apoptosis, proliferation and differentiation via managing a wide range of target proteins including NF-κB [14]. The phosphorylation of p65 and IκBα lead to NF-κB translocated into the nucleus and then binding to the specific sequences in the promoter regions of target genes encoding pro- inflammatory mediators such as PGE2, COX-2, MMPs, IL-6 and so on [15]. Hence, PI3K/Akt/NF-κB pathways is considered as a key target for the treatment of OA.
Angelica gigas Nakai is a traditional herb used to treat anemia, to promote blood flow, and is used as a tonic agent [16]. Decursin (DE) is a pyranocoumarin compound extracted from Angelica gigas Nakai. So far, several studies have shown that DE has many valuable biological ac- tivities, such as anti-inflammatory [17], anticancer [18,19], and anti- oXidant [20]. Previous studies demonstrated that DE exerts protective effects against neuroinflammation stimulated by amyloid-β in PC12 cells, which has something to do with the inhibition of NF-κB pathway [21]. Meanwhile, it was also reported that DE could restrain the secre- tion of inflammatory factors via restraining the excitation of PI3K/NF-κB pathway in HT1080 cells [22]. Besides, DE also remarkably restrained the produce of pro-inflammatory mediator and MMPs in cancer cells and suppressed the metastasis of cancer cells via blocking the activation of PI3K, ERK and NF-κB [23]. Athough the pharmacological properties of DE have been extensively studied, the anti-inflammatory function of DE in OA are still unclear. Thereby, we explored the possible mechanism of DE’s anti-inflammatory effect on mice chondrocytes after IL-1β induc- tion treatment, assessed the phylactic effect of DE on mouse OA model, and used the affinity of DE in molecular docking model to illustrate its potential anti-inflammatory mechanism.

2. Materials and methods

2.1. Reagents

Decursin (purity ≥ 99%) was obtained from MedChemEXpress (NJ, USA). Type II collagenase, recombinant human IL-1β and dime- thylsulfoXide were purchased from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies against p65, GADPH, Lamin B1 and COX-2 were obtained from Abcam (Cambridge, UK); iNOS antibodies was obtained from Sigma-Aldrich (St Louis, MO, USA); IκBα, P-PI3K,P-Akt,PI3K and Akt were acquired from Cell Signaling Technology (Danvers, MA,USA); Goat anti-rabbit and anti-mouse IgG-HRP were obtained from Boster (Wuhan, China). Alexa Fluor®488-labeled and Alexa Fluor®594-labeled goat anti-rabbit IgG (H L) second antibody was purchased from Jackson ImmunoReshearch (West Grove, PA, USA). The cell culture reagents were obtained from Gibco (Life Technologies Corp. Carlsbad, Calif, USA).

2.2. Primary mice chondrocyte isolation and culture

Ten C57BL/6 male mice under 2 weeks old obtained from the Animal Center of the Chinese Academy of Sciences (Shanghai, China), which were sacrificed with 10% chloral hydrate. Knee surgery is performed in a sterile environment to separate sterile articular cartilage and then cut the cartilage into fragments. The cartilage fragments were treated with 0.2% type II collagenase for 4–6 h and cleaned with PBS for 3 times.
Chondrocytes were acquired via centrifugation at 1000 rpm for 3 min and then cultured in a concentration of 1 105cells/ml on a SiX-well plate containing a miXture of DMEM/F-12 (with 10% fetal bovine serum and 1% antibiotic miXture of streptomycin and penicillin) and incubated with 5% atmospheric CO2 at 37 ◦C. The culture medium was changed every 1–2 days, and 0.25% trypsin – EDTA solution was used for cell passage. The second generation chondrocytes were selected in subsequent experiments to eliminate phenotypic loss.

2.3. Animal models

The male C57BL/6 wild-type (WT) mice aged ten weeks were ob- tained from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). As previously mentioned, the OA model was ob- tained via the surgical of destabilization of the medial meniscus (DMM) [24]. The general procedure was as follows: the mice were first anes- thetized after intraperitoneal injection of 2% (W/V) pentobarbital (40 mg/kg) and subsequent surgery was performed; then we used micro- surgical scissors to cut the connection of the medial meniscus to the tibial plateau. Intraoperative attention should be paid to the identifi- cation and protection of the lateral meniscotibial ligament. In the sham group, only the right knee arthrotomy was performed, excluding the severing of the medial meniscus tibial ligament. In short, the mice were divided equally into three groups based on random distribution: sham group, vehicle group and DE treatment group.

