Shikonin – Sr90090

Shikonin relieves osteoporosis of ovariectomized mice by inhibiting RANKL- induced NF-κB and NFAT pathways

Yong Chena, Zhong Xiea, Yangyang Zhanga, Chao Xiaa, Mingzhi Yanga, Xiongke Hub,∗
a Second Spinal Surgery Department of the First Aﬃliated Hospital of University of South China, Hengyang, Hunan, 421000, China
b Movement System Injury and Repair Research Center, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China

A R T I C L E I N F O

Keywords: Shikonin Osteoclast RANKL
NF-κB and NFAT pathway

A B S T R A C T

Postmenopausal osteoporosis is very common in women. Currently, many kinds of new drugs are being de- veloped for this disease. Postmenopausal osteoporosis is closely related to overactivity of osteoclasts in body. Shikonin is purple red naphthoquinone pigment extracted from lithospermum, which has anti-inﬂammation, antivirus, anticancer and other bioactivities. At the same time, it has been proved that shikonin can promote the proliferation and diﬀerentiation of osteoblasts, but its inﬂuence on osteoclasts and molecular mechanism are unknown. Our study showed that shikonin could inhibit the activity and formation of RANKL-mediated osteo- clasts depending on dose without aﬀecting the activity of bone marrow macrophages (BMM). In addition, we have also found that shikonin can inhibit the expression of speciﬁc marker gene of osteoclasts, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAcP) and calcitonin receptor. Shikonin also could promote the proliferation of MC3T3-E1, increasing the expression of mRNA related to osteogenesis, like the expression of bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). Luciferase re- porter gene assay and Western blot analysis further indicated that shikonin could inhibit the activity of RANKL- induced NF-κB and NFAT receptors. Moreover, shikonin can also slow down bone loss of ovariectomized (OVX) mice by inhibiting the activity of osteoclasts. This work explains the molecular mechanism of shikonin in RANKL-mediated formation of osteoclasts, and reveals the potential of further developing shikonin into a new drug for prevention and treatment of postmenopausal osteoporosis.

1. Introduction

Bone remodeling is the dynamic balance formed by close connection between bone formation of osteoblasts and bone resorption of osteo- clasts [1]. Postmenopausal osteoporosis is mainly osteolytic bone dis- ease caused by excessive bone resorption of osteoclasts [2]. The in- cidence of postmenopausal osteoporosis in women is as high as 30%, which can lead to pathological fracture, with high rates of disability and mortality, and bring heavy burden to family and society [3]. Therefore, the treatment of osteoporosis by inhibiting bone resorption seems to be very important. At present, drugs developed to inhibit bone resorption in the market mainly include estrogen [4], diphosphonate [5], calci- tonin [6], etc. However, these drugs often have potential side eﬀect, and are not ideal for treatment of postmenopausal osteoporosis [7]. Osteoclast are monocytes from hematopoietic stem cells/multi- nuclear giant cells of macrophage lineage. The stimulation and diﬀer- entiation of osteoclast is adjusted by the receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL). RANKL promotes the dif- ferentiation of osteoclast mainly by various signal paths such as NF-κB, AKT, MAPK and JNK, and is an important cytokine in the process of adjustment of bone metabolism [8,9]. In addition, NFATc1 has been proved to be the key transcription factor of RANKL-mediated diﬀer- entiation of osteoclast [10]. NFATc1 can induce the transcription of various genes related to diﬀerentiation of osteoclast, such as Ctsk, tartrate-TRAcP and calcitonin receptor [10]. Therefore, inhibiting RANKL-mediated signal path can help to relieve and treat osteolytic bone disease.

