Effects of Artemisia annua L. Essential Oil on Osteoclast Differentiation and Function Induced by RANKL

Objective This study aimed to assess the main components of Artemisia annua L. essential oil (AEO) and determine their effect on the proliferation and differentiation of RAW264.7 cells induced by receptor activator for nuclear factor-ligand (RANKL) in vitro. Then, we tried to explain part of the function of its possible mechanisms. Materials and Methods Essential oil was extracted from Artemisia annua L. Osteoclasts were induced in vitro by RANKL in mouse RAW264.7 cells. The experimental group was treated with different concentrations of AEO, while the control group was not treated with AEO. CCK8 was used to detect osteoclast proliferation. The osteoclasts were stained with TRAP. Western blot was used to detect protein in the MAPK pathway and the NF-κB pathway after treatment with different concentrations of AEO. RT-PCR was used to determine the expression of osteoclast-related mRNA in cells. Results The GC-MS analysis was used to obtain the main components of AEO, including camphor, borneol, camphor, borneol, terpinen-4-ol, p-cymene, eucalyptol, deoxyartemisinin, and artemisia ketone. The CCK8 results showed that the AEO volume ratio of 1 : 4000, 1 : 5000, and 1 : 6000 did not affect the proliferation of RAW264.7 cells. However, TRAP staining showed that AEO decreased osteoclast formation. Western blot results showed that the expression of protein TRAF6, p-p38, p-ERK, p-p65, and NFATc1 decreased in the MAPK pathway and the NF-κB pathway affected by AEO. Furthermore, RT-PCR results showed that the expression of osteoclast resorption-related mRNAs (MMP-9, DC-STAMP, TRAP, and CTSK) and osteoclast differentiation-related mRNAs (OSCAR, NFATc1, c-Src, and c-Fos) also decreased in the experimental group. Conclusions AEO inhibits osteoclast differentiation in vitro, probably by reducing TRAF6 activation, acting on the MAPK pathway and NF-κB pathway, and inhibiting the expression of osteoclast-related genes.


Introduction
Artemisia annua L. belongs to the genus artemisia in Asteraceae and has a fragrance. e plant name has been checked on http://www.theplantlist.org mentioning the website's data. In the past, it was decocted with water to treat malaria, bone steaming hot flashes, summer heat, jaundice, etc. Artemisia annua L. contains monoterpenes (artemisia ketone, isoartemisia ketone, cineole, and camphor), sesquiterpenes (artemisinin, artemisinin A, artemisinin B, artemisinin C, and artemisia acid), and triterpenes (β-artemisinin acetate) [1]. Artemisinin is used as an antimalarial drug for the clinical treatment of malaria. Artesunate may play a role in treating malaria, autoimmune diseases, cancer, diabetes, etc. [2]. Research on the effect of Artemisia annua L. on bone disease has gradually increased in recent years. Artesunate can reduce the level of inflammatory factors in the knee joint cavity [3], inhibit osteoclast secretion of axon guide factor netrin-1, and improve sensory nerve-mediated osteoarthritis pain [4]. It also inhibits the expression of vascular endothelial growth factors in fibroblast-like synovial cells of rheumatoid arthritis and has a therapeutic effect on synovitis [5]. An in vitro experiment showed that artemisinin and its derivatives could inhibit osteoclast differentiation [6].
Essential oil (EO) is a mixture of many chemical components in aromatic plants. EO is widely used as a perfume, food seasoning, and skincare product. It is also being developed in the pharmaceutical field since it has many pharmacological effects, such as antibacterial, antiinflammatory, antioxidant, analgesia, and antitumor. For instance, EO has antibacterial activity against various Grampositive and Gram-negative bacteria. Inhalation therapy with EO can treat sinusitis, acute and chronic bronchitis, and other diseases. Inhalation of EO can treat chronic illnesses that do not respond to conventional treatments, such as headaches, anxiety, depression, or epilepsy. Moreover, EO can improve the efficacy of various chemotherapeutic drugs, such as docetaxel. It can also enhance the immune system function of cancer patients [7].
Artemisia annua L. essential oil (AEO) combines aromatic volatile secondary metabolic molecules isolated from Artemisia annua L. by distillation. AEO has various effects, including antioxidation [8], antibacterial [9], anti-α-amylase, and anti-α-glucosidase [10]. erefore, AEO can treat diseases caused by microbial drug-resistant infections, diabetes, and oxygen-free radicals. Monoterpenes are the most abundant compounds in essential oils. Paeoniflorin [11] found that monoterpenes suppress the proinflammatory cytokines in RAW 264.7 cells induced by lipopolysaccharide. Monoterpenes also suppress mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. e MAPK and NF-κB signaling pathways are essential in differentiating RAW264.7 into osteoclasts. erefore, the active components of AEO can be further explored in different application fields. However, the AEO effect on osteoclast differentiation has not been elucidated. e RAW264.7 cell line is mouse mononuclear macrophages and can be fused and differentiated into osteoclasts only via receptor activator for nuclear factor-κB ligand (RANKL) [12,13]. RANKL is a cytokine encoded by the tumor necrosis factor ligand family gene TNFSF11. It is an inducing factor for the differentiation and fusion of osteoclast precursors to form multinucleated osteoclasts [14]. It can bind to the receptor receptor activator for nuclear factor-κB (RANK) on the surface of osteoclast progenitor cells and activate tumor necrosis factor-associated factor 6 (TRAF6) in the cells. TRAF6 activates some osteoclast-related pathways and cytokines [15] and can trigger the MAPK and NF-κB pathways [16]. ese pathways induce the expression and activation of nuclear factor of activated T-cell 1 (NFATc1), the main transcription factor of osteoclast differentiation [17,18]. Transferring NFATc1 to the nucleus can promote the expression of genes related to osteoclast formation, thus enhancing osteoclast differentiation.
Herein, cellular and molecular biological methods were used to observe the effect of AEO on the proliferation and differentiation of RANKL-induced RAW264.7 cells. e possible mechanism of AEO inhibiting osteoclast differentiation by interfering with the RANKL signal pathway was also assessed. erefore, this study may provide an experimental basis for preventing and treating metabolic osteopathy.