2.4. The design of experiments

In vitro experiments, in order to evaluate the anti-inflammatory outcomes of different doses of DE (1, 5, and 10 µM), cells were given with 10 ng/ml IL-1β alone or in association with DE pretreatment. The control group simply renewed the medium without treatment. In vivo experiments, the medial meniscus was surgically removed in the mice according to the above methods. After DMM surgery, the DE treatment groups were given intragastric injection of DE 20 mg kg—1day—1 for eight consecutive weeks. In the meantime, DMM group mice were given a same amount of normal saline. Ultimately, all animals were sacrificed eight weeks after the surgery, and the samples of cartilage tissue were taken for iconographic and histological analysis.

2.5. Cell viability and cytoxicity assays

The Cell counting kit-8 (Dojindo Co, Kumamoto, Japan) was used to measure the Cell viability. To put it simply, the mice chondrocytes were seeded into wells of a 96-well plates at a density of 5X103 cells per well, and cultured for 24 h under the condition of 37 ◦C, 5% CO2. Chondrocytes were then pretreated with DE at varioust concentrations (0, 0.1, 1, 5, 10 or 30 µM) for 24 h or 48 h. After that, the cells were cleaned with phosphate buffered saline (PBS), and 100ul medium containing 10% Cell counting kit-8 (CCK-8) solution was added to each well, and incubated for 4 h at the condition of 37 ◦C, 5% CO2. Then microplate spectrophotometer (Leica Microsystems, Germany) was used to detect the absorbance of the well at a wavelength of 450 nm. All experiments were carried out five times.

2.6. Griess reaction and ELISA

As mentioned earlier, the NO in the medium was determined with Griess reactant [25]. We relied on the ELISA kits (R&D Systems, Min- neapolis, MN) to detect the concentrations of TNF-α, PGE2, collagen II (Col II), IL-6, ADAMTS-5, aggrecan, MMP3, MMP9 and MMP13 in the supernatant. Moreover, the level of TNF-α, IL-1β and IL-6 in the plasma of mice were also detected with ELISA kits. All assays were conducted five times.

2.7. Western blotting analysis and protein extraction

The entire protein was abstracted from chondrocytes via a miXture of RIPA lysis buffer and PMSF (100:1). The protein concentration of each group were measured via the BCA protein assay kit (Beyotime). Equal quantities of 40 μg of total protein were divided via SDS-PAGE and transferred to PVDF membrane. The membrane was then sealed by 5% nonfat milk for 2 h. After three washes with TBST (TBS containing 0.1% Tween-20), the membranes were incubated with the primary antibody against IκBα(1:1,000), p65(1:1,000), COX-2(1:1,000), iNOS(1:1,000), PI3K(1:1,000), AKT(1:1,000), P-AKT(1:1,000), P-PI3K(1:1,000), GADPH(1:5,000) and Lamin-B(1:5,000) at 4 ◦C overnight. The mem- branes were cleaned three times with TBST for min and then incubated with corresponding secondary antibody (1:3000) for 2 h at room tem- perature. Ultimately, cleaning the membrane for three times using TBST, the membrane’s signals were tested via Image Lab 3.0 software (Bio-Rad Laboratories Inc., Hercules, CA, USA).

2.8. Immunofluorescence microscopy

For MMP-13 and collagen II staining, mice chondrocytes were seeded on a glass coverslips of a 6-well plate (3X105 cells/ml) and then treated with 10 ng/ml IL-1β alone or co-treated with 10 μM DE for 24 h in medium. For p65 staining, the time of IL-1β and DE treatment was only to 2 h. After removing the supernatant, the coverslips with a monolayer of chondrocytes were cleaned with PBS for 3 times and then fiXed via 4% paraformaldehyde for 15 min under the condition of room temperature. Then the samples were treated via 0.5% Triton X-100 for 15 min. After cleaning with PBS, cells were overlaid with 5% goat serum for 1 h at 37 ◦C. The cell samples were again cleaned via PBS and then incubated with primary antibodies against p65 (1: 200), Type II collagen (1: 200), and MMP-13 (1: 200) under the condition of 4 ◦C all-night. The following day, the coverslips were cleaned thrice and incubated via Alexa Fluor®594, Alexa Fluor®488 labeled conjugated secondary anti- bodies (1:200) for 45 min under the condition of room temperature, and then labeled via DAPI for 10 min. Ultimately, images of the cells were obtained via the fluorescence microscope (Olympus Inc., Tokyo, Japan). And then Image J Software 2.1 (Bethesda, MDUSA) was used to detect the fluorescence intensity.

