Apigenin neutralizes the inhibitory effect of inammation on the osteogenic differentiation of human mesenchymal stem cells

Background: The stimulating effects of apigenin on mesenchymal stem cells (MSCs) osteogenesis, as well as the anti-inammatory effect of this avonoid, have been identied. In this study, osteogenic differentiation was investigated under inammatory conditions and treatment with apigenin. Methods and Results: Along with osteogenic differentiation of MSCs, they became inamed with LPS/PA, and treated simultaneously with apigenin. The degree of differentiation was assessed by alizarin red staining and alkaline phosphatase (ALP) activity. Also, gene expression of NLRP3 and RUNX2 was performed along with protein expression of IL-1β. Signicant increase in NLRP3 and IL-1β were observed in MSCs when exposed to LPS/PA (p<0.01). Also, the osteogenesis was signicantly decreased (p<0.01). Apigenin treatment induced signicantly higher gene expression of RUNX2, the activity of ALP, and cell staining (p<0.01) which were also associated with reduced inammation in these cells. Conclusions: The effectiveness of apigenin on osteogenesis under inammatory conditions was cautiously observed.


Introduction
Improve general health and reduced fertility, have increased life expectancy. Therefore, ageassociated diseases such as cardiovascular and neurodegenerative diseases, cancer, osteoporosis, and other bone-related diseases are more threatening today [1]. Osteoporosis, as a diverse genetic and metabolic bone disease, is one of the most important public health issues characterized by bone loss, bone density, and bone mass [2]. This disorder, commonly referred to as postmenopausal osteoporosis (PMOP) in women over fty years, is associated with fracture and fat in ltration [3]. The real cause of it is due to an imbalance between the activity of osteoblasts (bone-forming cells) [4] and osteoclasts (bone cells) and a decrease in the differentiation potential of bone marrow mesenchymal stem cells [5]. In fact, in this case, this differentiation potential goes toward adipogenesis, which is associated with a signi cant change of cells from osteocytes to adipocytes [6].
Age, gender, inactivity, smoking, and alcohol are factors affecting osteoporosis [3]. The process of this disorder is associated with increased in ammation, especially the activation of nlrp3 In ammasome [7]. If aging is accompanied by an increase in the basal level of in ammation, potentially impairs bone formation [8]. Thus, activation of NLRP3 in ammatory as an intracellular protein complex involved in the innate immune response increases adipogenesis and suppresses osteogenesis [7]. As a result, by controlling the in ammatory mechanism, including Nlrp3 In ammasome, it seems that the behavior of stem cells can be regulated.
Anti-in ammatory effects of polyphenolic compounds have been reported with their effects in the treatment of diseases such as osteoporosis [9]. Among them, apigenin (4', 5, 7-Terry Hydroxy avone) is a member of the avonoid family that has potential anti-cancer, anti-in ammatory, antimicrobial and antioxidant properties [10].
Anti-osteoclastogenic effects of this compound [13] have also been reported to be associated with increased expression of osteoblast differentiation genes such as alkaline phosphatase, collagen, osteopontin, and BMP in osteoblasts [14]. Apigenin was also able to induce osteogenesis of hMSCs by increasing the expression of Runt-related transcription factor 2 (RUNX2) and osterix (Osx) [15,16]. These results suggest that apigenin enhances osteogenesis of hMSCs from osteoblast precursors to terminal differentiation states.
According to the above, the anti-in ammatory effect of apigenin along with its effect on stimulating osteogenesis has been identi ed. Due to this, and the role of in ammation in inhibiting osteogenesis, the protective effect of this compound against LPS/PA-induced osteoporosis, as well as its osteogenic stimulatory effect, was investigated.