Extraction of Essential Oil from Artemisia annua L.
e leaves of Artemisia annua L. were collected, cleaned, and dried. Approximately 1 kg of the extract was extracted from dried leaves using petroleum ether (3 × 7 L). Organic extracts were combined, concentrated under reduced pressure, and then subjected to steam distillation at 100°C. Finally, the obtained mixture was separated using a separatory funnel, and the essential oil of the upper layer was collected (1∼2 mL).

Gas Chromatography-Mass Spectrometry (GC-MS) Analytical Conditions.
e raw materials of essential oil from Artemisia annua L. were dried using anhydrous sodium sulfite before analysis. About 50 mg of dried oil was weighted and prepared in n-hexane at a 1 mg/mL concentration for sampling. e standard solution of n-alkenes was diluted in n-hexane to obtain a final testing concentration of 50 μg/mL.
An Agilent 8860GC equipped with a 5977B quadrupole mass spectrometer was used for GC-MS analysis. A fused silica HP-5 MS capillary column was used to separate the components. Helium (purity >99.999%) was used as the carrier gas at a constant flow rate of 1.0 mL/min and a split ratio of 10 : 1. e oven temperature was as follows: an initial temperature of 50°C, ramped at 10°C/min to 120°C, at 1°C/min to 135°C, and then at 3°C/min to the final 240°C maintained for 8 min. e injection volume was 1 μL. MS detection was conducted in the electron ionization (EI) mode (70 eV). e ion source, injector, and transfer line temperatures were 230°C, 250°C, and 250°C, respectively. MS analyzer was performed in the full scan mode (m/z 45-650), and the solvent delay time was set at 4 min. MassHunter GC-MS Data Acquisition (Version 10.0) and Qualitative Analysis (Version 10.0) were applied to control the equipment and acquire and treat raw data.

Cell
Culture of Murine RAW264.7. RAW264.7 cells (Chinese Academy of Sciences TCM13) were cultured with DMEM complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in CO2 incubator at 5% CO2, 37°C and saturated humidity. RAW264.7 cells were inoculated in a culture plate with 5 × 10 3 cells/cm 2 when inducing osteoclast differentiation. After adhering to the plate, the cells were replaced by complete culture medium MEM and then induced with RANKL 50 ng/ml for five days. e culture medium was changed every two days.