Lithospermum is a kind of herbal medicine widely used in China and other countries/regions. Shikonin is a kind of purple red naph- thoquinone compound extracted from the root of lithospermum. Shikonin has been proved to have anti-inﬂammation [11], antivirus [12], antibacterial [13], anticancer [14,15] and other bioactivities. Moreover, studies have shown that shikonin can promote the pro- liferation and diﬀerentiation of MC3T3-E1 cells through BMP-2/Smad5 signal path [16]. Shikonin can relieve joint swelling and cartilage de- struction caused by osteoarthritis [17,18]. In conclusion, shikonin has good bioactivity and can protect bones, but its eﬀect on osteoclasts and molecular mechanism are unknown. Our study showed that shikonin could inhibit the diﬀerentiation of osteoclasts. In addition, we have determined that shikonin can inhibit marker gene of RANKL-mediated diﬀerentiation of osteoclasts and NF-κB and NFAT signal path. Shikonin can also obviously reduce the bone loss of OVX mice. Our results show that shikonin has potential value for treatment of osteolytic bone di ease.

2. Results

2.1. Shikonin inhibits RANKL-induced generation of osteoclasts

In order to study the eﬀect of shikonin on diﬀerentiation of osteo- clasts, shikonin with diﬀerent concentrations was added for interven- tion in the process of RANKL-induced diﬀerentiation of osteoclasts of BMMs. After cell ﬁXation, TRAP activity staining was completed. It was found that shikonin inhibited the diﬀerentiation of osteoclasts de- pending on dose (negative, 0, 0.1, 0.2, 0.4 and 0.8 μM)（Figture 1B,C) And then, we detected the eﬀect of shikonin on the cell activity of BMMs and found that obvious cytotoXicity was not observed at the dose of 0.8 μM (Figture 1D).

2.2. Shikonin inhibits RANKL-induced expression of related genes of osteoclasts

Transcription of NF-κB and NFAT activates the speciﬁc gene ex- pression of osteoclasts, such as NFATc1, TRAcP, Ctsk and calcitonin re- ceptor. In order to study shikonin’s inhibition of the expression of marker mRNA related to diﬀerentiation of osteoclasts, we added dif- ferent concentrations (0.2, 0.4 μM) of shikonin for intervention in the process of RANKL-induced diﬀerentiation of BMMs, collected cells 5 days after induction, and extracted RNA for real-time PCR analysis. Our results showed that shikonin could signiﬁcantly inhibit the level of mRNA of NFATc1, TRAcP, Ctsk and calcitonin receptor (Figture 2A-D).

2.3. Shikonin inhibits RANKL-induced activity of NF-κB and NFAT

In order to study the eﬀect of shikonin on the activity of RANKL- mediated NF-κB and NFAT, we did the luciferase reporter gene assay experiment. The results showed that shikonin could signiﬁcantly inhibit the transcriptional activity of NF-κB and NFAT when its dose reached 0.2 μM and the dose of IC 50 was about 0.2 μM (Figture 3A-B). And then we detected the eﬀect of shikonin on RANKL-mediated NF-κB and NF signal path by Western blot, and the expression of IκBα protein and NFATc1 protein. The results showed that when shikonin could inhibit the degradation of IκBα protein at the dose of 0.2 μM. The inhibition was the most obvious when RANKL had induced for 20 min (Figture 3C). We found that shikonin could signiﬁcantly inhibit the expression of NFATc1 at the dose of 0.2 μM (Figture 3D).

2.4. Shikonin promoting osteoblast proliferation and expression of gene related to osteogenesis

In order to study the impact of shikonin on osteoblast, shikonin of diﬀerent concentrations were used to intervene MC3T3-E1 cell. The results showed that shikonin could promote the proliferation of MC3T3- E1 cells (Figture 4 A). Subsequently, shikonin with diﬀerent solubility (0.2, 0.4 μM) was used to interfere with MC3T3-E1 cells for 48 h, and RNA was extracted for real-time PCR analysis. The results showed that shikonin could increase mRNA levels of BMP-2, Ranx2, ALP and OCN (Figture 4B-E).

2.5. Shikonin slowed OVX-induced bone loss in mice

And then we explored whether shikonin could treat bone loss of mice caused by OVX. We divided the mice into sham-operated group, OVX group and OVX + shikonin group. The right femur of mice was scanned by Micro-CT. We found that shikonin could signiﬁcantly re- duce the bone loss of OVX mice (Figture 5A). The further analysis showed that shikonin could increase the ratio of bone volume over tissue volume (BV/TV) (Figture 5B), trabecular thickness (Tb·Th) (Figture 5C), and trabecular number (Tb. N) (Figture 4D) as well as decreases in trabecular separation (Tb.Sp) (Figture 5E). TRAP activity staining of tissue section of femur of mice showed that shikonin treat- ment group had less TRAP positive osteoclasts than OVX group (Figture 5F-G). Immunoﬂuorescence staining showed that shikonin could in- crease quantity of osteoblasts in the vicinity of bone trabecula of thighbone in the mice (Figture 5H-I).