2.9. Molecular modeling

Firstly, we mapped the molecular structure of DE using ChemBio- Draw and used ChemBio3D to minimize its energy. According to the needs of current molecular docking experiments, we downloaded the corresponding PI3K (PDB code 5IS5) [26] from the PDB website (htt ps://www.rcsb.org/). After being treated via PyMoL (version 1.7.6), the lowest energy conformations for docking were decided by default parameters. And the molecular structure of DE and PI3K required for docking analysis is ready, subsequent molecular docking shall be star- ted. The docking analysis was performed by the AutoDockTools (version 1.5.6). The eventual pictures of 3D views were performed via the PyMoL.

2.10. X-ray imaging

After 8 weeks of the surgery, the three groups of mice were subjected to the X-ray checkup. All mice were x-rayed via a digital X-ray machine (Kubtec Model XPERT.8; KUB Technologies Inc.) to evaluate the space of articular cavity and the degree of osteophyte formation and the calci- fication of cartilage surface. And the above indicators are devoted to the degree of articular cartilage degeneration.

2.11. Histopathologic analysis

First, the obtained knee specimens were soaked in 4% para- formaldehyde and placed at 4 ◦C for 24 h for fiXation. Then 10% EDTA was used for decalcification at 37 ◦C for 4 weeks. The paraffin-embedded specimens were cut into 5 μm thick sections, which were used for subsequent Safranin O-fast Green (S-O) staining to evaluate the extent of cartilage damage. The destruction of joint cartilage is assessed via the Osteoarthritis Research Society International (OARSI) scoring system for the medial femoral condyle and medial tibial plateau [27]. We used the OARSI score (0–12) to represent the extent of articular cartilage damage. Besides, the explanation of subchondral bone thickness has been illus- trated in previous studies [28,29].

2.12. Statistical analysis

All the studies were carried out independently five times. The experimental results were presented as mean standard deviation (SD). Statistical analysis of all experimental data was performed using SPSS 20.0 statistical software. Comparisons between different groups were analyzed via ANOVA followed by Tukey’s test. And the P value below 0.05 was deemed statistically significant.

3. Results

3.1. Effect of DE on mice chondrocyte viability

The Fig. 1 A exhibit the molecular structure of DE. The cytotoXicity of DE on chondrocyte was measured via a CCK-8 kit. Briefly, chondrocytes were cultured at elevated DE (0, 0.1, 1, 5, 10, and 30 μM) concentrations for 24 or 48 h, and then CCK-8 analysis was performed. See Fig. 1 B and C, the results of CCK-8 analysis displayed that DE had the most ideal effect on cell viability at a concentration of 10 μM. Consequently, we choose 1, 5, and 10 μM DE to continue the follow-up experiment.

3.2. Effect of DE on IL-1β-mediated secretion of iNOS, COX-2, PGE2, NO, TNF-α and IL-6 in mice chondrocyte

To determine whether DE suppresses IL-1β-mediated inflammation in mice chondrocytes, we measured several inflammatory cytokines. The iNOS and COX-2 protein expression levels were evaluated via Western blot analysis. By observing A and B in Fig. 2, we know that when the concentration of DE is 5 and 10 μM, it can well reduce the effect of IL-1β stimulation on INOS and COX excessive expression. However, there was no statistical difference between the 1 μM DE treatment group and IL-1β single treatment group. Under the stimulation of IL-1β, the secretion levels of PGE and NO also increased abnormally. Interestingly, the overexpression of PGE2 and NO decreased in a dose- dependent form after pretreatment with different concentrations of DE (Fig. 2C). The suppression of DE on TNF and IL-6 expression also showed a dose-dependent pattern (Fig. 2D). And the difference was statistically significant when the concentration was 1, 5 and 10 μM.

3.3. Effects of DE on extracellular matrix (ECM) of the mice chondrocytes

The main component of cartilage extracellular matriX (ECM) is collagen II (Col II) and aggrecan. And ADAMTS and MMPs are related to the degradation of ECM. By observing Fig. 3 A, we conclude that DE can promote the secretion of aggrecan and Col II and suppress the protein levels of ADAMTS-5, MMP-3, MMP-9 and MMP-13 in a dose-dependent form. In addition, DE activates the synthesis of Col II and inhibits the secretion of MMP13, which has been verified in immunofluorescence staining (Fig. 3B,C, D, E). In short, these experimental data show that DE is able to reduce the collapse of ECM in mice OA chondrocytes.