Cell Counting Kit-8 (CCK-8) Proliferation Assay.
RAW 264.7 cells were inoculated in a 96-well plate (2 × 10 3 cells/well). After the cells were attached, different culture mediums were added to the experimental groups. e drug concentration (volume ratio 1 : 4000, 1 : 5000, 1 : 6000) with no statistical differences on cell survival rate was determined to assess the nontoxic effect of AEO on cells. e experimental group was treated with different concentrations of AEO and 50 ng/ml RANKL, while the control group was not treated with AEO. e CCK-8 staining solution (10 μl) was added to each well at 0 h, 24 h, 48 h, 72 h, and 96 h after the intervention and then incubated for 2 h. e absorbance OD value of 450 nm was read using an enzyme labelling instrument, and cell activity was observed.

Tartrate-Resistant Acid Phosphatase (TRAP) Dyeing.
Cells were inoculated in a 24-well plate (1 × 10 4 cells / well) following the instructions of the TRAP staining kit and then treated. e experimental group was treated with 50 ng/ml RANKL and AEO (1 : 4000, 1 : 5000, 1 : 6000), while the control was not treated with AEO. Cells were stained after five days of intervention. e TRAP-positive cells with three or more nuclei in each well were counted.

Real-Time Quantitative Polymerase Chain Reaction (RT-PCR) Quantitative
Analysis. RAW 264.7 cells were inoculated in a 6-well plate (5 × 10 4 cells/well) and then treated. e experimental group was treated with 50 ng/ml RANKL and AEO (1 : 4000, 1 : 5000, 1 : 6000), while the control was not treated with AEO. AEO and RANKL were not added to the blank group. Total RNA was extracted using Trizol after 24 hours of cell culture. A spectrophotometer was used to quantify the mRNA, and 1 μm RNA was reverse transcribed into cDNA via PCR. Forty cycles of two-step polymerase chain reaction amplification were performed on the Applied Biosystems real-time polymerase chain reaction system (95°C 5 s, 60°C 30 s) using the TB Green Premix Ex Taq kit. e primer sequence was as follows: e ΔCt (Ct target gene-Ct GAPDH) and −ΔΔCt (ΔCt mean −ΔCt) were calculated, and 2 −ΔΔCt was used to express the analyzed genes.

Western Blot
Analysis. RAW 264.7 cells were inoculated in a 6-well plate (5 × 10 4 cells/well) and then treated. e experimental group was treated with 50 ng/ml RANKL and AEO (1 : 4000, 1 : 5000, 1 : 6000), while the control was not treated with AEO. Similarly, AEO and RANKL were not added to the blank group. Cells were collected using RIPA lysate containing 1% protease and 5% phosphatase inhibitors and then centrifuged at 4°C, 12000g for 20 min. Proteins (20 ug) were separated on 10% SDS-polyacrylamide gel by electrophoresis. e separated proteins were then imprinted on the PVDF membrane, blocked with 5% skim milk for 1 h, and then incubated with the appropriate dilution of the first antibody at 4°C overnight. e membrane was washed with TBST buffer containing 0.05% tween-20 and then incubated with HRP labelled secondary antibodies for 1 h. e membrane was washed using TBST, and the target protein band was obtained via a gel analyzer. e grey level of the band was analyzed using Image J, and the relative protein expression was calculated via the normalization of the actin protein.
Evidence-Based Complementary and Alternative Medicine 2.9. Statistical Analysis. All data were expressed as mean-± standard deviation (SD). A single-factor analysis of variance (ANOVA) was used for multiple comparisons. P < 0.05 was considered the statistically significant level. e quantitative data of TRAP-positive cells represented six experiments in each group, while all the PCR and WB experiments data represented three groups. GraphPad Prism9 software (GraphPad Software Inc.) was used for all statistical mapping analyses.

