Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K‐AKT‐NFATc1 pathway

Abstract Bone loss (osteopenia) is a common complication in human solid tumour. In addition, after surgical treatment of gynaecological tumour, osteoporosis often occurs due to the withdrawal of oestrogen. The major characteristic of osteoporosis is the low bone mass with micro‐architectural deteriorated bone tissue. And the main cause is the overactivation of osteoclastogenesis, which is one of the most important therapeutic targets. Inflammation could induce the interaction of RANKL/RANK, which is the promoter of osteoclastogenesis. Triptolide is derived from the traditional Chinese herb lei gong teng, presented multiple biological effects, including anti‐cancer, anti‐inflammation and immunosuppression. We hypothesized that triptolide could inhibits osteoclastogenesis by suppressing inflammation activation. In this study, we confirmed that triptolide could suppress RANKL‐induced osteoclastogenesis in bone marrow mononuclear cells (BMMCs) and RAW264.7 cells and inhibited the osteoclast bone resorption functions. PI3K‐AKT‐NFATc1 pathway is one of the most important downstream pathways of RANKL‐induced osteogenesis. The experiments in vitro indicated that triptolide suppresses the activation of PI3K‐AKT‐NFATc1 pathway and the target point located at the upstream of AKT because both NFATc1 overexpression and AKT phosphorylation could ameliorate the triptolide suppression effects. The expression of MDM2 was elevated, which demonstrated the MDM‐p53‐induced cell death might contribute to the osteoclastogenesis suppression. Ovariectomy‐induced bone loss and inflammation activation were also found to be ameliorated in the experiments in vivo. In summary, the new effect of anti‐cancer drug triptolide was demonstrated to be anti‐osteoclastogenesis, and we demonstrated triptolide might be a promising therapy for bone loss caused by tumour.


| INTRODUC TI ON
In human solid tumour, Bone loss (osteopenia) is one of the most common complications.1,2 Surgical treatment is an effective therapy for the treatment of gynaecological tumour, such as cervical cancer, ovarian cancer and endometrial cancer.3-5 However, after complete hysterectomy and bilateral ovariectomy (OVX), osteoporosis often occurs due to the oestrogen deficiency. 6,7 Osteoporosis is characterized by decreased bone density and strength due to excessive loss of bone protein and mineral content. 8 The specific pathogenesis mechanism of osteoporosis is still not completely clarified. However, it has been demonstrated by multiple researches that the imbalance between bone formation and bone resorption is the major cause.9 During the pathogenesis of osteoporosis, inflammation-induced osteoclastogenesis overactivation after menopausal contributed most to the bone metabolism imbalance.10,11 Thus, one of the important strategies to prevent and treat osteoporosis is to suppress inflammation. [12][13][14] The origination of osteoclastogenesis is RANK/RANKL interaction.
After activated by RANKL, RANK recruits receptor-associated factor 6 (TRAF6) and other TRAFs. 15 Following, multiple downstream signalling pathways, including NF-κB, AKT and MAPKs, are activated. Through these pathways, bone resorption is induced by the transcription and expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metalloproteinase 9 (MMP-9) and nuclear factor of activated T cells, c1 (NFATc1).
Recently, several researches have demonstrated their new finding of anti-osteoporosis medicine from traditional Chinese herbs. 16 Triptolide is isolated from the traditional Chinese herb lei gong teng (thunder god vine, Tripterygium wilfordii Hook.f). It has been reported that triptolide could suppress cell growth and induce apoptosis in multi-range of cancer cells in human,17,18 and presented anti-inflammation functions. 19 Thus, we hypothesized that triptolide could suppress the proliferation and bone resorption ability of osteoclast, which could be a promising therapy for the treatment of bone loss caused by tumour.

