Professor Jun Liu, Department of Orthopaedics, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 55 Inner Ring West Road, Guangzhou, Guangdong 510006, P.R. China
Psoralen, one of the active ingredients in
Osteoporosis is a common systemic skeletal bone disease that is mainly caused by age-related bone loss (
As understanding on the pathophysiology of osteoporosis deepens in the field of Traditional Chinese Medicine (TCM), psoralen has been reported to exert an effect on the osteogenic differentiation of BMSCs (
Therefore, the present study explores the effect of varying concentrations of psoralen on the osteogenic differentiation of human BMSCs (hBMSCs) and investigates the association between psoralen and the TGF-β/Smad3 pathway, with aims of providing a reference for the development of treatment strategies for osteoporosis.
hBMSCs (cat. no. BNCC338194) were obtained from BeNa Culture Collection. hBMSCs were cultured in DMEM (Thermo Fisher Scientific, Inc.) with 100 mg/ml streptomycin and 100 U/ml penicillin (Wuhan Boster Biological Technology, Ltd.) and 10% FBS (Sigma-Aldrich; Merck KGaA). The cells were grown in an incubator at 37˚C and 5% CO2. The medium was replaced every 3 days. The cells were digested using 0.05% EDTA and 0.25% trypsin (Sigma Aldrich; Merck KGaA) and passaged when they filled ~80% of the culture flask. hBMSCs at passage three were used in the present study.
The hBMSCs were seeded into a 96-well plate at a density of 1x104 cells/well and cultured in complete medium. After 24 h the complete medium was replaced with serum-free medium and psoralen (purity >99%; cat. no. 110739-201416; China Food and Drug Administration) was added and dissolved in DMSO. The concentrations used were 0.1, 1, 10 and 100 µmol/l, with each concentration added to 6 replicate wells per experimental repeat. The 0 µmol/l psoralen condition was used as the negative control (NC) group. When SB431542 was required, 5 μmol of SB431542 and different concentrations of psoralen (0.1, 1, 10 and 100 µmol/l) were administered at the same time and co-cultured with hBMSCs at 37˚C for 3, 7 or 14 days.
Third-passage hBMSCs were collected and seeded into 96-well plates at a density of 1x104 cells/well and co-cultured with different concentrations (0.1, 1, 10 and 100 µmol/l) of psoralen. Following cell culture for 12, 24, 36, 48 and 72 h at 37˚C, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added into each well. Cells were then incubated in the dark at 37˚C for 2 h. Absorbance values were detected at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). hBMSCs cultured with 0 µmol/l psoralen were used as a NC group. A total of 6 replicate wells were established for each group before the average values were calculated.
Third-passage hBMSCs were seeded into 96-well plates at 1x104 cells/well. Following cell adherence to the culture flask, the original medium (DMEM containing 10% FBS) was discarded and replaced with serum-free medium. Following 24 h of incubation at 37˚C, hBMSCs were co-cultured with psoralen (0.1, 1, 10 and 100 µmol/l) for 24, 48 and 72 h at 37˚C. A total of 20 µl 5 g/l MTT was added to each well and cells were incubated at 37˚C for 4 h. Subsequently, the medium was discarded, 150 µl of DMSO was added before shaking for 10 min and absorbance was measured using a microplate reader at 570 nm. A total of 6 replicate wells were established for each group and the average values were calculated.
Third-passage hBMSCs were seeded into 48-well plates. Following cell adherence to the culture flask, the DMEM was discarded and hBMSCs were incubated with different concentrations (0.1, 1, 10 and 100 µmol/l) of psoralen and osteogenic differentiation medium (cat. no. RASMX-90021; Cyagen Biosciences, Inc.) for 7 days at 37˚C. An osteogenic differentiation inducer without any treatment was used as a NC group. The medium was changed every 2 days. Subsequently, the cells were rinsed three times with PBS and treated with 150 µl 0.05% Triton X-100 for 10 min at room temperature. An ALP kit (cat. no. AP0100; Sigma-Aldrich; Merck KGaA) was used to detect the ALP activity of hBMSCs according to the manufacturer's instructions. The wavelength was set to 520 nm and a total of 6 replicate wells were established in each group. The average value was calculated.