3.4. Effects of DE on IL-1β-mediated NF-κB excitation and nuclear translocation in mice chondrocytes

The activation of NF-κB pathway is essential in the inflammatory response. We relied on Western Blot and immunofluorescence to assess the protein levels of IκBα and p65 to further explore the potential anti- inflammatory mechanisms of DE. Under the induction of IL-1β, IκBα was going to degradation and p65 underwent nuclear translocation. Nevertheless, these disadvantages were reversed by DE pretreatment (Fig. 4A, B). The nuclear translocation of p65 in chondrocytes in each group was evaluated by immunofluorescence staining. The experimental design of immunofluorescence staining is as follows: (a) control group, (b) IL-1β stimulation group, and (c) IL-1β DE (10 μM) treatment group.
Apparently, in the control group, the p65 was normally distributed in the cytoplasm. But in the IL-1β stimulation group, a large number of p65 active proteins were found to be transferred from cytoplasm to nucleus. Meanwhile, in the IL DE group, the translocation of p65 in chon- drocytes pretreated with DE was significantly reduced (Fig. 4C). The research results showed that DE has the ability to block the nuclear translocation of p65 mediated via IL-1β in chondrocytes.

3.5. Effect of DE on IL-1β-mediated PI3K and Akt phosphorylation in mice chondrocytes

In fact, many studies have shown that the PI3K/Akt pathway is an significant signaling pathway in the beginning of the inflammatory re- action mediated via IL-1β. Consequently, in order to farther study the anti-inflammatory mechanism of DE in mice OA chondrocytes, we detected the phosphorylation levels of PI3K and AKT with Western blot analysis. Pretreatment of OA chondrocytes with DE significantly sup- pressed IL-1β-mediated PI3K and Akt phosphorylation in a dose- dependent form (1, 5, and 10 μM), as is shown in Fig. 5(A,B). In addition, the above results were statistically significant (P < 0.05). Alto- gether, the results suggested that DE substantially restrained IL-1β- mediated PI3K and AKT phosphorylation at 10 μM.

3.6. Molecular docking

Molecular docking studies were conducted to determine whether DE interacts directly with the corresponding upstream proteins in the PI3K/ Akt/NF-κB pathway. The molecular structure of DE was docked with the binding bag of antagonist in PI3K [26]. We discovered that DE was able to dock well at the binding site of PI3K and formed some stable connections via observing all the models returned. In the picture, macro and local perspectives of the mutual effect between PI3K protein resi- dues and DE are represented by the Ribbon model. At the same time, the space filling model intuitively displays the spatial position of DE binding on PI3K (Fig. 6). Through molecular docking study and analysis, we discovered that there are some important hydrogen bonds between the molecular structure of DE and the amino acid residue of PI3K (GLU- 826), with an affinity of 7.2 kcal /mol. All figures were obtained via the PyMOL software.

3.7. DE ameliorates the progression of OA in the mouse model

We evaluated the inhibitory effect of DE on the development of osteoarthritis in vivo through animal experiments. The mouse model of osteoarthritis was built via DMM surgery, and 20 mg/kg DE was administered daily by gavage for eight weeks. In terms of X-rays, by comparing with the sham group, we found that the OA group had obvious joint space stenosis, severe osteophyte formation and dense subchondral bone plates. Surprisingly, compared with the OA group, it was found that osteophytes and calcification were significantly reduced in the DE treatment group, in spite of the joint space remained narrow (Fig. 7A). The severity of OA mice was assessed via Safranin O staining. And we used OARSI scoring system to conduct quantitative analysis of OA stained tissue. After roughly observing the Safranin O staining of