Cell culture
Mesenchymal stem cells were enzymatically isolated from abdominal adipose tissue isolated from women who underwent liposuction procedure. Due to the use of disposable abdominal fat, this was con rmed by the ethics committee of Isfahan University of Medical Sciences. The MSC isolation steps included washing the adipose tissue with PBS (0.1 M, pH=7.4) -penicillin-streptomycin (10000 U-10000 mg/ml) three times and digesting the tissue with collagenase type I (1 mg/ml in PBS) (Thermo Fisher Scienti c, USA) followed by lysis of erythrocytes by RBC lysis buffer (155 mM NH 4 Cl, 12 mM NaHCO 3 , and 0.1 mM EDTA) and centrifugation (800 × g for ve minutes). The isolated cells were maintained at 37˚C and 5% CO 2 in self-renewal medium (DMEM-F12 + 10% FBS + 1% P/S (Thermo Fisher Scienti c, USA). The medium was changed every three days and the cells were passaged after each lling of the ask. After four passages, the cells were differentiated under the following conditions. For osteogenesis, the self-renewal medium was changed by an osteogenic medium contained 100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM L-ascorbic acid-2-phosphate. For chondrogenesis, the chondrogenic medium was used which contained the same complete medium and dexamethasone (100 nM), L-proline (40 µg/ml; Merck Millipore Corporation, Istanbul, Turkey), L-ascorbic acid 2-phosphate (50 µg/ml; Sigma-Aldrich, Germany), and TGF-β3 (10 ng/ml; BioLegend Ò , Istanbul-Turkey). For adipogenesis, the adipogenic medium contained dexamethasone (1 µM), indomethacin (100 µM), insulin (10 µg/mL), and 3-isobutyl-1-methylxanthine (IBMX; 0.5 mM) (All from Sigma-Aldrich Co. Ltd., Dorset, UK) [17,18].
hMSCs immunophenotyping and multipotency assays The cell identi cation was done by three lineage differentiation and FACS Immunophenotyping with positive (CD73, CD90, CD105) and negative (CD34 and CD45) CD markers include. For isotype controls, rabbit polyclonal IgG and rat IgG2b were used (all BD Biosciences, San Jose, USA) [17]. In summary, the cells from passage four were trypsinized and transferred to a 15 ml tube and xed with 4% paraformaldehyde. Finally, surface CD markers were characterized using a ow cytometry device (Partec, Germany) [18].

MTS assay
The toxicity effects of apigenin in the whole period of treatment was done by cell viability assay using an MTS assay kit (Abcam, Cambridge, UK). Accordingly, after the growth of cells in a 96-well plate at the density of 5 × 10 3, they were treated with apigenin (1-100 µM) for 1, 2, 3, 7, and 14 days. Then, using 20 μL/well MTS reagent for four hours at 37°C the absorbance was read at 490 nm in a microplate reader (BioTek Instruments, USA) [17].

Osteogenesis induction and treatment
To evaluate the effect of apigenin on the expression of NLRP3 and interleukin induced by LPS and PA, MSCs were exposed to 0.1 mg.ml -1 LPS/0.25 mM PA and 1 mg.ml -1 LPS/0.25 mM PA for 72 hours. Then, different concentrations of apigenin (25 and 50 μM) were added to the medium for 14 days. To compare the effect of apigenin on osteogenesis in in ammatory conditions, cells were rst exposed to LPS/PA for 72 hours and then induced osteogenesis for 21 days while being treated simultaneously with apigenin for 14 days. The protocol for inducing osteogenesis, in ammation, and treatment has been based on the assessment of cell viability and previous studies [7,19,20] Alizarin red staining Brie y, cells cultured on a 24 well plate were washed with PBS, xed with 4% formaldehyde, and then stained with 0.5% Alizarin Red in deionized water (pH = 4.1) for 30 minutes at room temperature [15].

Evaluation of ALP activity
The cells cultured on the plate were washed with PBS and lysed using lysis buffer (20 mM Tris -HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100). ALP activity was measured by laboratory kits and protein content was measured by the bicinchoninic acid (BCA) method [21].

Measurement of interleukin-1β by ELISA
The amount of interleukin-1β released in the culture medium was measured using a human IL-1β ELISA kit in cells cultured on a 24 well plate (Carmania Pars Gene, Iran).