The Alizarin Red staining method (AR-S) for calcium nodules was used to evaluate the extent of bone formation in hBMSCs. Third-passage hBMSCs were inoculated into a 6-well plate with different concentrations of psoralen (0.1, 1, 10 and 100 µmol/l) and osteogenic differentiation medium. Six replicate wells were established for each concentration. An osteogenic differentiation inducer without any treatment was used as a NC group. The medium was replaced every 2-3 days for 2 weeks at 37˚C. The cells were fixed with 4% formaldehyde for 15 min at room temperature, adjusted to pH 4.2 and stained with 0.1% Alizarin Red (Sigma-Aldrich; Merck KGaA). Cells were incubated overnight at room temperature and subsequently washed three times with PBS. Stained cells were photographed using a digital camera (Nikon Corporation) under a light microscope (magnification, x100). In order to detect calcium deposits, cells were then treated with 10% cetylpyridinium chloride (Sigma-Aldrich; Merck KGaA) in 10 mM sodium phosphate for 15 min at room temperature. A multifunctional microplate reader (Varioskan LUX; Thermo Fisher Scientific, Inc.) was used to measure the absorbance value at 560 nm in each well. The experiment was repeated three times and the average value was taken.
Third-passage hBMSCs were inoculated into a 6-well plate and co-cultured with osteogenic differentiation medium and different concentrations of psoralen (0.1, 1, 10 and 100 µmol/l) or 5 µmol SB431542 (cat. no. SF7890; Beyotime Institute of Biotechnology) for 72 h at 37˚C. Following 72 h of co-cultivation of hBMSCs with different concentrations of psoralen or SB431542, RT-qPCR was used to detect the expression of osteogenic differentiation-related genes of hBMSCs. Total RNA from the hBMSCs was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.). NanoDrop2000 was used to measure RNA concentration and purity. PrimeScript™ RT Master Mix (cat. no. RR036A; Takara Bio, Inc.) was used for reverse transcription according to the manufacturer's instructions. The following protocol was used: 37˚C for 15 min (reverse transcription reaction) and 85˚C for 5 sec (reverse transcriptase inactivation reaction). The cDNA obtained by reverse transcription was amplified on StepOne Plus (Thermo Fisher Scientific, Inc.) using the SYBR™ Green PCR Premix kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The thermocycling conditions used were as follows: 50˚C for 2 min and 95˚C for 10 min; followed by 95˚C for 15 sec and 60˚C for 60 sec for 40 cycles; and final extension at 72˚C for 1 min. Relative mRNA levels were quantified using the 2-ΔΔCq method (
After 7 days of treatment with different concentrations of psoralen or 5 µmol of SB431542, hBMSCs were lysed with RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitors, and centrifuged at 14,000 x g at 4˚C for 20 min. The supernatant was the total protein and was used to determine the protein concentration using the BCA method. Subsequently, 40 µg of the extracted protein was added per lane and separated by SDS-PAGE on a 10% gel. Proteins were then transferred onto PVDF membranes and blocked with 5% non-fat milk in PBS with 0.1% Tween-20 at 4˚C overnight. The membranes were then probed using the following primary antibodies: Osteopontin (OPN; cat. no. bs-0019P; 1:1,000; BIOSS), bone morphogenic protein 4 (BMP4; cat. no. GP20102; 1:1,000; GlpBio Technology), runt-related transcription factor 2 (Runx2; cat. no. ab114133; 1:2,000; Abcam), Osterix (cat. no. ab209484; 1:2,000; Abcam), TGF-β1 (cat. no. E1A0340C; 1:1,000; EnoGene Biotech Co., Ltd.), TGF-β Receptor I (RI; cat. no. E1A1126B-2; 1:1,000; EnoGene Biotech Co., Ltd.), Smad3 (cat. no. E1A0031B; 1:1,000; EnoGene Biotech Co., Ltd.), phosphorylated (p-) Smad3 (cat. no. E8ET1609-41; 1:1,000; EnoGene Biotech Co., Ltd.) and internal reference GAPDH (cat. no. ab8245; 1:20,000; Abcam) at room temperature for 2 h. Subsequently, the blot was incubated with the diluted secondary antibody (1:5,000; cat. no. ab205718; Abcam) and labelled with HRP at room temperature for 1 h, followed by a wash step with 0.1% TBST. Protein expression was detected using a SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Roche Applied Science). The relative value of the target protein was calculated by comparing with the corresponding internal reference. Densitometry was conducted using the ImageJ software (v1.8.0; National Institutes of Health).
SPSS 21.0 software (IBM Corp.) was used for statistical analysis. Metrological data was first evaluated for normal distribution and data conforming to a normal distribution were expressed as mean ± standard deviation. Mixed design ANOVA with Bonferroni post hoc test was used to compare differences between groups with multiple factors. One-way ANOVA with Tukey's post hoc test was used to compare differences among groups with a single factor. P<0.05 was considered to indicate a statistically significant difference.
