Different effects of total flavonoids from Arachniodes exilis on human umbilical cord mesenchymal stem cells in vitro

Abstract Traditional Chinese medicines are used in promotion of fractured bone healing and bone diseases. Some studies reported total flavonoids from plant can be used as an auxiliary source of exogenous. Use different methods to identify and verify effects of total flavonoids from Arachniodes exilis (TFAE) on human umbilical cord mesenchymal stem cells (HUCMSCs) in vitro. Concentrations of 1 and 5 μg/mL TFAE significantly increased ALPase activity in HUCMSCs compared to the other concentrations at days 3 and 7 (P < .05). RT-PCR showed that expression levels of osteogenic genes (Col1a1, OPN, Runx2 and Osx) were remarkably enhanced in HUCMSCs following treatment with different concentrations of TFAE for 9 days compared with 0 μg/mL TFAE group (control). The results showed that concentration < 5 μg/mL of TFAE induced osteogenic differentiation in HUCMSCs Alizarin red staining assays revealed that both TFAE and S1191 was significantly decreased (7.80 ± 0.66) compared with the TFAE group (16.00 ± 0.97) (P < .01). ALPase activity on days 3 and 7 was relatively lower in HUCMSCs grown in media supplemented with both S1191 and TFAE than that of in TFAE group only. The results indicated that osteogenic markers (Col1a1, OPN, Runx2 and Osx) were significantly downregulated in the TFAE + S1191 group in comparison to the control group. The expressions of Col1a and OPN in the TFAE + S1191 group decreased significantly (P < .01) by Western blotting. TFAE promotes the odonto/osteogenic differentiation of human UCMSCs via activation of ER.


Introduction
Traditional Chinese medicines (TCMs) are used in promotion of fractured bone healing and bone diseases such as osteoporosis. [1] Many studies have shown the positive effects of TCM on bone formation in vitro and in vivo. [2] The study showed that several TCMs have bone promoting properties,The mechanism of this may be due to the angiogenic effects. [3] Recently,some scholars reported the role of total flavonoids in anti-cancer and osteogenic differentiation.Zhou et al found that ethanol extracts from Arachniodes exilis have anti-oxidant and hepato-protective roles and could potentially be used in the treatment of liver disease. [4] We previously reported that total flavonoids from arachniodes exilis (TFAE) enhances the osteogenic capacity of mesenchymal stem cells via BMP signaling. [5] The roles of flavonoids and polyphenol derivatives of these extracts have been studied, but the biological effects of various extracts are not clear. The study showed that TFAE mediates apoptosis in HepG2 cells by activating MAPK signaling, but the proliferation effect of LO 2 cells is not obvious at appropriate concentrations. [6] Some researchers proved that flavonoids derived from epimedium pubescens can promote the differentiation of rat bone marrow mesenchymal stem cells into bone cells, [7] others found that epimedium pubescens flavonoids promotes MC3T3-E1 cell proliferation and osteogenic differentiation by activating ERK-JNK signaling mediated by the estrogen receptor (ER). [8] The study showed estrogen promotes early osteoblast differentiation. [9] Little is known about the direct impact of TFAE on human umbilical cord mesenchymal stem cells (HUCMSCs).
TCM have been used in the Chinese population for the treatment of bone diseases and in promoting bone healing for thousands of years. [10] Shalan NA et al reported Noni leaf and black tea enhance bone regeneration in estrogen-deficient rats. [11] If TCMs are found to promote bone formation, these can be used in bone grafting materials that could eliminate or decrease the need to use allografts, which may be a source of transmission of disease. In this study, we present evidence that TFAE induces osteogenic differentiation of HUCMSCs. Our study provides support for the use of exogenous growth factors as remedies that could potentially be used to treat bone defects.