| Cell viability assay
To investigate the cell viability of triptolide, the CCK-8 assay was carried out according to the manufacturer's instructions. BMMCs were cultured in 96-well plates at densities of 4 × 10 4 cells/well without or with triptolide (6.25, 12.5, 25.0, 50.0 or 100.0 nM) for 24 hours, 48 hours and 4 days. After co-cultural, 10 µL CCK-8 was added into the plate. After 4-hours incubation, the absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer.
Before the experiments, the mice were kept in a specific-pathogenfree (SPF) animal laboratory of our hospital (certification: SCXK (Shanghai) 2007-0003). FBS, DMEM low glucose medium and the mixture of penicillin-streptomycin were used for the BMMCs culture to enough number. Following, the BMMCs were induced to osteoclasts with RANKL (50 ng/mL) and M-CSF (20 ng/mL), without or with triptolide (3.125, 6.25, 12.5 or 25.0 nM) for 7 days. According to the manufacturer's instructions, osteoclasts were detected with TRAP staining. RAW264.7 cells were also induced to osteoclasts with RANKL (50 ng/mL) and M-CSF (20 ng/mL), without or with triptolide (3.125, 6.25, 12.5, or 25.0 nM) for 7 days before osteoclasts detection with TRAP staining. Multi-nucleated (BMMCs nuclei > 5 or RAW264.7 nuclei > 5) TRAP + cells were counted as osteoclasts. 21,22 For further investigation of the role of triptolide in the suppression of PI3K-AKT-NFATc1, SC79 (the AKT activator) and the overexpression plasmid of NFATc1 were also used during the cell culture.

| Western blot analysis
Immunoblot analysis was used for key protein quantification. CTR, MMP-9, Cathepsin K, TRAP and NFATc1 as osteoclastogenesis-related markers were detected for osteoclastogenesis evaluation. Key protein in PI3K-AKT-NFATc1 pathway and the phosphorylation were also detected. MDM2 expression was detected for mechanism investigation.

| Animal preparation
All procedures related to animal experiments of this study were ap-

| Ovariectomy animal model
The mice were randomly assigned to four groups including the following: sham group, ovariectomized (Model) group, ovariectomized mice treated with triptolide (Trip group), or treated with DMSO (DMSO group). Each group consists six mice. The OVX surgery were carried out as described in our former study. 24 The mice in Trip group were intraperitoneally (i.p.) given the triptolide (66.7 µg/ kg), which was dissolved in DMSO. And the mice in DMSO group were injected the same dose of DMSO. The treatment procedure lasted for 6 weeks. After the treatment, mice were executed with chloral hydrate. Serum was collected for biochemistry examination, and bilateral femurs were excised and fixed in 4% paraformaldehyde solution for histologic and micro-CT analysis.

| Histomorphometric examination
After the 4-day fixation of right femurs, it took 2 weeks to decalcify the femurs with 10% tetracycline-EDTA aqueous solution. Then, haematoxylin and eosin (H&E) staining and TRAP staining were carried out after the femurs were paraffin-embedded to prepare 4-mm sections. H&E-stained sections were used for observing the bone trabecula and TRAP-stained sections were used for osteoclasts observation. The region of the metaphysis was selected to count the number of osteoclasts. The count procedure was done with the software Image-Pro Plus 6.0.

| Serum biochemistry
Blood was collected from the left ventricular, followed with centrifugation process of 1000 g for 5min. ELISA kits were used to determine the serum concentration of bone generation biomarker osteocalcin (OCN), and bone resorption biomarkers IL-6, TNF-α and TRAcp5B. The ELISA procedure was done according to the manufacturer's instructions of the ELISA kits (Anogen).

| Statistical analysis
IBM SPSS Statistics 22.0 was used to perform all the statistical analysis. The results are presented as mean ± SD. The Student's t test was done to compare differences between two groups, and the one-way ANOVA was done for the differences among more than two groups. If P < .05, the differences were regarded as statistically significant.

| Triptolide inhibits expressions of osteoclastogenesis-related markers
The osteoclastogenesis was reflected by expressions of several biomarkers, including Cathepsin K, CTR, MMP-9, TRAF6 and TRAP.
The results are shown in Figure 2A-E, which indicated that RANKL treatment significantly enhanced the expression of these biomarkers (P < .01), and triptolide (>6.25 nM) inhibited the RANKL-induced expression (P < .01).

| Triptolide suppresses PI3K-AKT-NFATc1 pathway activation
Key proteins and the phosphorylation were detected by Western blot assays, and the results are shown in Figure 3A

| Triptolide promotes MDM2 expression in RANKL-induced osteoclast in vitro
According to previous study,25 PI3K-AKT pathway could regulate cell cycle through regulating MDM translocation. MDM2 was also detected with Western blot assay, and the results were shown in Figure 3A,E. As the results indicated the expression of MDM2 was decreased after treated with RANKL and enhanced with triptolide treatment.