Results from the CCK-8 assay demonstrated that elevated cell proliferation was exhibited by hBMSCs treated with psoralen at concentrations of 0.1, 1 and 10 µmol/l, with 1 µmol/l demonstrating the highest elevation in proliferative activity compared with that in the NC group. There was no significant difference in cell proliferation between concentrations of 0 and 100 µmol/l, although cell proliferation was higher at 100 µmol/l (
The effects of different concentrations of psoralen on the osteogenic differentiation of hBMSCs were evaluated by measuring ALP activity and AR-S staining of calcium deposition. The results demonstrated that low concentrations of psoralen (0.1, 1 and 10 µmol/l) significantly promoted the formation of calcified nodules and enhanced ALP activity in hBMSCs compared with those in the NC cell group. However, there was no significant difference in these two parameters tested between the high concentration (100 µmol/l) and NC groups (0 µmol/l). The most potent effect on the osteogenic differentiation of hBMSCs was found at 1 µmol/l psoralen (
The results of qPCR and western blot analysis revealed that low concentrations of psoralen promoted the expression of genes associated with osteogenic differentiation. Among these, at concentrations of 0.1, 1 and 10 µmol/l, psoralen significantly promoted the expression of Osterix and BMP4 compared with those in the 0 µmol/l NC group, whilst at concentrations of 1, 10 and 100 µmol/l, psoralen significantly promoted the expression of OPN compared with those in the 0 µmol/l NC group. In terms of Runx2, only 0.1 and 1 µmol/l of psoralen significantly promoted expression of Runx2 compared with that in the 0 µmol/l NC group (
As indicated by the aforementioned results, 1 µmol/l psoralen demonstrated the optimal effect on the osteogenic differentiation of hBMSCs and the components of the TGF-β/Smad3 pathway. Therefore, 1 µmol/l psoralen was selected for subsequent experiments. AR-S results revealed that psoralen promoted calcium deposition in hBMSCs compared with all the other groups. By contrast, SB431542 significantly inhibited the calcium deposition of hBMSCs compared with the control group, whilst psoralen markedly reversed this inhibitory effect (
The results of the western blot analysis and RT-qPCR revealed that SB431542 significantly inhibited the expression of osteogenic differentiation-related genes compared with the control group, whilst psoralen effectively reversed the inhibitory effect of SB431542 on the osteogenic differentiation of hBMSCs (
Previous studies have revealed that psoralen has a relaxant effect on smooth muscle, in addition to possessing the ability to stimulate bone formation and induce osteoblast differentiation without affecting cell proliferation (
In a previous study, psoralen was revealed to promote the osteogenic differentiation of BMSCs via the miR-488/Runx2 pathway (
ALP is an important marker of the osteogenic activity of hBMSCs and is a non-specific phosphomonoesterase that is used to determine the early osteogenic differentiation ability of hBMSCs (
To explore the underlying mechanism of psoralen in promoting osteogenic differentiation of hBMSCs further, RT-qPCR and western blot analysis were performed to probe the expression of genes associated with osteogenic differentiation, namely BMP4, OPN, Runx2 and Osterix. BMP4 belongs to the TGF-β family that has been reported to induce cartilage and bone formation and regulates mesoderm induction, tooth development, limb formation and bone fracture repair (
Signal transduction by the TGF-β superfamily of proteins serves an important role in the regulation of cell proliferation, differentiation and development in many biological systems (
To test this hypothesis, 5 mol/l TGF-β RI inhibitor SB431542(
In conclusion, the present study suggest that psoralen promotes hBMSC cell proliferation, maintain cell viability and increase the expression of genes associated with osteogenic differentiation in hBMSCs, which may be closely associated with the activation of the TGF-β/Smad3 pathway. However, the present study has some limitations, such as the blocking of TGF-β RI alone and not the Smad pathway, which may lead to insufficient results. A combination of blocking both TGF-β RI and Smad would more accurately prove that psoralen promotes the differentiation of hBMSCs into osteoblasts through the TGF-β/Smad3 pathway. In addition, it remains unclear how psoralen activates the binding of TGF-β to the receptor, therefore the precise molecular mechanism underlying this phenomenon remains to be fully elucidated and is therefore a matter for future research.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
QH, YH and JL conceived and designed the experiments; YH, LL and TJ performed the experiments; HS and XC contributed to the statistical analysis of the data. YH and QH confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effect of psoralen on cell activity. (A) Cell Counting Kit-8 assay demonstrated elevated cell viability in hBMSCs treated with psoralen at concentrations of 0.1, 1 and 10 µmol/l. (B) MTT assay revealed that with no psoralen treatment (0 µmol/l group), the cell viability of hBMSCs gradually decreased after 24, 48 and 72 h. In contrast, low concentrations of psoralen (0.1, 1 and 10 µmol/l) maintained the viability of hBMSCs following treatment for 24, 48 and 72 h. #P<0.05 vs. 0 µmol/l group. *P<0.05 vs. 1 µmol/l group. hBMSC, human bone marrow mesenchymal stem cells; OD, optical density.