Isolation and culture of HUCMSCs
The research protocol was approved by the ethics committee of the School of Basic Medical Science at Jiujiang University and by the Jiujiang Maternal and Child Health Hospital. Informed consent was obtained from delivering mothers. Umbilical cords (UCs) were processed within 24 hours after delivery. Adherent cells were cultured and isolated using the explant method. Briefly, HUCMSCs were longitudinally dissected from the UC, separating them from the Wharton's jelly tissue coating. HUCMSCs were isolated and cultured following the manufacturer's instructions (Cyagen Biosciences Inc, America).
2.3. Preparation of TFAE and determination of the total flavonoids content TFAE was prepared in Jiujiang Key Laboratory of Translational Medicine (China) according to a previously published method. [9] The roots of A. exiliswere collected from Lushan city, Jiangxi province, China. A voucher specimen (No. F1205) has been deposited in the Jiujiang Key Laboratory of Translational Medicine, Jiujiang, Jiangxi, P. R. China. Determination of the Total Flavonoids Content in TFAE. The total flavonoids in TFAE were estimated as rutin equivalent. Briely, a series of rutin solutions in the concentration range of 0 to 65.0 mg/mL were used for the calibration curve and were prepared as below: 25 mL of rutin samples at certain concentrations was prepared by step by step mixing appropriate volumes of rutin stock solution with 1 mL of 5% (w/v) NaNO 2 solution, 1 mL of 10% Al(NO 3 ) 3 solution, and 10 mL of a 10% (w/v) NaOH solution, and after each adding, the mixtures were allowed to stand for 6 min. Finally, the mixtures were made up to 25 mL with distilled water and mixed well. he absorbance was measured with a spectrometer (Purkinje General Instrument Co., Ltd, Beijing, China) ater 15 min. Similarly, the TFAE sample was prepared and analyzed as above. On the basis of the calibration curve, the total flavonoids content of TFAE was calculated.

Proliferation of HUCMSCs
A CCK-8 assay was performed to detect the effect of TFAE on the viability of HUCMSCs. For this assay, cells were seeded in 96-well plates at 5 Â 10 3 cells/well. After 24 hours, cells were treated with different concentrations of TFAE (1 mg/mL, 5 mg/ mL, 10 mg/mL, or 20 mg/mL) for 24 hours , 48 hours , and 72 hours. Control cells were cultured in DMEM/F12 not supplemented with TFAE. 10 mL CCK-8 assay reagent was added to each well, and the plate was incubated for 2 h at 37°C. The absorbance (A) at 450 nm was recorded for each well using a microplate reader. Cell viability (%) was calculated.

Osteogenic differentiation of HUCMSCs with treatment of TFAE and S1191
HUCMSCs were seeded at 3 Â 10 4 cells/cm 2 in 6-well plates precoated with a gelatin solution. After 24 h, cells were treated with different concentrations of TFAE. An osteogenic differentiation medium from the Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation Basal Medium kit (HUXUC-90021, Cyagen Biosciences Inc) was used. This medium consisted of DMEM/F12 base medium (Guangzhou Cyagen Biosciences Inc) supplemented with 10% fetal bovine serum (Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation Fetal Bovine Serum, Guangzhou Cyagen Biosciences Inc), penicillin-streptomycin (100 units/mL and 100 mg/mL, respectively, Guangzhou Cyagen Biosciences Inc), 10 nM dexamethasone (Guangzhou Cyagen Biosciences Inc), 10 mM b-glycerophosphate (Guangzhou Cyagen Biosciences Inc), and 50 mg/mL Ascorbate (Guangzhou Cyagen Biosciences Inc). Four treatment groups were analyzed in triplicate: a control group (cultured in DMEM/F12 not supplemented with the osteogenic differentiation medium), a 0 mg/mL TFAE group (cultured in the osteogenic differentiation medium not supplemented with TFAE), a 1 mg/mL TFAE group (cultured in the osteogenic differentiation medium supplemented with 1 mg/mL TFAE), and Yu et al. Medicine (2020) 99: 25 Medicine a 5 mg/mL TFAE group (cultured in the osteogenic differentiation medium supplemented with 5 mg/mL TFAE). The 0 mg/mL TFAE group was used as a positive control.
To determine the potential involvement of ER in TFAEmediated osteogenic differentiation of HUCMSCs, we used the ER inhibitor S1191. Four treatment groups were analyzed in triplicate: a 0 mg/mL TFAE group (cultured in the osteogenic differentiation medium not supplemented with TFAE), a 5 mg/mL TFAE group (cultured in osteogenic differentiation medium supplemented with 5 mg/mL TFAE), a 5 mg/mL TFAE + S1191 group (cultured in the osteogenic differentiation medium supplemented with 5 mg/mL TFAE and S1191 (Sigma-Aldrich, USA), and an S1191 group (cultured in osteogenic differentiation medium supplemented with S1191). Media was changed every 3 days. Osteogenic differentiation medium without the TFAE supplement was used as control.