3. Discussion

If the balance between bone formation of osteoblasts and bone re- sorption of osteoclasts is upset, osteoporosis will be caused.
postmenopausal osteoporosis is typically caused because bone resorp- tion of osteoclasts exceeds bone formation of osteoblasts. The excessive bone resorption of osteoclasts is caused by higher level of RANKL and enhanced expression of related downstream genes. This pathway has also become the main target for the treatment of osteolytic bone disease [19]. Diphosphonate protects bones by inhibiting functions of osteo- clasts, but there are risks such as osteonecrosis of the jaw and kidney failure [20,21]. At present, it is reported that many kinds of active in- gredients of Chinese herbal medicine can inhibit the diﬀerentiation of osteoclasts and relieve osteoporosis [22,23]. Our study showed that shikonin could inhibit the formation and diﬀerentiation of osteoclasts, reduce the activity of RANKL-induced NF-κB and NFAT, and slow down the bone loss of OVX mice, indicating that shikonin could be used as the potential medicine for prevention and treatment of postmenopausal osteoporosis.

Shikonin is a kind of purple red naphthoquinone compound ex- tracted from the root of lithospermum. A lot of literatures have proved that shikonin can inhibit the growth of many kinds of malignant tumors such as leukemia [15], lymphoma [24], gastric cancer [14], colon cancer [25] and breast cancer [26], and has good analgesia [27], anti- inﬂammatory [11] and antibacterial functions [13]. For the bone system, Fang et al. reported that shikonin promotes osteoblast pro- liferation and diﬀerentiation through BMP-2/Smad5 signaling pathway [16]. Our study also found that shikonin can promote the proliferation of MC3T3-E1 cells and increase the expression of proteins related os- teogenic, such as BMP-2, ALP, Runx2 and OCN, which are consistent with previous studies. In vitro experiment proved that shikonin could increase quantity of o osteoblasts in the vicinity of bone trabecula of thighbone in the mice. At the same time, studies have proved that shikonin can have functions such as inhibits lymphangiogenesis and inducing tumor cell apoptosis by inhibiting NF-κB signal path [28,29].
NF-κB signal path is an important way of early diﬀerentiation of osteoclasts. Thus, we infer that shikonin not only promote bone forma- tion, but also slow down diﬀerentiation of osteoclasts and relieve os- teolytic bone disease by inhibiting NF-κB signal path.

To discuss about the mechanism of shikonin’s inhibition of diﬀerentiation of osteoclasts, we ﬁrst extracted bone BMM of mice. We found that shikonin inhibited the diﬀerentiation of BMM into osteoclasts de- pending on dose, but did not aﬀect the cell activity of BMM. RANKL belongs to tumor necrosis factor, and is the key cytokine for formation of osteoclasts [30]. After combining with receptor RANK, RANKL can
activate signal paths such as NF-κB, TRAFs and NFATc1, and adjust the diﬀerentiation of osteoclasts [8,9]. NF-κB signal path is an important way for RANKL to induce the diﬀerentiation of osteoclasts. After combining with RANK, RANKL promotes the phosphorylation of IKKα and β, thus inducing the degradation of IκB and causing activation of NF-κB [31]. Yang et al. reported that shikonin could signiﬁcantly in- hibit the expression of IκBα and p65 in the NF-KB pathway, thus slowing down lipopolysaccharide-induced mastitis [32].

