osteonecrosis of the femoral head (onFH) is a debilitating and progressive disease which can lead to femoral head collapse and subsequent osteoarthritis. This disease is the major cause of total hip arthroplasty in young adults (1) . it is reported that more than 1 million new patients are affected with this disease annually and the annual incidence is 15 to 20 million according to a nationwide survey (2) . accumulating evidence has alluded to several etiologic and pathogenic mechanisms for onFH (3) . The principal mechanism involves interruption of osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMScs) (4) . Thus, investigation of the disordered mechanism of osteogenic differentiation in BMScs and development of effective methods of prevention and early therapy are crucial.
a number of previous studies have reported that osteogenic differentiation-related genes are expressed periodically (5, 6) . Brain and muscle arnT-like 1 (BMal1) is the most important component of the molecular biological clock, expression of which has been found to have 24-h periodicity in bone. The circadian system rhythmically regulates cellular proliferation, physiology, and behavior (7, 8) . Moreover, it is implicated in drug efficacy, metabolism, detoxification and toxicity (9) (10) (11) . The core transcriptional activator of the clock network is BMal1 which is regulated by delicate systems in mammals (7) . increasing numbers of studies have demonstrated that BMal1 and other circadian clock genes could be regulated by drugs and toxicants. Miranda et al (12) showed that resveratrol could reverse circadian disruption induced by a high-fat diet in rats. chen et al (13) demonstrated that carbon tetrachloride altered circadian rhythms of liver clock genes in a mouse model of hepatic fibrosis, while Gabás-Rivera et al (14) reported that dietary oleanolic acid supplementation mediated circadian clock gene expression in an animal model. icariin is extracted from Herba epimedii which is widely used as a major active ingredient to prevent osteonecrosis induced by glucocorticoids in patients with severe acute respiratory syndrome. our previous study revealed that icariin regulated cell proliferation and osteogenic differentiation in Mc3T3-e1 cells (15) and acted as an effective ingredient for preventing the progression of onFH induced by glucocorticoids in a rat model (16) . However, the mechanism underlying the association between icariin and BMal1 in osteogenic differentiation of BMScs remains unclear.
in the present study, it was demonstrated that icariin could alter BMal1 and induce osteogenic differentiation of BMScs in vivo. Furthermore, it was also shown that icariin enhanced the bone morphogenetic protein 2 (BMP2)/runX family transcription factor 2 (runX2) signaling pathway through upregulation of BMal1 expression.
Cell culture and treatment. according to the method described by Gong et al (17) , a total of 20 3-week-old female Sprague-dawley rats (laboratory animal center of Huazhong University of Science and Technology) were sacrificed by the intraperitoneal injection of 100 mg/kg sodium pentobarbital and BMScs were extracted from femurs and tibias by aseptic manipulation. cells were expanded in minimum essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin G-streptomycin and cultured in 5% CO2 at 37˚C. BMSCs at a density of 80-90% were passaged and cells at passage three to six were used in the following experiments. For osteogenic induction, basic medium was supplemented with 10 -7 mol/l dexamethasone, 10 -2 mol/l sodium β-glycerophosphate and 50 µg/ml l-ascorbic acid. The medium was changed every 3 days for BMSc differentiation. each experiment was performed in triplicate. all the experimental protocols involving animals were approved by the institutional animal care and use committee of Tongji Medical college, Huazhong university of Science and Technology (iacuu no. S496; date of permission, october, 31, 2017). icariin (purity >98%; abcam) was dissolved in dimethyl sulfoxide (DMSO) and then stored at -20˚C without light. Subsequent experiments used a dMSo concentration of ≤0.1%. All experiments were performed in triplicate.
Cell Counting Kit-8 (CCK-8) assay. The effect of icariin on cell proliferation was evaluated using the ccK-8 assay. Briefly, BMScs (5x10 3 cells/well) were plated in 96-well plates and treated with icariin (10 -10 -10 -3 mol/l) for 48 h at 37˚C. Subsequently, cell proliferation was assessed using the ccK-8 reagent (nanjing Jiancheng Bioengineering institute), according to the manufacturer's instructions, at a wavelength of 450 nm using an elx800 multifunctional microplate reader (BioTek instruments, inc.). The concentration of icariin used to treat the cells that were analyzed by rT-qPcr and western blotting was based on the results of the ccK-8 assay.
