Effects of Bakuchiol on chondrocyte proliferation via the PI3K‐Akt and ERK1/2 pathways mediated by the estrogen receptor for promotion of the regeneration of knee articular cartilage defects

Abstract Objectives Cartilaginous tissue degradation occurs because of the lack of survival of chondrocytes. Here, we ascertained whether bakuchiol (BAK) has the capability of activating chondrocyte proliferation. Materials and methods The effect of BAK on the proliferation of rat chondrocytes at a concentration of 10 and 20 µmol/L was investigated. The molecular mechanisms involving target binding and signalling pathways were elucidated by RNA‐sequencing, qPCR, molecular docking and Western blotting. Matrigel mixed with bakuchiol was implanted locally into rat knee articular cartilage defects to verify the activation of chondrocytes due to bakuchiol in vivo. Results Bakuchiol implantation resulted in the activation of rat chondrocyte proliferation in a dose‐dependent manner. RNA‐sequencing revealed 107 differentially expressed genes (DEGs) with 75 that were up‐regulated and 32 that were down‐regulated, indicating increased activation of the PI3K‐Akt and cell cycle pathways. Activation of the phosphorylation of Akt, ERK1/2 and their inhibitors blocked the proliferative effect of bakuchiol treatment, confirming its direct involvement in these signal transduction pathways. Molecular docking and siRNA silencing revealed that estrogen receptor‐α (ERα) was the target of bakuchiol in terms of its cell proliferative effect via PI3K activation. Two weeks after implantation of bakuchiol, the appearance and physiological structure of the articular cartilage was more integrated with abundant chondrocytes and cartilage matrix compared to that of the control. Conclusions Bakuchiol demonstrated significant bioactivity towards chondrocyte proliferation via the PI3K‐Akt and ERK1/2 pathways mediated by estrogen receptor activation and exhibited enhanced promotion of the remodelling of injured cartilage.


| INTRODUC TI ON
Dietary supplements and traditional herbal medicines are considered to be forms of complementary and alternative medicine that are extensively used globally and widely used for the prevention of cartilage degeneration in Asia and other developed countries. It has been demonstrated that Fructus psoraleae (FP), the seeds of Psoralea corylifolia L. (Leguminosae), exhibits powerful effects against diseases of the skeletal system. [1][2][3] It has been established that bakuchiol, a natural meroterpenoid compound extracted from Fructus psoraleae, exhibits antioxidant, 4 antitumour, 5 antimicrobial 6 and anti-inflammatory activity. 7 We have previously demonstrated that components of extracts of fructus psoraleae demonstrate the potential to treat cartilage degeneration induced by chondrocyte death. 8 In that study, we found that bakuchiol was present at a concentration as high as 84.5% of the active extract and that it was the principal cartilage protective component within the extract. This discovery has motivated us to continue to explore the therapeutic possibilities of bakuchiol against cartilage diseases.
Osteoporosis (OP) and osteoarthritis (OA) are high-risk diseases of the elderly, whose main features are degeneration of the cartilaginous matrix and apoptosis of functional chondrocytes. 9 In addition to anti-inflammatory treatments, it is more important in OP and OA to activate autologous chondrocytes to proliferate and produce cartilaginous extracellular matrix so as to replenish the degenerated matrix. 10 Therefore, promotion of chondrocyte growth is a key factor in the treatment of that form of cartilage damage.

Lim et al compared different compounds from Psoralea corylifolia
L. and demonstrated that bakuchiol is a key component with oestrogenic activity due to high estrogen receptor-binding affinity. 11 Several studies have confirmed that oestrogen and similar active compounds regulate the cell cycle and therefore proliferation and growth. 12,13 These findings provide the basis for the hypothesis that bakuchiol, as a compound with oestrogenic activity, has a potential role as a therapy of degenerative diseases of cartilage.
For the first time, in this study and in accordance with our initial hypotheses and experimental results, we have demonstrated that bakuchiol, a bioactive natural product, caused significant stimulation of chondrocyte proliferation via the PI3K-Akt and ERK1/2 signal transduction pathways mediated by estrogen receptors. It also usefully exhibited promotion of injured cartilage remodelling. The addition of BAK to medications and food supplements has the potential to treat cartilage degeneration and osteoarthritis.