At the same time, Liang et al. reported that shikonin can also eﬃciently decreased NF-κB activation by inhibiting the degradation and phosphorylation of IκBα, Thus play an anti-inﬂammatory properties in lipopolysaccharide- mediated acute lung injury [33]. Shikonin has a variety of good eﬀects, and studies have proved that it can also improve the spinal cord edema in the rat spinal cord injury model and the neurological deﬁcit in pa- tients with ischemic stroke by inhibiting the NF-κB signaling pathway [34,35]. In our study, we found that shikonin could inhibit the tran- scriptional activity of NF-κB by preventing the degradation of IκB-α, proving that shikonin can block classic NF-κB path. NFATc1 has been proved to be the key transcription factor for the diﬀerentiation of os- teoclasts, and induces after activation by NF-κB [10]. NFATc1 also adjusts and controls the expression of marker genes of the diﬀerentiation of osteoclasts such as Ctsk, TRAcP and V-ATPase d2 [36]. Our study showed that shikonin could inhibit the transcriptional activity of RANKL-mediated NFATc1 and protein level of NFATc1. At present we can’t determine that shikonin directly inhibits the activity of NFATc1 or indirectly inhibits through NF-κB. However, shikonin can signiﬁcantly reduce mRNA expression of osteoclast associated proteins, NFATc1, Ctsk, TRAcP and calcitonin receptor. These results show that shikonin can inhibit the diﬀerentiation of osteoclasts by NF-κB and NFATc1- mediated pathway.

4. Materials and methods

4.1. Animals and reagents

8-week C57BL/6 mice were purchased from hunan Silaike labora- tory animal company and cultured in the department of experimental animals at university of south China. Shikonin was purchased from Sigma (USA) and diluted with phosphate buﬀered brine (PBS). α- minimum essential medium (α-MEM) was purchased from Hyclone (USA), fetal bovine serum (FBS) from Gibco (USA), macrophage colony- stimulating factor (M-CSF) and RANKL from R&D (USA). Primary an- tibodies for NFATc1, IκBα， osteocalcin and β-actin were purchased from Abcam (USA). Trap active staining kit was purchased from Sigma (USA) and ﬂuorescein enzyme analysis reagent was purchased from Promega (Australia).

4.2. Cell culture

RAW264.7 and MC3T3-E1 cells were obtained from America Type Culture Collection (USA) and grown in α-MEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. BMMs were isolated by washing the bone marrow of the femur and tibia of C57BL/6 mice. BMMs were inoculated on a-MEM (including 10% FBS, 2 mM L-gluta- mine and 100 U/mL penicillin/streptomycin) and M-CSF was added. The concentration (0.1–0.8 μM) of shikonin intervention cells was based on published literatures [16,38].

4.3. Osteoclastogenesis assay In vitro

BMMs were inoculated on a 96-well plate (8 × 103 in each well). M- CSF (25 ng/ml) and RANKL (50 ng/ml) was given to a-MEM culture medium. And then diﬀerent concentrations of shikonin were used for intervention. The culture solution was changed every 2 days. Cells were ﬁXed and stained with trap kit on the 5th day. The number of positive cells of osteoclasts was recorded (with more than 3 cell nucleuses).

4.4. Cytotoxicity assay

BMMs were inoculated on a 96-well plate (6 × 103 in each well) with M-CSF (25 ng/ml). And then diﬀerent concentrations of shikonin were used for intervention. After intervention for 1–4 days in each hole, CCK-8 (Beyotime, China) was added and it was incubated for 2 h in a
dark place. OD value of absorbance was measured at 450 nm on the microplate reader.

4.5. Real time Polymerase Chain reaction (real-time PCR)

BMMs were inoculated on a 6-well plate (1 × 105 in each well). M- CSF (25 ng/ml) and RANKL (50 ng/ml) were given for induction.
0.2 μM and 0.4 μM of shikonin was added at the same time for inter- vention of 5 days. MC3T3-E1 cells were inoculated on a 6-well plate and added with 0.2 and 0.4 μM shikonin to intervene for 48 h. Total RNA was extracted with the Trizol reagent (Invitrogen, Australia), 1 μg
of the total RNA was used for reverse transcription using a commercial kit (Fermentas, Burlington, Canada).