Flow cytometry. After reaching confluence, BMSCs at passage 3-4 were used for flow cytometric analysis. First, the cells were incubated with trypsin for 1 min at 37˚C, and then the cell pellet was collected by transient centrifugation at 400 x g at room temperature for 5 min. next, the cell pellet was washed with ice-cold PBS and then centrifuged at 400 x g at room temperature for 5 min. Subsequently, the cell pellet was resuspended in 100 µl ice-cold PBS supplemented with 0.5% BSA (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at 4˚C. Additionally, the cells were treated with Rat Fc block (cat. no. 550271; BioLegend, Inc.) for 20 min at 4˚C. Finally, the cells were identified by negative expression of CD34 [phycoerythrin (Pe)-labeled; cat. no. 128611; Biolegend, inc.] and cd45 (FiTc-labeled; cat. no. 202205; Biolegend, inc.) and positive expression of cd73 (FiTc-labeled; cat. no. 127219; Biolegend, inc.), cd90 (Pe-labeled; cat. no. 205903; Biolegend, inc.) and cd105 (Pe-labeled; cat. no. 120407; Biolegend, inc.) using a FACSscan flow cytometer (Beckton Dickinson) and analyzed by FlowJo software (version X; FlowJo llc). The reaction conditions were as follows: 95˚C for 15 sec, 60˚C for 20 sec and 75˚C for 10 sec after denaturing DNA templates at 95˚C for 10 min; 40 cycles. The 2 -ΔΔcq method (18) was used to determine the relative expression levels.
Western blotting. Total protein was extracted with riPa buffer (Sigma-Aldrich; Thermo Fisher Scientific, Inc.) according to standard protocols. cell lysates with 50 µg of total protein (quantified using the bicinchoninic acid assay) were separated by 10% SDS-PAGE, transferred onto PVDF membranes, and then blocked in TBST with 5% non-fat dried milk for 1 h at room temperature. The membranes were incubated with primary monoclonal mouse antibodies targeted against: cat. no. ab34710; abcam) and β-actin (1:1,000; cat. no. ab8227; Abcam) at 4˚C overnight, and then followed by secondary antibodies conjugated with horseradish peroxidase (1:10,000; cat. no. aSP00001; dako; agilent Technologies, inc.) for 1 h at room temperature after washing. The immunoblots were visualized and detected using the ecl system (Pierce; Thermo Fisher Scientific, Inc.). Protein expression was quantified using Fusion Solo software (version 4; Vilber Lourmat Deutschland GmbH).
Transient transfection and luciferase assay. BMal1 short hairpin rna (shrna) interference lentiviral vector was constructed and synthesized by Shanghai Genechem Co., Ltd. The BMAL1 shRNA interference target sequence was 5'-ACA CGC AAT AGA TGG GAA A-3', and a scramble sequence 5'-TTC AAG ATC CTC AAT TAT A-3' was used as a negative control. BMSCs were cultured in 6-well plates with specific media to appropriate confluence and then transfected with the virus assisted with 6 µg/ml polybrene for 12 h at 37˚C. The viral supernatants were removed and the culture was continued in complete medium. BMSCs were grown to 30-70% confluence and then transfected with BMal1, BMal1-shrnas or empty vector overnight. The following day, the cells were co-transfected with pRL-SV40 Renilla luciferase reporter plasmids (10 ng; Promega Corporation) and firefly luciferase reporter vectors (50 ng), using lipofectamine ® 2000 (invitrogen; Thermo Fisher Scientific, Inc.) as the delivery system and a control. Cells were lysed after a 48-h transfection and firefly and Renilla luciferase activities were measured using the dual luciferase reporter assay system (Promega corporation).