| Cell culture and treatment
Rat chondrocytes were isolated using a method adapted from a previous study. 9 Briefly, articular cartilage was retrieved from the knees of healthy Sprague-Dawley rats (male, n = 5, weight: 150 g).
The cartilage was minced, and pieces digested with 2 mg/mL of collagenase type II (Gibco) for 2 hours. After removal of undigested tissue, the cells were cultured in DMEM/F12 (1:1) supplemented with 10% FBS. Passage 2-3 (P2-3) cells were used in the experimental studies. The bakuchiol was purchased from Selleck (S3813). Animal experiment protocol was reviewed and approved by the Institutional Review Board (IRB) of Chongqing University.

| Cell viability testing
Cell viability in response to BAK was evaluated using a cell counting kit-8 (CCK8) assay which was also utilized to assess cell viability. Cells were seeded at a density of 1 × 10 4 cells/well in a 24-well plate and the cytotoxicity of BAK tested using concentrations of 1 to 100 μmol/L for 3 days. The cells were then treated with 10 μmol/L BAK for 7 days. The cells were then washed with PBS and incubated in serum-free medium containing 10% CCK8 reagent for 2-3 hours at 37°C in an atmosphere containing 5% CO 2 . Aliquots were pipetted into a 96-well plate and absorbance at 490 nm measured using a plate reader (Bio-Rad).

| Cell proliferation assay
Chondrocyte proliferation was quantified using an ethynyl deoxyuridine (EdU) DNA in vitro proliferation detection kit (Guangzhou RiboBio) using flow cytometry, in accordance with the manufacturer's instructions. 14 Images of cells were captured by light with microscopy using Image-Pro Plus 6.0 software (Media Cybernetics).
The number of EdU-labelled cells was calculated from fields randomly selected in each well. Numbers of cells in each phase of the cell cycle were calculated using a BD FACS Calibur Flow Cytometer at a 488 nm excitation wavelength.

| qRT-PCR
Cells were harvested using Trizol reagent (Invitrogen), followed by RNA isolation. Each sample was reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).
The reverse transcription product was then used as a template to per-

| Western blotting
Western blotting analysis was performed as described previously. After separation using SDS-PAGE then transfer to PVDF

| RNA-sequencing for detection of DEGs and pathways
RNA-sequencing (RNA-seq) analysis and quantification were utilized to investigate changes in cell mRNA profiles among the different treatments performed. Isolated RNA was sent to BGI Co., Ltd.
for conducting RNA-seq, which was performed on a BGISEQ-500 (Shenzhen, China). All samples were replicated three times for confirmation purposes. Sequencing results were further analysed using R programming language and software (version 3.5.1), in order to identify differentially expressed genes (DEGs), and perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

| Target prediction and molecular docking
The online tool "SwissTargetPrediction" was used to predict potential targets of BAK compounds. 15 Simplified molecular input line entry specification (SMILES) was used and the molecule identified within the NCBI PubChem Compound database. Molecular docking analysis was performed using Autodock 4 16 and protein information identified using the RCSB PDB protein data bank.

| Evaluation of the effect of BAK on PI3K-Akt and ERK pathways via estrogen receptor activation
The PI3K-Akt and ERK signalling pathways were involved in the regulation of cell proliferation by BAK. Two methods of analysis were conducted to verify this. Firstly, the cells were treated with BAK or BAK plus the Akt or ERK inhibitors PI-103 and SCH772984, respectively.
Following culture of the cells, Western blotting was used to ascertain if Akt or ERK1/2 were phosphorylated and to verify the association between BAK and the activation of the Akt and ERK1/2 pathways.
Secondly, ERα siRNA (ERα si-R) was used to knockdown estrogen receptor in the target cells, which were then further cultured for 48 hours with BAK. Meanwhile, the non-treatment group was used as the control. Finally, the protein expression levels of CDK1 and

| Animal experiments
Animal experiments were conducted in accordance with protocols approved by the Chongqing University Animal Care and Use Committee.
For the cartilage injury model, a 0.5-mm-deep thickness defect was created through the articular cartilage and subchondral bone of the patellar groove of Sprague-Dawley (SD) rats (200-250 g) using a micro-electrical drill. Matrigel mixed with bakuchiol (100 µmol/L) was implanted locally to fill the cartilage defect. Articular cartilage samples were harvested after 1 and 2 weeks post-implantation for macroscopic and histological evaluation, with analysis by micro-CT.

| Histological analysis
The harvested samples were embedded in paraffin, sectioned for 10 μm thickness and then stained with haematoxylin and eosin (H&E), Safranin-O green and Alcian blue. The sections were scored histologically in accordance with previous studies. 9,17,18 The relative area of each defect ( Figure 6: area within the dashed line) in the different groups was measured using Image J. The relative content of cartilage matrix was ascertained from the degree of Safranin staining (Red area) and GAG content by Alcian blue staining (Positive blue area). All morphological measurements were performed using Image J. The mean histological scores for each group were statistically analysed, with t tests performed between the BAK-treated group and control. In addition, the histological immunofluorescent staining for Ki67, p-Akt, and p-ERK was performed to verify the in vivo mechanisms of BAK on cell proliferation.