| NFATc1 overexpression reverses triptolide effects on osteoclastogenesis
To further investigate the mechanism of triptolide suppressing The reverse effect of NFATc1 overexpression indicated that triptolide targeted to the upstream of NFATc1.

| AKT agonist reverses triptolide effects on osteoclastogenesis
The AKT phosphorylation agonist SC79 was also used to investigate the mechanism of triptolide effects on osteoclastogenesis. SC79 (5 μg/mL) was used to treat RAW264.7 with M-CSF, RANKL and triptolide (25 nM). As TRAP staining results showed in

| Triptolide inhibits ovariectomy-induced bone loss in vivo
Animal experiments on OVX mice were carried out to simulate the clinical patients with complete hysterectomy and bilateral OVX. And the effects of triptolide on anti-osteoporosis were explored. As H&E staining showed in Figure 6A, there was significant trabecular bone loss after OVX (P < .05). Triptolide significantly prevented the OVX-induced trabecular bone loss (P < .01), however DMSO did not. As shown in Figure 6B Consistent with this, the serum levels of TRAcp5B, IL-6 and TNF-α induced by OVX were also reduced by M54 treatment (Figure 6I-K). There is no significant influence on the serum levels of OCN ( Figure 6L). In order to clarify how triptolide suppress PI3K-AKT-NFATc1 pathway, NFATc1 overexpression plasmid and AKT phosphorylation activator SC79 were used in the experiments in vitro. 57 The results

| D ISCUSS I ON
showed that the overexpression of NFATc1 and activation of AKT both ameliorated the effects of triptolide on osteoclastogenesis, which indicated that triptolide targeted to the upstream section of AKT.
According to previous researches, the anti-cancer effects of triptolide was proved with cancer cells originated from different tissues, including breast, prostate and kidney. 19 In our study, triptolide showed new biological effects of suppressing osteoclastogenesis, and protecting ovariectomized mice from bone loss.
We hypothesize that the use of triptolide of cancer patients also prevents the common complication osteoclast-mediated bone destruction.
Limitations within our study indicate the need for future work.
Firstly, in our study, we demonstrated triptolide inhibit osteoclastogenesis through suppressing PI3K-AKT-NFATc1 pathway and clarified the target point located upstream of AKT. However, the exact mechanism how triptolide interact with the famous signal pathway still need to be defined. Secondly, we demonstrated the new effect F I G U R E 5 SC79 partially ameliorate the effect of triptolide. A, Formation of tartrate-resistant acid phosphatase (TRAP)-positive cells from RAW264.7 cells. B, The resorption area on the bone biomimetic synthetic surface. C, Western blot detected expressions of osteoclastogenesis-related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP-9, TRAF6 and TRAP with beta-actin as reference. D, Expression of phosphorylation of AKT, PI3K and NFATc1 detected by Western blot assay (**P < .01, *P < .05 vs RANKL-induced group; ##P < .01, #P < .05) | 6159 CUI et al.
of anti-cancer drug triptolide to prevent osteoporosis and hypothesized the effect on cancer patients to prevent the complication of bone reconstruction. The hypothesis still needs to be confirmed with clinical trials.
In summary, this study proved that triptolide can suppress osteoclastogenesis in vitro, and prevent OVX-induced bone loss in vivo.
We demonstrated that the mechanism of triptolide effects is to inhibit PI3K-AKT-NFATc1 pathway by targeting to upstream section of AKT, and the MDM2-p53-induced cell death also contributed. In conclusion, the traditional Chinese herb derived anti-cancer drug triptolide might be a promising therapy for the treatment of osteoporosis caused by tumour.

ACK N OWLED G EM ENTS
We thank the Clear-Medtrans studio for language polishing.

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

AUTH O R CO NTR I B UTI O N
CJ, LXQ and SJC designed this study. CJ, ZX and SYM finished the animal studies. CJ and LXQ finished BMMCs isolation. CJ, WSC and LXQ performed Western blotting. CJ and CX wrote the manuscript, and SJC reviewed the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.