ALP and Alizarin red S (ARS) staining. BMScs were seeded at a density of 1x10 5 cells/well in 6-well plates and incubated with osteogenic medium. According to the manufacturer's instructions, alP staining was assessed using an alP staining kit (nanjing Jiancheng Bioengineering institute) on day 7.
after osteogenic induction for 14 days, the cells were washed three times with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. Then paraformaldehyde was removed and the cells were washed three times with ddH 2 o. Finally, the cells were stained with 0.04 M arS for 30 min at room temperature, and then rinsed twice with ddH 2 o and visualized under a light microscope (magnification, x40). For quantitative analysis of arS staining, the absorbance at 540 nm was measured using a UV spectrophotometer. Results of arS are expressed as µg/mg protein with average and standard deviation.
Statistical analysis. data are presented as the mean ± Sd. Statistical analyses were performed using SPSS software (version 17.0; SPSS, inc.). differences between groups were analyzed using an unpaired Student's t-test or one-way ANOVA followed by Dunnett's post hoc test. P<0.05 was considered to indicate statistical significance.
Flow cytometry was performed to identify BMScs by expression of cd34, cd45, cd73, cd90 and cd105 in the isolated cells. The percentages of cd73-positive cells, cd90-positive cells and cd105-positive cells were 99.8, 99.7 and 99.9%, respectively; while the percentages of cd34-positive cells and cd45-positive cells were 3.66 and 1.2%, respectively, indicating that the isolated cells were BMScs (Fig. 1) . 
The proliferative activity of BMScs in the presence of different concentrations of icariin (10 -10 -10 -3 M) was determined using the ccK-8 assay (Fig. 2a) . The results showed that cell proliferation was significantly increased by lower concentrations of icariin (10 -9 -10 -5 M; P<0.05), with 10 -7 M being the most effective.
rT-qPcr and western blotting demonstrated that icariin significantly increased the expression of BMP2, RUNX2, ALP and oc (Fig. 2B and c) .
To investigate whether icariin induces BMal1 expression in BMScs, BMal1 expression of 10 -7 M icariin-treated cells was evaluated by RT-qPCR and western blotting. Icariin significantly increased BMal1 mrna and protein levels (Fig. 3) . These results suggested that icariin induced BMal1 expression in BMScs.
BMSCs. To evaluate whether BMal1 regulates osteogenic differentiation via BMP2, BMScs were transduced with BMal1. BMal1 overexpression increased expression of osteogenic differentiation-associated genes. Moreover, BMal1 overexpression enhanced both mrna expression and the promoter activity of BMP2 ( Fig. 4e and F) .
BMSCs. in order to determine whether BMal1 is essential for osteogenic differentiation induced by icariin, the osteogenic differentiation-inducing effects of 10 -7 M icariin treatment were further investigated in BMal1-knockdown (BMal1 -/-) BMScs. compared with the effects of icariin in wild-type (WT) BMScs, expression of osteogenic genes was reduced in the BMal1 -/-BMScs evaluated by Pcr analysis (Fig. 4e ). in accordance with the Pcr results, western blotting also showed that levels of BMP2, runX2, alP and oc were lower in the BMal1 -/-BMScs compared to the WT BMScs (Fig. 4F) . Furthermore, arS of BMal1 -/-BMScs indicated that BMal1 was required for mineralized nodule formation during osteogenic differentiation ( Fig. 4B and d) . in addition, less positive staining was detected in BMal1 -/-BMScs ( Fig. 4a and c) .
Taken together, icariin increased the BMal1, BMP2 and runX2 expression in BMScs. BMal1 overexpression also upregulated BMP2 and runX2 expression compared with the control group. However, these levels of expression were significantly downregulated after BMAL1 knockdown whether icariin treatment was administered or not. The results suggested the icariin could enhance the BMP2 signaling pathway by upregulating BMal1 expression in vitro.
in the present study, it was demonstrated that icariin promoted osteogenic differentiation by upregulating BMal1 expression through BMP signaling in BMSCs. These findings suggested that BMal1 plays a critical role in icariin-mediated osteogenic differentiation.