Effect of psoralen on the osteogenic differentiation of hBMSCs. (A) AR-S revealed that low concentrations (0.1, 1 and 10 µmol/l) of psoralen can markedly promote the formation of calcium nodules in hBMSCs (magnification, x100). (B) Comparison of calcium deposition in different groups. (C) ALP activity was significantly enhanced by psoralen at low concentrations (0.1, 1 and 10 µmol/l). #P<0.05 vs. 0 µmol/l group. *P<0.05 vs. 1 µmol/l group. hBMSC, human bone marrow mesenchymal stem cells; ALP, alkaline phosphatase; AR-S, alizarin red S; OD, optical density.
Effect of psoralen on the expression of genes associated with osteogenic differentiation and the TGF-β/Smad3 pathway of hBMSCs. Osteogenic differentiation-related gene and protein expression analysis using (A) western blotting and (B) RT-qPCR. (C) TGF-β/Smad3 pathway activity and expression analysis using western blotting. (D) The expression of TGF-β1 and TGF-β RI using RT-qPCR. (E) Comparison of the p-Smad3/Smad3 ratio. #P<0.05 vs. 0 µmol/l group. *P<0.05 vs. 1 µmol/l group. hBMSC, human bone marrow mesenchymal stem cells; RT-qPCR, reverse transcription-quantitative PCR; RI, Receptor I; p-, phosphorylated; Runx2, runt-related transcription factor 2; BMP4, bone morphogenetic protein 4; OPN, osteopontin.
Effect of SB431542 on the osteogenic differentiation of human bone marrow mesenchymal stem cells. (A) Alizarin red S staining revealed formation of calcified nodules in the different groups (magnification, x100). (B) Comparison of calcium deposition among the different groups. #P<0.05 vs. psoralen group. *P<0.05 vs. SB431542 group. OD, optical density; NS, not significant.
Effect of SB431542 on the expression of osteogenic differentiation-related genes in human bone marrow mesenchymal stem cells. (A) The expression of BMP4, OPN, Runx2 and Osterix in hBMSCs was measured using western blot analysis. (B) The results of reverse transcription-quantitative PCR were consistent with the western blot analysis. #P<0.05 vs. psoralen group. *P<0.05 vs. SB431542 group. BMP4, bone morphogenetic protein 4; OPN, osteopontin; Runx2, runt-related transcription factor 2; NS, not significant.
Proposed model of psoralen-induced osteogenic proliferation and differentiation through the TGF-β/Smad3 pathway in human bone marrow mesenchymal stem cells. BMP4, bone morphogenetic protein 4; OPN, osteopontin; Runx2, runt-related transcription factor 2; TβR, TGF-β receptor; p, phosphorylated.
Sequences of each primer used in the present study.
Gene name | Primer pairs |
---|---|
Runx2 | F: 5'-TCTTAGAACAAATTCTGCCCTTT-3' |
R: 5'-TGCTTTGGTCTTGAAATCACA-3' | |
Osterix | F: 5'-AGAGATCTGAGCTGGGTAGAGG-3' |
R: 5'-AAGAGAGCCTGGCAAGAGG-3' | |
BMP4 | F: 5'-CTCCAAGAATGGAGGCTGTAGGAA-3' |
R: 5'-CCTATGAGATGGAGCAGGCAAGA-3' | |
OPN | F: 5'-ATCTCCTAGCCCCACAGAAT-3' |
R: 5'-CATCAGACTGGTGAGAATCATC-3' | |
TGF-β1 | F: 5'-CTGCTGACCCCCACTGATAC-3' |
R: 5'-AGCCCTGTATTCCGTCTCCT-3' | |
TGF-β RI | F: 5'-AAGATGACCGCT CTGACATCA-3' |
R: 5'-CTTATAGACCTCAGCAAAGCGAC-3' | |
GAPDH | F: 5'-ATTTGGTCGTATTGGGCG-3' |
R: 5'-TGGAAGATGGTGATGGGATT-3' |
Runx2, runt-related transcription factor 2; BMP4, bone morphogenetic protein 4; OPN, osteopontin; RI, Receptor I; F, forward; R, reverse.