Qualitative Analysis of Essential Oil via GC-MS. NIST
Mass Spectral Library search identified the components of AEO. Further identification was confirmed by comparing their retention indices with data from the literature. e typical total ion chromatography obtained from the GC-MS analysis is presented in Figure 1. Fifty-one volatile compounds were identified using the mass spectrum search and retention index, and the main volatile components were terpenoids, including camphor, borneol, eucalyptol, and piperitone. e name, retention time, CAS number, and the retention index (from experiments and literature) of each volatile compound identified from the AEO are shown in Table 1.

Effect of AEO on the Proliferation of RANKL-Induced
RAW264.7 Cells. RAW264.7 cells were fused and differentiated into osteoclasts (Figure 2(a)) after induction with RANKL for five days. e expansion of RANKL-induced RAW264.7 cells accelerated and slowed after 72 h (Figure 2(b)). Cell survival rates in the experimental group were higher than 90% after 24 h, 48 h, 72 h, and 96 h of intervention. Furthermore, cell survival rates in the experimental and control groups were not statistically different (Figure 2(c)). Western blot did not show significant differences in PCNA (proliferating cell nuclear antigen) between the experimental and control groups (Figure 2(d)).

AEO Inhibits Osteoclast Resorption-Related Protein
Expression. TRAP staining showed that almost no osteoclasts were observed when the concentration was 1 : 4000 and 1 : 5000, while a few osteoclasts formed when the concentration was 1 : 6000 (Figures 3(a) and 3(b)).
RT-PCR showed that the osteoclast bone resorptionrelated protein matrix metalloproteinase-9 (MMP-9) mRNA expression more significantly decreased after the AEO intervention (1 : 4000, 1 : 5000 and 1 : 6000) than in the control group. e terms of the osteoclast bone resorption-related protein dendritic cell-specific transmembrane protein (DC-STAMP) mRNA, the osteoclast specific marker enzyme cathepsin K (CTSK), and TRAP [19] were lower in the experimental group than in the control group. However, the expression was not significantly different among the three experimental groups (Figure 3(c)). erefore, these results show that AEO inhibits the number of osteoclasts in a dose-dependent manner. AEO also inhibits the expression of osteoclast-related specific mRNA.

AEO Inhibits the RANKL-Induced p-38/MAPK, ERK/ MAPK, and NF-κB Pathways in RAW264.7 Cells.
Western blot showed that TRAF6 decreased significantly after AEO intervention at 1 : 4000 than in the control group, while it was not different between the 1 : 5000 and 1 : 6000 groups. TRAF6 was lower in the 1 : 4000 group than in the 1 : 5000 and 1 : 6000 groups (Figure 4(a)). e amount of NFATc1 was significantly decreased after AEO intervention at 1 : 4000, 1 : 5000, 1 : 6000 than in the control group. NFATc1 was lower in the 1 : 4000 group than in the 1 : 5000 and 1 : 6000 groups (Figure 4(b)). Phosphorylation of extracellular signal-regulated kinase (ERK) and p38 in the MAPK subfamily can promote osteoclast differentiation and prolong survival time [20]. e amount of p-p38 protein decreased significantly after AEO intervention at 1 : 4000 than in the control group. e amount of p-p38 significantly reduced in the 1 : 4000 group than in the 1 : 6000 group in a dose-dependent manner (Figure 4(c)). e amount of p-ERK significantly reduced in the 1 : 4000 group than in the control group, while it increased in the 1 : 6000 group. However, p-ERK was not different among the three experimental groups (Figure 4(d)).
erefore, AEO has different effects at various concentrations on the content of TRAF6, p-p38, p-ERK, and NFATc1 proteins. Herein, AEO (1 : 4 000) was used to detect cell protein for 24 h, 48 h, 72 h, and 96 h. e results showed that the protein contents of TRAF6, p-p38, p-ERK, and NFATc1 were not significantly different in cells treated with AEO for 24 hours and in the control group. However, the protein of TRAF6, p-p38, p-ERK, and NFATc1 decreased at 48 h, 72 h, and 96 h (Figure 4(e)). e activation of the NF-κB pathway can produce NF-κB/RelA [21], such as NF-κB p65. e degradation of IκBα (I-kappa-B kinase alpha) can transfer NF-κB to the nucleus to promote the transcription of the target gene. e amount of IκBα protein decreased significantly in the control group. However, IκBα increased after AEO intervention than in the control group ( Figure 5(a)). Compared to the control group, there was no significant increase of IκBα in the experimental groups at 24 h, 48 h, 72 h, and 96 h ( Figure 5(b)). e amount of p-p65 protein decreased significantly in the 1 : 4000 group compared to the control group.
ere is no significant difference of p-p65 in the 1 : 4000 group between the 1 : 5000 and 1 : 6000 group ( Figure 5(c)). Similar results also found that the protein of p-p65 decreased at 48 h, 72 h, and 96 h after AEO intervention ( Figure 5(d)).
e RT-PCR results showed that the mRNA levels of osteoclast differentiation-related protein NFATc1 were significantly downregulated in the experimental group than in the control group ( Figure 6). e translocation of NFATc1 to the nucleus can promote the mRNAs expression of osteoclast formation-related genes, such as neuronal protooncogene tyrosine-protein kinase Src (c-Src), osteoclastassociated immunoglobulin-like receptor (OSCAR), and cellular oncogene Fos (c-Fos). In this article, the level of c-Src mRNA expression was lower in the 1 : 4000 group than in the 1 : 6000 group. e mRNA expression level of OSCAR was lower in the 1 : 4000 group than in the 1 : 5000 group. However, the mRNA levels of NFATc1 and c-Fos were not significantly different among the three experimental groups. erefore, these results show that AEO inhibits osteoclast differentiation-related pathways and mRNA expression.