4.6. Luciferase reporter gene assay for NF-κB and NFAT

The analysis on the activity of luciferase was made according to instructions for use (Promega). RAW264.7 cells stably transfected with NF-κB luciferase reporter gene and NFATc1 luciferase reporter gene were inoculated on a 48-well plate. And then, the cells were pretreated
for 1 h with diﬀerent concentrations of shikonin. RANK was added for intervention of 6 h and 24 h. Cells were lysed to detect the activity of NF-κB luciferase and NFATc1 luciferase.

4.7. Western blot assay

BMMs were inoculated on a 6-well plate and treated with shikonin at diﬀerent times. After cell lysis, protein was extracted. The sample was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis. And then it was transferred to PVDF membranes. Under the room temperature it was blocked for 1 h in 5% skim milk. And then it was incubated over night with primary antibodies at the temperature of 4 °C under vibration. After the membrane was washed for 3 times, the sample was incubated for 1 h with secondary antibodies conjugated with horseradish peroXidase. And then it was exposed and developed.

4.8. Animals

Eighteen 8-week C57BL/6 mice were divided into sham-operated group, OVX group and OVX + shikonin group. The in vivo dose de- signed for the experiment was relatively safe dose which was se- lected by referring to relevant reference [38–40]. One week after ovariectomy, 3 mg/kg shikonin was administered to the treatment group by intravenous injection. The same dose of PBS was administered to other groups for intervention. Two times a week. After continuous treatment for 2 months, femur of the same side was taken for Micro-CT scan and TRAP activity staining.

4.9. Micro-CT analysis

Femur samples were ﬁXed with 4% paraformaldehyde for 24 h, and then scanned by Micro-CT (Skycan, Aartselaar, Belgium). The voltage, current, and resolution were set to 50 kV, 400 μA, and 8.88 μm per piXel, respectively. The images of the whole femurs were reconstructed
by NRecon and visualized by μCTVol v2.2.

4.10. TRAP staining

The femur was embedded by paraﬃn after ﬁXation, and then cut into pieces. The slides were dewaxed with Xylene and hydrated with
graded ethanol. The slide and TRAP dye were preheated in water bath at the temperature of 37 °C. And then TRAP dye was added to the slide. The sample was incubated for 5 min at the temperature of 37 °C in a dark place. It was observed under microscope. Observe under the mi- croscope and adjust the incubation time according to the color devel- opment. At the room temperature, methyl green dye liquor was added for 10 min. And then it was rinsed in water for 15 min.

4.11. Immunoﬂuorescence histochemistry staining

Thighbone of mice was performed with OTC bury after dehydration, then sliced. Glass slide was taken from refrigerator and placed at
ambient temperature for 20min. The slide was added with 0.1% Triton for rupture of membranes at ambient temperature for 10min. It was washed by PBS for 3 times, 3min once. The slide was added with sheep serum for tissue close for 30min. The slide was added with OCN pri- mary antibodies and placed into 4 °C refrigerator to stay the night. It was washed by PBS for 3 times, 3min once. Second antibody was di- luted and dripped on the slide for incubation at ambient temperature without light for 1 h. It was washed by PBS for 3 times, 3min once. The slide was sealed by DAPI for taking the pictures.

4.12. Statistical analysis

All data were shown as the mean ± SD. Statistical analysis used the student’s t-test or ANOVA to assess statistical diﬀerences, P < 0.05 was considered statistically signiﬁcant. 5. Conclusion Our study has proved that shikonin can inhibit the formation of osteoclasts by RANKL-mediated NF-κB and NFATc1 pathway, and re- duce the bone loss of OVX mice. At the same time, it was conﬁrmed that shikonin could promote bone formation. In summary, our study has indicated that shikonin is a kind of potential medicine for the treatment of osteoporosis. Declaration of competing interest No conﬂict of interest. Acknowledgments This work was supported by the Natural Science Foundation of Hunan Province, China (Grant No. 2019JJ50553) and the China Postdoctoral Science Foundation (Grant No. 2018M632998). References [1] G.A. Rodan, Bone homeostasis, Proc Natl Acad Sci USA 95 (1998) 13361–13362, https://doi.org/10.1073/pnas.95.23.13361. [2] H. 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