Alizarin red S staining
Alizarin red S staining was performed to determine bone nodule formation. After 10 days of treatment, cells were washed twice with 1 Â PBS and then fixed with 4% formaldehyde solution for 30 minutes. Next, cells were washed twice with 1 Â PBS and stained with 1 mL alizarin red solution for 5 minutes. Lastly, cells were rinsed 3 times with 1 Â PBS and visualized and imaged under a microscope.

Alkaline phosphatase activity(ALP)
An ALP assay kit (Maccura Biotechnology Co., Ltd. China,No: CH0101203)was used to determine alkaline phosphatase activity. After 3 or 7 days of treatment, media was aspirated off of the cells, 0.5 ml 0.1% TritonX-100 was added to the cells, and then, the cell lysate was collected using the ALP assay kit according to the manufacturer's recommended protocol. Absorbance values (OD) were recorded at 450 nm, the average absorbance values (DA/min) were determined, and ALP activity was calculated as follows: ALP(DU/L) = DA/min Â 2713.

Western blot
The cells were lysed with immunoprecipitation assay buffer containing protease inhibitors. The protein concentrations were examined using the BCA Protein Assay kit (Vazyme Biotech, China).40 mg of proteins were separated by sodium dodecyl

j Statistical analysis
Statistical analyses were performed using the SPSS 19.0 statistical software package (SPSS Inc.). The data was expressed as the mean ± standard deviation (SD). Differences between groups were analyzed using the Student t-test or 1-Way ANOVA. P < .05 was considered to be statistically significant.

Isolation and culture of HUCMSCs
HUCMSCs migrated from the adherent UC tissue fragments after 3 to 5 days. We successfully isolated HUCMSCs from UCs after culturing for 10 to 14 days. The cells displayed similar fibroblastlike spindle-shaped morphology and were not significantly different from the source material ( Fig. 1A-D).

Identification of HUCMSCs
HUCMSCs at passage 5 were dissociated using pancreatin, stained with isotype controls and labeled with antibodies against CD19, CD29, CD90, and CD105 for flow cytometry. The results showed that over 95% of the cells were positive for the CD90, CD29, and CD105 surface markers. Over 98% of the cells were negative for CD19 (Fig. 2).

The effect of TFAE on proliferation of HUCMSCs
The effect of TFAE on the proliferation of HUCMSCs was evaluated using the CCK-8 assay. Cell viability was also examined using the CCK-8 assay. The viability reflected the metabolic activity of living cells, and the data was expressed as a percentage of the untreated control. HUCMSCs were treated with TFAE (1 mg/mL, 5 mg/mL, 10 mg/mL, or 20 mg/mL) for 24 hours, 48 hours, and 72 hours. The results indicated that concentrations of TFAE >5 mg/mL significantly inhibited cell proliferation (P < .05), while concentrations of TFAE < 5 mg/mL significantly promoted the proliferation of HUCMSCs (Fig. 3A).

Effects of TFAE on osteogenic differentiation of HUCMSCs
To study the effect of different concentrations of TFAE on osteogenic differentiation of HUCMSCs, cells were treated with TFAE for 10 days and then stained with alizarin red to analyze calcification levels. Alizarin red staining revealed that calcification was increased in a concentration-dependent manner after treatment. 5 mg/mL TFAE was determined to be the optimal concentration and was used for the following experiments (Fig. 3B, C).
In agreement with the alizarin S staining results, ALPase activity was initially detected on days 3 and 7 after treatment with different concentrations of TFAE in HUCMSCs. Concentrations of 1 and 5 mg/mL TFAE significantly increased ALPase activity in HUCMSCs compared to the other concentrations at days 3 and 7 (P < .05) (Fig. 4A). RT-PCR showed that expression levels of  osteogenic genes (Col1a1, OPN, Runx2 and Osx) were remarkably enhanced in hUCMSCs following treatment with different concentrations of TFAE for 9 days compared with 0 mg/ mL TFAE group (control). In particular, expression levels of Col1a1, OPN, Runx2 and Osx in HUCMSCs treated with 5 mg/ mL TFAE were significantly higher on day 9 than those of 0 mg/ mL TFAE group (control). Expression levels of Col1a1, OPN, Runx2 and Osx could be upregulated by treatment with as little as 5 mg/mL (Fig. 4B, C). Western blotting was employed to detect HUCMSC Col1a1 and OPN protein levels 7 days after treatment with different concentrations of TFAE (1 mg/mL and 5 mg/mL). Col1a1 and OPN protein levels were normalized to the internal control GAPDH (Fig. 5A, B). The results showed that the expressions of Col1a1 and OPN protein was significantly increased (P < .01) in the groups treated with 1 and 5 mg/mL TFAE compared with the control group. Compared with the positive control, the Col1a1 and OPN protein levels were further increased after 7 days of treatment with 1 or 5 mg/mL TFAE, (P < .01). The results showed that concentration <5 mg/mL of TFAE induce osteogenic differentiation in HUCMSCs (P < .01). The results showed that concentration <5 mg/mL of TFAE induced osteogenic differentiation in HUCMSCs