Discussion
Herein, AEO inhibited osteoclast formation. It also reduced the expression of osteoclast marker proteins by reducing the activation of TRAF6, inhibiting the p38/MAPK and ERK/ MAPK pathways, and reducing NFATc1 synthesis (Figure 7). ese results suggest that AEO inhibits osteoclast differentiation.  11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 8.5 8 7.5 7 6.5 6 5.5  Evidence-Based Complementary and Alternative Medicine 5 Osteoclasts decompose the collagen matrix during bone metabolism to form bone lacunas by secreting acid hydrolase [22]. In addition, osteoblasts form new bone to fill bone lacunae to maintain bone homeostasis. e imbalance of bone homeostasis may lead to metabolic bone diseases, such as osteoporosis [23] and periodontitis [24]. e function of osteoclasts is also closely related to the immune system. e number and activity of osteoclasts increase in inflammatory diseases, such as rheumatoid arthritis and Crohn's disease [25,26]. Mature multinucleated osteoclasts are formed via proliferation, migration, cell adhesion, and fusion of progenitor cells [27]. erefore, it is necessary to control the number and activity of osteoclasts to reduce bone loss and inhibit joint inflammatory diseases. us, negative regulation of osteoclast differentiation induced by RANKL can regulate bone metabolism and prevent or treat metabolic osteopathy.
Camphor is the main component of many aromatic plant essential oils. It has anti-inflammatory and analgesic properties [29]. Dr. Duke's Phytochemical Databases and Ethnobotanical Databases (https://www.ars-grin.gov/duke/) show that Artemisia annua contains camphor (concentration in the leaves, 6460 ppm). Borneol and its derivatives have antibacterial, anti-inflammatory, antiviral, antiproliferation, and antiedema effects [30]. ey can also relieve mild muscle and joint pain caused by arthritis or sprain [31]. Brand [32] showed that terpinen-4-ol could inhibit t inflammation via lipopolysaccharide-stimulated monocytes. Eucalyptol controls airway mucus hypersecretion and asthma by inhibiting anti-inflammatory cytokines [33]. Zhong [29] found that p-cymene can significantly inhibit the activation of EKR, p38, and c-Jun N-terminal kinases in RAW 264.7 cells by reducing proinflammatory cytokines. e AEO monoterpenes, such as p-cymene, can also minimize osteoclast differentiation.
We found that AEO inhibited the differentiation of RAW264.7 cells into osteoclasts induced by RANKL. As a shared signal sensor of TNF, Toll-like/IL-1, and the cellular  Evidence-Based Complementary and Alternative Medicine inflammatory receptor family, TRAF6 is located upstream of the MAPK and NF-κB pathway [34]. e MAPK and NF-κB pathways play an essential role in osteoclast differentiation. MAPK pathway includes p38, JNK, and ERK pathways. Western blot images show that TRAF6 protein increased significantly after RANKL-induced RAW264.7 cells, and it increased with the induction time, consistent with other studies [35,36]. An in vitro experiment [6] showed that artemisinin and its derivatives could decrease the amount of TRAF6 protein and inhibit the activation of TRAF6 recruitment in RAW264.7 cells. In this paper, the amount of intracellular TRAF6 protein decreased after the AEO intervention. Furthermore, the proteins p-p38 and p-ERK in the MAPK pathway increased significantly after RANKL induction and increased with induction time. A study has shown that Dihydroartemisinin in Artemisia annua L. inhibits RANKL-induced ERK phosphorylation of Pathways [37]. We also observed that p-p38 and p-ERK protein decreased after the AEO intervention. e AEO could inhibit the phosphorylation of p38 and ERK in a dosedependent manner. e main transcription factor of osteoclast differentiation, NFATc1, began to accumulate and increase after 24 h-48 h after induction [38], similar to our western blot results. In western blot images, AEO can inhibit NFATc1 in a dose-dependent manner. e ability to inhibit NFATc1 is an integral part of the mechanism of AEO that inhibits osteoclast formation induced by RANKL. e NFAT signal is located downstream of the MAPK pathway and downstream of the NF-κB pathway. AEO may be also achieved by suppressing the NF-κB signal by suppression of NFATc1.
e RANKL-induced NF-κB pathway is an important signal pathway activated during osteoclast formation. e interaction between RANK and RANKL results in proteasome degradation of IκBa, followed by the release of NF-κB/RelA dimers, such as NF-κB p65, which are then transported from the cytoplasm to the nucleus to initiate osteoclast-specific gene transcription [39][40][41]. In the previous studies, Artesunate and Dihydroartemisinin inhibited the activation of NF-κB and osteoclast formation induced by RANKL by regulating the degradation of IκBα protein and the expression of downstream genes in vitro [37,42]. e effect of AEO on the NF-κB pathway is yet to be reported. Results show that AEO inhibits the phosphorylation of NF-κB p65 and the degradation of IκBα, thus inhibiting the activation of NF-κB induced by RANKL. AEO can also inhibit osteoclast differentiation by inhibiting the NF-κB pathway.
AEO decreased the expression of osteoclast-related gene mRNA, including c-Fos, c-Src, and OSCAR, thus reducing osteoclast differentiation. e c-Fos is located downstream of MAPK and can form a close nuclear phosphoprotein with Jun/AP-1 transcription factors, essential for bone cell development and maintenance. Kinase c-Src can phosphorylate the tyrosine residues of c-Fos [43]. e OSCAR is an immunoglobulin-like receptor associated with osteoclast differentiation [44]. As a costimulatory signal needed for RANKL-mediated activation of NFATc1, OSCAR is the direct target of NFATc1 [40]. erefore, the mRNA expression of c-Src, c-Fos, and OSCAR in cells can reflect the formation mechanism of osteoclasts.
AEO also decreased the expression of DC-STAMP, MMP-9, TRAP, and CTSK, thus decreasing the bone resorption function of osteoclasts. After RANKL stimulation, the DC-STAMP moves from the surface of osteoclast progenitor cells to the cytoplasm [45], which is one of the fusion mediators directly regulated by NFATc1 [36]. DC-STAMP is related to bone resorption and osteoclast fusion and participates in TRAP expression in osteoclast precursors [46,47]. MMP-9 is associated with the migration of RAW264.7 cells [48].
e CTSK and TRAP are specific markers of osteoclasts [19]. Besides, the mRNA expression of CTSK, TRAP, MMP-9, and DC-STAMP genes can reflect the bone resorption function of osteoclasts. erefore, the differentiation of mouse osteoclasts is inhibited by AEO in vitro. Besides, this study can provide insights into preventing and treating metabolic bone diseases. However, the effect of AEO on osteoclast differentiation has not been tested in vivo. e specific mechanism of AEO deserves further exploration.

Conclusions
ese results suggest that AEO can inhibit osteoclast formation and bone resorption by inhibiting RANKL-induced activation of the MAPK pathway and the NF-κB pathway in vitro.