| Statistical analysis
RNA-sequencing results were further analysed using R programming language and software (version 3.5.1). All other data were analysed and expressed as means ± standard deviation (SD). Molecular docking was analysed using Autodock 4. Differences among groups were
Concentrations of 5, 10 and 20 µmol/L increased proliferation significantly (*P < .05) for chondrocytes vs the control group (0.1% DMSO; Figure 1A). After 7 days of culture, supplementation with 10 µmol/L BAK promoted cell viability in comparison with the control group (*P < .05; Figure 1B). Fluorescent staining of EdU in the proliferation assay indicated that 10 and 20 µmol/L BAK treatments significantly promoted cell proliferation after 24 and 48 hours ( Figure 1C and 1). Notably, the numbers of cells involved in genome replication (S-phase) were clearly increased when F I G U R E 4 BAK promotes cell proliferation via PI3K-Akt and ERK1/2 pathways mediated by ERα. (A) Western blotting for CDK1, phosphorylated Akt (p-Akt), and total Akt (t-Akt) of the cells with 10 µmol/L BAK and 1 µmol/L PI-103 (PI3K inhibitor) treatment, statistical analysis of CDK1 and Akt were collected according to the grey semi-quantification and presented ratio to GAPDH, (*) P < .05 (vs control) and (#) P < .05 was accepted as statistically significant. (B) Western blotting for CDK1, phosphorylated ERK1/2 (p-ERK1/2), and total ERK1/2 (t-ERK1/2) of the cells with 10 µmol/L BAK and 1 µmol/L SCH772984 (ERK1/2 inhibitor) treatment, statistical analysis of CDK1 and ERK1/2 collected according to the grey semi-quantification and presented ratio to GAPDH, (*) P < .05 (vs control) and (#) P < .05 was accepted as statistically significant. (C) Western blotting for detecting ERα expression to verify ERα si-RNA (ERα si-R) knockdown efficiency for 72 h. (D) Western blotting for detecting p-Akt, CDK1 with BAK plus ERα knockdown, (E) the grey intensity was measured, (*) P < .05 (vs control) and (#) P < .05 were accepted as statistically significant. (F and G) EdU assay for verifying cell proliferation effect with BAK plus ERα knockdown, (*) P < .05 and (#) P < .05 were accepted as statistically significant, n = 10 (random fields) chondrocytes were incubated in 10 and 20 µM BAK-conditioned culture medium after 24 and 48 hours (*P < .05; Figure 1E,F).  Figure 2C). Our results demonstrated that these DEGs were highly enriched in functions related to PI3K-Akt, FoxO and cell cycle signalling pathways, among others ( Figure 2D). and MKI67 (*P < .05) after 48 hours. Similar to the gene expression results described above, BAK not only stimulated CDK1 protein synthesis but also up-regulated E2F1 at the translation level ( Figure 3B and 3).

| Bakuchiol regulates proliferative gene/ protein expression
According to immunofluorescent staining ( Figure 3D), 10 µmol/L BAK clearly promoted MKI67 protein synthesis (*P < .05) relative to the control. SwissTargetPrediction provided suggestions of more than 15 potential molecular targets for BAK ( Figure 3E). When combined with a literature analysis of BAK, Esr1 was selected for further molecular docking analysis ( Figure 3F). The results demonstrated that BAK can successfully bind to Esr1 using the residues GLU353 and ARG394 (green dashed line) via hydrogen bond interactions and residues LEU391, PHE404, LEU387, LEU346, MET421, ILE424, LEU525 and GLY521.