Estrogen receptor(ER) involvement in TFAE-mediated osteogenic differentiation of HUCMSCs
To determine the involvement of ER potential in TFAE-mediated osteogenic differentiation of HUCMSCs, we investigated the effects of an inhibitor of ER (S1191, a special inhibitor of ER) on osteogenic differentiation of HUCMSCs by Alizarin red S staining, ALP activity assays and analysis of expression levels of odonto/osteogenic genes. Calcification levels in HUCMSCs treated with TFAE for 10 days were identified by alizarin red staining. Four groups were included in the staining assays: a control group (0 mg/mL TFAE), a TFAE group (5 mg/mL TFAE), a TFAE + S1191 group (5 mg/mL TFAE) and an S1191 group (0 mg/ mL TFAE). Alizarin red staining assays revealed that staining in the TFAE group (16.00 ± 0.97) was higher than in the control group (4.80 ± 0.41) and that the staining in hUCMSCs treated with both TFAE and S1191 was significantly decreased (7.80 ± 0.66) compared with the TFAE group (16.00 ± 0.97) (P < .01). The staining level in the S1191 group (5.10 ± 0.62) was found to be lower than that of the TFAE + S1191 group (7.80 ± 0.66) (P < .05) (Fig. 6A, B). ALPase activity was initially detected on day 3 in HUCMSCs and was increased on day 7 for all treatment groups. The ALPase activity for HUCMSCs was significantly higher in the TFAE group than that in the control group on days 3 and 7 (P < .05). However, ALPase activity on days 3 and 7 was relatively lower in HUCMSCs grown in media supplemented with both S1191 and TFAE than that of in TFAE group only. No significant differences were observed between HUCMSCs treated with S1191 only and the control groups. These data indicate that ER is involved in TFAE-mediated osteogenic differentiation of HUCMSCs (Fig. 6C).
Next, we examined the expressions of Col1a1 and OPN by Western blotting. The cells incubated with TFAE after 14 days, the expression levels of Col1a1 and OPN were significantly increased in the TFAE group compared with the control group (P < .01). Compared with the TFAE group, the expressions of Col1a and OPN in the TFAE + S1191 group decreased significantly (P < .01). However, there was no difference between the control group and the S1191 group. These results demonstrate that ER is involved in TFAE-mediated osteogenic differentiation of HUCMSCs (Fig. 8A, B).

Effect of ER inhibitor on ER expression in TFAEmediated osteogenic differentiation of HUCMSCs
Since TFAE has been shown to promote the osteogenic differentiation of HUCMSCs we further examined the effect of the ER inhibitor (S1191) on TFAE-mediated osteogenic differentiation of HUCMSCs. We observed the effects of TFAE on the mRNA expression of ERa and ERb in TFAE-mediated odonto/osteogenic differentiation of HUCMSCs. The results showed that there was no significant effect of TFAE on ERa and ERb mRNA expression levels after the cells treated with 1 or 5 m g/mL TFAE, which suggested that TFAE does not change the mRNA expressions of ERa and ERb, and that the ER protein is involved in TFAE-mediated osteogenic differentiation of HUCMSCs (Fig. 8C, D).