in fact, a circadian clock exists in every cell of the human body. The central players in these are BMal1 encoded by the gene arnTl and clock circadian regulator (clocK) by the gene clocK (19) . BMScs are stem cells with the ability to differentiate in vitro into adipocytes, osteoblasts and cartilage-forming chondroblasts (20) . regardless of its exact developmental origin, the function of the circadian clock gene is related to the behavior of various stem cells involved in homeostasis and repair of bone and adipose tissue (21) . a recent report revealed impairment of osteogenic differentiation of BMScs from BMal1 -/mice (5) . This finding was in line with our present study as well as an observational study that showed that the numbers of active osteocytes and osteoblasts were reduced in BMal1 -/mice in vivo (22) . However, a previous study showed that the amount of osteoblastic activity was increased in BMal1 -/mice (21) . These differences may due to the different ages of the mice. in this study, we clearly evaluated the positive regulatory mechanism of BMal1 on osteogenic differentiation. our results indicated that BMal1 could accelerate osteogenic differentiation through BMP2 signaling. BMP2, a member of the transforming growth factor (TGF)-β superfamily, is considered to be a key regulator of cell growth and differentiation (23) (24) (25) . a number of studies have suggested that the activity of clock genes is closely correlated with BMPs. one study reported that regulation of clock genes controlled BMP2 expression in osteoblasts (26) . Meanwhile, BMAL1 regulation of TGF-β and BMP signaling in the control of fat adipogenesis has also been reported (27) . a recent study showed that BMal1 silencing-induced BMP2 was found in rat uterus endometrium stromal cells (28) . Samsa et al (5) recently reported that BMal1 -/mice had reduced osteoblast differentiation in vitro and they proposed some potential molecular mechanism of BMal1-dependent control of osteogenic differentiation including oxidative stress response, the mTor signaling pathway and translation. However, the precise mechanism via which BMal1 induces osteogenic differentiation of BMScs remains unclear. in the present study, it was demonstrated that BMAL1 deficiency decreased BMP2 expression during osteogenic differentiation of BMScs.
The disorder and downregulation of clock genes may be the cause of disease, and in some cases could also be used as a target of drug action to improve disease symptoms and treat disease. recently, in order to alleviate circadian clock-related disease, there has been significant progress in the development of chemical compounds which are capable of manipulating clock genes as novel preventive and therapeutic agents (29) . For example, resveratrol, which is considered a natural antioxidant polyphenol compound, has also been demonstrated to ameliorate circadian disorders of lipid metabolism (30) . Moreover, a recent study revealed that glucocorticoids altered the circadian clock in the iris-ciliary body in a mouse model (31) . However, few studies have yet demonstrated the potential mechanism by which osteogenic differentiation is mediated between icariin and BMal1.
The results of the present study suggested that icariin at a concentration range between 10 -9 and 10 -5 mol/l significantly promoted the proliferation of BMScs, especially at 10 -7 mol/l. a number of previous studies have reported that icariin increased cell proliferation at different concentrations. Yang et al (32) showed enhanced proliferation of human neural stem cells in vitro at a concentration of 10 -5 mol/l. in addition, Mok et al (33) demonstrated that icariin stimulated the proliferation of uMr 106 cells at concentrations ranging from 10 -14 to 10 -6 mol/l. These differences may be attributable to the variety of cells used in the experiments.
icariin has been demonstrated to have therapeutic properties, with reports of antitumor, anti-hepatotoxic, immune-enhancing, anti-inflammatory, neurite outgrowth activity and erection dysfunction-improving effects over the past decades among chinese researchers (34) (35) (36) . Furthermore, the bone-strengthening activity of icariin has attracted much recent attention. However, there have been few reports on the effect of icariin on the circadian clock in BMScs. The present study suggested that icariin increased BMal1 expression. Furthermore, reduced expression of BMP2/runX2 was observed in the present study during osteogenic differentiation of BMal1-deficient cells. in addition, mineralized nodule formation was suppressed both in BMal1 -/-BMScs and in icariin-mediated BMal1 -/-BMScs. Taken together; these findings demonstrated that icariin promoted osteogenic differentiation by upregulating BMal1 expression through BMP signaling in BMScs. However, as the effects of icariin on BMScs in the present study were mainly observed in vitro, further studies will be needed to confirm these results in animal models.
in conclusion, to the best of our knowledge, the present study is the first to provide evidence that icariin promoted osteogenic differentiation through BMal1-BMP2/runX2 signaling.