Statistical analyses
Data analyses were performed using SPSS software (SPSS version 16.0) and GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA). Statistical signi cance was performed using one-way analysis of variance (ANOVA) followed by post hoc Tukey and p<0.05 was considered as statistically signi cant.

Results
Mesenchymal stem cell con rmation Mesenchymal stem cell con rmation was performed by examining surface CD markers as well as the ability to differentiate cells into three categories: adipocytes, osteocytes, and chondrocytes (Fig. 1).
According to Fig. 1, the results showed high expression of positive markers (CD73; 86.9%, CD90; 88.7% and CD105; 88.3%), and low expression of negative markers (CD34; 2.41% and CD45; 2.06%), which along with their high ability to differentiate into these three categories by speci c staining con rmed cell type.

Cell viability assay
Initially, a cell viability test was performed by multiple doses of apigenin (1 to 100 μM) by the MTS method (Fig.2). The results showed that in a 14-day period of cell treatment, different doses had no toxic effect.

LPS/PA caused in ammation in the MSCs.
To evaluate the response of MSCs to in ammatory agents, they were in amed by LPS/PA at different doses and the gene expression of NLRP3 along with the protein level of IL-1β was examined (Figure 3).
LPS / PA-induced in ammation was observed as a signi cant increase in NLRP3 gene expression and IL-1β levels compared with controls (p<0.001). The anti-in ammatory effect of apigenin was also investigated by examining NLRP3 gene expression and IL-1β levels under the in uence of this compound.
Statistically apigenin in both concentrations signi cantly decrease protein level of IL-1β and downregulated gene expression of NLRP3 compared to cells that didn't receive the compound (p<0.001).

LPS/PA-induced in ammasome suppresses osteogenesis in MSCs.
The effect of apigenin and LPS/PA on osteogenesis were subsequently examined (Figure 3). For this purpose, ALP activity, RUNX2 and NLPRP3 gene expression, and IL-1β protein were determined. As shown in Figure 3A, the stimulatory effect of apigenin on osteogenesis in non-in ammatory conditions was observed with increasing ALP activity (p<0.01), and increased RUNX2 expression (p<0.001), which was statistically signi cant. In ammation, induced by LPS/PA was associated with a signi cant increase in the expression of the NLRP3 gene and IL-1β protein (p<0.001; group OSX+LPS/PA v.s OSX). These in ammatory conditions were also associated with a signi cant reduction in osteogenesis, which was observed by measuring RUNX2 expression and ALP activity in comparison with the control group (p<0.001). Interestingly, also in in ammatory conditions, apigenin was able to improve ossi cation at both concentrations, citing a signi cant increase in RUNX2 expression and ALP activity, compared with the untreated group (p<0.01 v.s OSX+LPS/PA). To con rm their effects on the degree of osteogenesis, the differentiation of mesenchymal stem cells into osteocytes was evaluated by evaluating alizarin red staining. In staining, increased bone calcium staining was observed in treatment with 50µM apigenin compared to the untreated group in both in ammatory and non-in ammatory conditions.