Discussion
In recent years, human umbilical cords have become an attractive source of mesenchymal stem cells (MSCs) for tissue regeneration because of no ethical considerations. MSCs are considered a promising therapeutic tool for diseases including heart failure, kidney injury, and myasthenia gravis. [12,13] We previously reported that TFAE enhances the osteogenic capacity of mesenchymal stem cells. [5] The potential beneficial biological effects of dietary flavonoids should obviously be a key concept in studies of these compounds. [14] However, the effects of TFAE on the osteogenic differentiation of HUCMSCs is unclear. Although effects of flavonoids have been extensively studied in vitro and in vivo, this study is the first to demonstrate that TFAE promotes the differentiation of HUCMSCs into bone cells.
In the present study, high TFAE concentrations (>10 mg/mL) significantly inhibited proliferation of HUCMSCs, while low concentrations of TFAE regulated cell proliferation in a dosedependent manner. To elucidate the effects of TFAE on the osteogenic differentiation of HUCMSCs, we used TFAE at a concentration <5 mg/mL to stimulate differentiation of HUCMSCs in vitro. Under such conditions, HUCMSCs can retain a stable capacity for growth, guaranteeing sustainable differentiation and enhancing ALP activity as well as the odonto/ osteogenic potential of the HUCMSCs without affecting their proliferation. As osteoblastic-specific markers, Runx2 and Osx are early-stage markers of osteoblastic differentiation, [15][16][17] the expressions of Runx2 and Osx have been found to be increased in TFAE-treated HUCMSCs, indicating that TFAE concentration under 5 mg/mL promote osteoblastic differentiation.
Runx2 overexpression can induce the differentiation of MSCs into osteoblast lineages in vitro, [18,19] while Osx is highly expressed in functional odonto/osteoblasts. [20] Thus, upregulation of Runx2 and Osx in HUCMSCs after TFAE treatment (>5 mg/mL) suggests that TFAE induces the biological differentiation changes in HUCMSCs. Our results are consistent with previous reports. [5] Col1a1 and OPN are considered to be endstage markers of bone formation, closely related to bone maturation. [21] The upregulation of Col1a1 and OPN further suggested that TFAE promotes the differentiation of HUCMSCs into bone cells. Western blotting for Col1a1 and OPN confirmed these observations. Understanding the effects of TFAE on the differentiation of HUCMSCs provides us with the opportunity to explore the molecular mechanisms of TFAE-mediated osteoblastic differentiation. Okazaki et al [22] reported that estrogen can promote the differentiation of rat BMSCs through ER. Studies have found that some plant sources of phytoestrogens have the same effect as estrogen did. Some flavonol compounds have a phytoestrogens effect [23,24] Song [9] et al found that epimedium promotes MC3T3 E1 cell proliferation and can be mediated by ER via activation of ERK and JNK signaling pathways, promoting osteogenesis differentiation.
In this study, we found that a specific ER inhibitor (S1191) can reverse the osteogenic differentiation of HUCMSCs caused by TFAE treatment. The results of Alizarin red S staining and ALP activity assays suggest that ER is activated in TFAE-treated HUCMSCs. Furthermore, inhibition of ER by S1191 treatment dramatically suppressed the mRNA expressions of the odonto/ osteogenic markers Runx2, Osx, Col1a1, and OPN. To this regard, we next analyzed protein levels of the osteogenic Col1a1 and OPN after ER suppression by S1191. The results were consistent with the results of the mRNA analyses. Some reports have shown that S1191 competitively inhibits binding of oestradiol to the ER. S1191 blocks nuclear localization of ER by impairing receptor dimerization, [25] and binds to ERa leading to decreased ERa stability and increases turnover of the ERa protein through enhanced proteasomal degradation. [26,27] In addition, S1191 down-regulates androgen receptor expression and diminishes androgenic responses in LNCaP human prostate cancer cells. [28] It is interesting to note the effects of S1191 on the expression of osteogenic markers in this study. These data suggest that ER is involved in TFAE-mediated osteogenic differentiation of HUCMSCs. However, we found that mRNA expression level of ER was not affected in TFAE-treated HUCMSCs. Some studies indicated that flavonoids may reduce estrogen receptor binding capacity by binding to nuclear type II estrogen binding sites. [29,30] Studies have reported that the majority of ER a and b are found in the cytoplasm and nucleus. The cytoplasmic estrogen-ER complex translocates into the nucleus and binds to estrogen response elements located in the target gene promoters, stimulating gene transcription. [31,32] Our results indicate that the binding of ER to TFAE influences gene transcription in TFAEmediated osteogenic differentiation of HUCMSCs but that TFAE does not affect ER expression at the mRNA level. Further investigation of interactions between TFAE and ER is warranted. Although the underlying mechanism remains unclear, this information may provide new insights. Based on the present findings, it can be inferred that ER plays a pivotal role during committed differentiation of TFAE-treated HUCMSCs.

Conclusions
TFAE promotes the odonto/osteogenic differentiation of human UCMSCs via activation of ER. The findings provides a strong evidence that the therapeutic application of TFAE resultes in the HUCMSCs's osteogenic differentiation. Further studies are warranted to investigate other potential mechanisms associated with TFAE-mediated osteogenic differentiation of HUCMSCs.
Author contributions