4. Discussion

Osteoarthritis (OA) is essentially a degenerative disease character- ized by inflammatory changes in synovium, wear and tear of articular cartilage, increased subchondral bone density, and osteophyte forma- tion [30]. With the increasing aging of the population, the prevalence of knee osteoarthritis presents an obvious rising trend, which has brought great burden to public health [31]. The cause of arthritis is not fully understood and there are no effective preventive treatments [32,33]. Though non-steroidal anti-inflammatory drugs are diffusely applied clinically to treat OA, these drugs only temporarily relieve clinical symptoms and cause some significant side effects [34,35]. Therefore, there is an urgent need to explore mild drugs with specific molecular targets to inhibit the cartilage degradation of OA.
As a traditional herbal medicine, Angelica gigas Nakai can improve atopic dermatitis by blocking the activation of MAPKs and NF-κB pathways to inhibit the secretion of inflammatory factors [36]. Decursin (DE), an active pyranocoumarin compound obtained from the roots of Angelica gigas. DE can significantly induce expression of PKR, ATF4, and CHOP and synergize with doXorubicin-induced apoptosis in HeLa cells [37]. In addition, DE is able to promote dorsal hair growth in mouse by inhibiting the expression of pro-inflammatory cytokines and enhancing the expression of anti-inflammatory cytokines [38]. Although many studies have shown that DE exhibits anticancer and anti- inflammatory effects in a variety of diseases, the accurate mechanism of its potential inhibitory effect on OA remains unclear. Thus, we con- ducted a study of the anti-inflammatory mechanism of DE in osteoarthritis.
More and more studies have shown that NF-κB pathway serves an important role in the inflammatory response, which has something to do with the occurrence and development of OA. In the normal state, the NF- κB dimers are retained in the cytoplasm via interaction with IκBα. Fol- lowing IL-1β stimulation, the phosphorylated IκBα and then degraded by the proteasome, which leads to the phosphorylation of p65 and the subsequent NF-κB complexes translocating to the cell nucleus [39,40]. NF-κB binds to the corresponding response elements in the nucleus and activates the expression of pro-inflammatory cytokines, including COX- 2, iNOS, NO, PGE2, IL-6, TNF-α, MMPs and ADAMTS [41,42]. In the midst of these overexpression cytokines, iNOS is able to catalyze NO to stimulate the secretion of MMPs and inhibit the synthesis of type II collagen and proteoglycan, which eventually result in ECM degradation [43]. And the overexpressed COX-2 is a critical enzyme of PGE2 syn- thesis. PGE2 is a pro-inflammatory factor that affects articular cartilage degeneration via increasing MMPs and ADAMTS5 produce [44].
It is certain that these inflammatory factors play a critical role in the progression of osteoarthritis. By analyzing the study data, we found that DE suppressed the overproduction of COX-2, iNOS, PGE2, NO, TNF and IL-6 at the protein levels. Furthermore, we found that DE suppressed the overexpression of ADAMTS-5 and MMPs in mice chondrocytes and the degeneration of aggrecan and type II collagen. The experimental data showed that DE could alleviate the IL-1β-mediated inflammatory reac- tion in OA chondrocytes via restraining the excitation of NF-κB pathway. The intracellular PI3K/Akt pathway has also been shown to be associated with the changes of ECM during the pathogenesis of OA [45]. EXcitation of the PI3K/Akt pathway leads to activation of the down- stream NF-κB pathway, resulting in phosphorylation of IκBα and p65 that boost the overexpression of inflammatory mediators in chon- drocytes [46,47]. Besides, it is widely believed that restraining the excitation of PI3K/Akt is one of the important ideas for the treatment of osteoarthritis. Based on western blot analysis, DE significantly inhibited
the phosphorylation of PI3K/Akt in mouse chondrocytes induced by IL- 1β. Previous studies have shown that DE has an inhibitory effect on inflammatory activation of cancer cells via restraining the PI3K/Akt pathway, which partly encourages this view [23]. In summary, our re- sults suggest that DE is able to restrain IL-1β-mediated inflammatory reactions via restraining the activation of PI3K/Akt/NF-kB pathway in mice chondrocytes. And the brief description of this potential mecha- nism is given in the picture (Fig. 8). However, the exact mechanism by which DE regulates inflammatory processes in chondrocytes remains to be investigated.
Molecular docking analysis is often used to explore intermolecular interactions. Docking analysis shows that DE can be stably attached to the PI3K inhibitory binding pocket via the interaction force with the amino acid residue (GLU-826), with a high affinity. In other words, DE occupies the binding site and thus inhibits the phosphorylation of PI3K, which prevents the separation of PI3K from the NF-κB dimer. There may be other potential mechanisms that have not been explored. However, the current results suggest that DE is able to restrain the excitation of PI3K/Akt/NF-κB via blocking PI3K phosphorylation
DMM-induced mouse OA model is similar to human OA, which has been previously accepted. And this model has been diffusely used to value the effectiveness of hypothesized therapeutic drugs [24]. In this study, the mice in the DMM group developed cartilage calcification and erosion, the reduction of chondrocytes and the degradation of extra- cellular matriX. Nevertheless, the DE therapy could offset these adverse effects to a certain extent, thus suggesting its potential to mitigate the progression of OA in vivo.
Obviously, current studies have shown that DE significantly sup- presses IL-1β-mediated inflammatory reaction and catabolism in chon- drocytes via blocking the excitation of the PI3K/Akt/NF-κB signal pathway. Secondly, through molecular docking analysis, it was also found that the inhibitory binding pocket of PI3K had a high affinity with DE, which intuitively verified the conclusions of the in vitro cell ex- periments. Furthermore, the protective effect of DE on articular cartilage was also shown in mice model. In a word, these experimental data may in the future encourage the application of DE as a promising medicine for the treatment of osteoarthritis.

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