Discussion
The hypothesis of this study was based on the relationship between in ammation and osteoporosis. Osteoporosis is a chronic disease that occurs in old age, especially in postmenopausal women, due to decreased estrogen secretion, which is associated with loss of bone mass and increased fat penetration into the bone marrow [23]. Also, the role of in ammation on bone turnover and osteoporosis has been identi ed [24], which is also strongly associated with menopause [25]. NLRP3-based in ammation has also been shown to play an important role in this process [7].
If so, targeted interventions to modulate in ammation are one of the potential strategies to optimize bone repair in the elderly [8]. Mesenchymal stem cells play an important role in bone modeling and remodeling by forming basic osteoblasts while maintaining a balance between bone formation and resorption [26].
Accordingly, in the present study, mesenchymal stem cells were in amed with LPS/PA in the osteogenic differentiation and their behavior under the in uence of in ammation was examined. As expected, in ammation disrupted the process of this differentiation. This was consistent with previous information about the association of aging with in ammation and subsequent osteoporosis [24]. Also, the effect of lipopolysaccharide-induced in ammation on inhibition of osteogenesis has been accurately expressed in other studies [27,7].
These changes in osteogenesis were characterized by changes in RUNX2 gene expression, ALP activity, and calcium deposition stained by Alizarin Red. RUNX2 transcription, as a major osteogenic factor, is a nuclear transcription factor and regulator of bone differentiation [28]. The essential role of the RUNX2 transcription factor in this process has been proven in various molecular and genetic studies [29]. ALP is an early marker of osteoporosis that plays an important role in bone formation and its peak activity indicates differentiation [30], Therefore, changes in these cases indicate an inhibitory effect of in ammation on the osteogenesis.
In the present study, the anti-in ammatory effects of apigenin were evaluated. Accordingly, while the stem cells were in amed with LPS/PA, the anti-in ammatory effects of this avonoid compound were observed via signi cant down-regulation of NLRP3 and IL-1β when compared with the non-treatment group.
The anti-in ammatory role of avonoid derivatives by inhibiting NLRP3 in ammasome has been well demonstrated [31]. Apigenin in particular has an anti-in ammatory effect by inhibiting oligomerization of in ammasome components by blocking Syk and Pyk2 signaling pathways and other pathways [31]. NLRP3 as the most important member of this complex, and sensors of pathogen-associated molecular patterns (PAMPs), facilitates interaction with caspase-1 and is necessary for IL-1β processing [32].
Therefore, down-regulation of its expression as well as IL-1β secretion showed its anti-in ammatory activity.
Apigenin, as a plant avone, has also shown its osteogenic effects by inhibiting osteoclasts, prevent bone loss, and stimulation of osteogenic differentiation of hMSCs in various studies [33,34,15]. On the other hand, as mentioned, the anti-in ammatory effect of this avonoid has been identi ed in various studies [35,31], which is done by inhibiting the NLRP3 in ammasome [35,36,31]. In the present study, the effect of in ammation on bone differentiation and the effect of apigenin on ossi cation stimulation were investigated, most likely through inhibition of in ammation. These effects were based on altering the expression of the NLRP3 in ammasome gene and the amount of IL-1β along with the stimulation of ossi cation based on the study of RUNX2 gene expression, ALP activity, and Alizarin Red staining. Therefore, the stimulatory effect of apigenin on osteogenesis can be exerted in two ways: reducing NLRP expression and in ammation, and the intrinsic effect of apigenin on differentiation.
According to previous reports, in 2005, more than 2 million people suffered from osteoporotic fractures, which will bring a lot of treatment costs by 2025 due to the aging of the population [37]. The drugs developed have two functions: inhibiting bone resorption or stimulating bone formation, which lead to many problems, from inhibiting bone formation in long-term use, gastrointestinal and hormonal problems to muscle pain, and nally osteosarcoma [38][39][40]. Therefore, the importance of developing a safer compound based on natural origin is important. Due to the limitations of the present study concerning the evaluation of adipogenic pathway, as well as using pathway inhibitors, more detailed assessments have been considered to obtain more complete results in subsequent studies.
In conclusion, our study showed the stimulatory effect of apigenin on osteogenesis in in ammatory conditions. This could be due to the anti-in ammatory effect of this compound, as well as the inherent stimulatory effect of apigenin on osteogenesis. It also con rms the role of in ammation in osteogenesis and the importance of inhibiting in ammation in this process. The importance of using avonoids with a focus on the anti-in ammatory role of these compounds was also identi ed in this study. All authors whose names appear on the submission 1) made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; 2) drafted the work or revised it critically for important intellectual content; 3) approved the version to be published; and 4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Funding
This work was supported as a master's thesis by Deputy for Research and Technology of Isfahan