| Bakuchiol promotes remodelling of injured knee articular cartilage defects
After 1 week, the central regions of the defect sites were not filled with cartilage matrix in either the control or BAK group, indicating that cartilage repair remained limited. However, according to the micro-CT imaging, the patellar fossa was significantly larger in the control group than in the BAK-treated group at this time point ( Figure 5). The defects in the BAK-treated group were filled with a shiny, smooth, hyaline-like tissue after 2 weeks compared with the control group ( Figure 6). Compared with the controls after 1 and 2 weeks, the histological scores of the BAK-treated group defects were 3.60 ± 0.54 (*P < .05) and 5.40 ± 0.55 (*P < .05) respectively. It is worth noting that not only were the histological scores significantly different, but so also was the content of GAG, with BAK treatment clearly different (*P < .05) from the control (Figure 6). In addition, with BAK treatment, no significant depression was observed after 2 weeks in comparison F I G U R E 6 Histological staining of H&E, Alcian blue and Safranin-O/green for 1 and 2 wk after defective injury on knee articular cartilage of rats with or without BAK treatment, cs: cartilage surface, d: defective injury (by dashline), b: bone. Black arrows indicate degenerated cartilage matrix, blue arrows indicate abundant glycosaminoglycan, and red arrows indicate constructive cartilage matrix. For the statistical analysis, (*) P < .05 and (#) P < .05 were accepted as statistically significant, bar: 500 µm with its appearance after 1 week (#P < .05), suggesting that the articular cartilage repair was complete. Taken together, these data suggest that BAK protected in situ chondrocyte viability, prevented degradation of the articular cartilage, and provided optimal regeneration of the cartilage and bone defects.
In addition, the expression of Ki67, p-ERK and p-Akt was mea-

| D ISCUSS I ON
In a previous study, we demonstrated that components in extracts of fructus psoraleae demonstrated the potential to treat cartilage degeneration induced by chondrocyte apoptosis. In that study, we found that the percentage content of bakuchiol (BAK) in the active fructus psoraleae extract was as high as 84.5%, as measured by HPLC analysis and the principal active compound in the extract. 8 Combined with the results of the cell proliferation experiments, we believe that the substance that actively promotes chondrocyte proliferation is bakuchiol. In addition, in a previous study of FP extract on chondrocyte proliferation, we found that petroleum ether extracts of FP at concentrations greater than 0.1 mg/mL revealed significant toxicity. At first, we suspected that this phenomenon was due to residual petroleum ether, but this was not the case. In this study, we treated chondrocytes directly with different concentrations of BAK, the results indicating that with BAK concentrations >20 μmol/L in the medium, significant cytotoxicity was experienced. our preliminary experiments, we tested the release profile of BAK in F I G U R E 7 Immunofluorescent staining of p-Akt, p-ERK1/2 and MKI67 (Ki67) for 1 and 2 wk after defective injury on knee articular cartilage of rats with or without BAK treatment. A, Dashline indicates cartilage surface of defective injury site, and arrows indicate positive staining. B, Semi-quantitative analysis, (*) P < .05 (vs control in 1 wk) and (#) P < .05 (vs control in 2 wk) were accepted as statistically significant Matrigel ( Figure S2), with an in vivo implantation concentration of 100 μmol/L BAK showing therapeutic effects.
We conducted transcriptome high-throughput sequencing to explore possible signal transduction pathways in order to ascertain the molecular mechanisms for the promotion of proliferation in chondrocytes. The cell cycle, PI3K-Akt and ERK1/2 pathways were analysed by enrichment of sequencing data. Subsequent studies also confirmed that BAK promoted chondrocyte proliferation and was indeed associated with the pathways described above.
However, it was not apparent what the specific target molecule of BAK was. The convenient online tool "SwissTargetPrediction" predicted that the most likely potential target of BAK was the estrogen receptor. Lim et al compared different compounds from Psoralea corylifolia L. and demonstrated that bakuchiol is a key compound with oestrogenic activity due to its high estrogen receptor-binding affinity. 11 Several studies have confirmed that oestrogen and similar active compounds regulate the cell cycle and so proliferation. [19][20][21] Therefore, the hypothesis that "bakuchiol as an active compound demonstrating oestrogenic activity may be used as a therapy for cartilage degenerative diseases" has been proposed. However, the results of the animal experiments presented above suggest that BAK has clearly positive pharmacological effects.
Studies have shown that, for cartilage regeneration, in addition to the proliferation of chondrocytes, it is more important to regulate the microenvironment around the cartilage such as inflammation, 22 angiogenesis 23 or niches appropriate for stem cells. 24 It has been reported that BAK has certain anti-inflammatory effects. 25 For diseases of the cartilage, osteoarthritis is the most common degenerative disease. Its principal pathological feature is a large number of inflammatory reactions. 9,26 For future cartilage tissue regeneration and repair strategies, the specific mechanism that BAK is capable of promoting that requires additional exploration is inhibition of the inflammatory response.

CO N FLI C T O F I NTE R E S T
We declare that the authors have no conflict of interest.

DATA AVA I L A B I L I T Y
All data included in this study are available upon request by contact with the corresponding author.