Isopsoralen Ameliorates H2O2‑Induced Damage in Osteoblasts Via Activating the Wnt/Β‑Catenin Pathway

EXPERIMENTAL AND THERAPEUTIC MEDICINE 18: 1899-1906, 2019 Isopsoralen ameliorates H2O2‑induced damage in osteoblasts via activating the Wnt/β‑catenin pathway YU-PENG LI, BIN WU, JIE LIANG and FEI LI Department of Orthopedics, The People's Hospital of China Three Gorges University, Yichang, Hubei 443000, P.R. China Received July 23, 2018; Accepted May 16, 2019 DOI: 10.3892/etm.2019.7741 Abstract. Osteoporosis is a disease with a worldwide preva- osteoporosis and menopause (4), whilst one in five men will lence that involves a severe loss of bone mineral density and suffer from osteoporotic fracture during their lifetime (5). decreased microarchitecture, which increases the risk of Therefore, the treatment of osteoporosis has attracted intensive bone fracture. The present study evaluated the effects of attention worldwide. isopsoralen on osteoblastic OB-6 cells following hydrogen To date, teriparatide, strontium and bisphosphonates have peroxide (H2O2)-induced damage and investigated the been widely used for the treatment of osteoporosis by reducing molecular mechanisms involved in this process. For in vitro bone resorption and increasing bone formation (6); however, experiments, OB-6 osteoblasts were treated with H2O2 or these synthetic medicines have several side-effects (7). H2O2 + isopsoralen then the cell viability, apoptosis, reactive Therefore, novel, more effective and safer agents that can be oxygen species (ROS) production and calcium accumulation used for the treatment of osteoporosis are highly required. were determined. Results demonstrated that treatment with Notably, isopsoralen and its isoformer psoralen are isolated H2O2 reduced cell viability, runt-related transcription factor 2 from a number of fruits, vegetables and traditional Chinese (RUNX2) and osteocalcin (OCN) expression levels, and medicines (8,9), and they both display antiosteoporotic effects calcium deposition, whilst markedly increasing cell apoptosis in ovariectomy-induced osteoporotic rats (10) and in vitro (11). and ROS production. However, isopsoralen (1 µM) provided In addition, isopsoralen demonstrates osteoprotective effects as significant protection against H2O2-induced alterations in well as stimulatory effects on bone formation (12,13) through osteoblasts. In addition, isopsoralen effectively upregulated increasing strength of trabecular bone microstructure (14), protein expression of tankyrase and β-catenin which are the inducing apoptosis or necrosis of osteosarcoma (15) and main transductors of the Wnt/β-catenin pathway. Of note, the promoting the differentiation of bone marrow stromal stem protective effects of isopsoralen against H2O2-induced damage cells (BMSCs) into osteoblasts (16). However, the underlying were attenuated in OB-6 cells treated with tankyrase inhibitor osteoprotective mechanisms of isopsoralen have not been XAV-939. In conclusion, the present findings provided widely investigated. evidence that isopsoralen attenuated oxidative stress-induced The Wnt/β-catenin signaling pathway is a conserved injury in osteoblasts via the Wnt/β-catenin signaling pathway. signaling pathway with its signal transduced by β-catenin and tankyrase proteins (17,18). The alterations of Wnt/β-catenin Introduction activity are often associated with developmental disorders and diseases such as cancer, neuronal diseases and skeletal disor- Osteoporosis is a skeletal disease where systemic destruc- ders (19). Increasing evidence indicates that the Wnt/β-catenin tion of bone mass and microarchitecture occurs, which often signaling pathway is involved in the regulation of osteogenesis increases the risk of bone fractures and other complica- and bone formation (��20��,21��). In the present study, the protec- tions (1,2). Osteoporosis typically affects the elderly, with tive effects and mechanisms of isopsoralen on oxidative women more at risk, and the disease negatively impacts the stress-induced osteoblastic OB-6 cells were investigated. patient's quality of life (3). There is a direct link between Results suggested that isopsoralen could ameliorate oxidative stress-induced damage through the Wnt/β-catenin signaling pathway, indicating that isopsoralen may be a novel treatment approach for osteoporosis. Correspondence to: Dr Yu-Peng Li, Department of Orthopedics, The People's Hospital of China Three Gorges University, 4 Hudi Materials and methods Street, Yichang, Hubei 443000, P.R. China E-mail: [email protected] Cell culture and treatments. OB-6 osteoblastic cells (Cell Bank of Shanghai Institute of Biological Science) were Key words: isopsoralen, oxidative stress, osteoblast, Wnt/β-catenin cultured in α-minimum essential medium (α-MEM; Thermo pathway Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.), and incubated at 37˚C 1900 LI et al: ISOPSORALEN PROTECTS AGAINST H2O2-INDUCED DAMAGE IN OSTEOBLASTS with 5% CO2. OB-6 cells were divided into four groups Western blot analysis. Western blotting was used to deter- including the control group with α-MEM only, an isopso- mine the protein expression levels of tankyrase, Wnt3 and ralen group treated with 1 µM isopsoralen (Shanghai Meilun β-catenin in accordance to a previous study (11). Total protein Biotechnology Co., Ltd.) for 12, 24 and 48 h, a hydrogen was extracted from cells using RIPA lysis buffer (Beyotime peroxide (H2O2) group treated with 100 µM H2O2 for 12, Institute of Biotechnology) and their concentrations were 24 and 48 h or a isopsoralen + H2O2 group pretreated with determined using the BCA method. A total of 20 µg protein 1 µM isopsoralen and 100 µM H2O2 for 12, 24 and 48 h. was separated using 10% SDS-PAGE and transferred to PVDF Subsequently, cell viability, apoptosis, runt-related transcrip- membranes. The membranes were subsequently blocked with tion factor (RUNX2) and osteocalcin (OCN) expression, 5% non-fat milk for 2 h at room temperature and incubated reactive oxygen species (ROS) production, mitochondrial at 4˚C overnight with primary antibodies against tankyrase respiratory chain complex I activity, tankyrase protein (1:2,500; cat. no. sc-365897; Santa Cruz Biotechnology Inc.) expression, Wnt3, β-catenin, and calcium accumulation Wnt3α (1:2,500; cat. no. 2391; Santa Cruz Biotechnology Inc.), were analyzed, as described below. β-catenin (1:1,000; cat. no. 8480; Cell Signaling Technology, Inc.) and β-actin (1:2,500; cat. no. sc-47778; Santa Cruz Cell viability assay. OB-6 osteoblastic cells at a density of Biotechnology Inc.). Finally, membranes were incubated with 5x103 cells/ml were seeded in 96-well plates and cultured in horseradish peroxidase-conjugated anti-rabbit IgG antibody α-MEM with 1 µM isopsoralen, 100 µM hydrogen peroxide (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at (H2O2) or 1 µM isopsoralen + 100 µM H2O2. Following 37˚C for 1 h. Image J2X software version 2.1.4.7 (Rawak 12, 24, 48 h of culture, cell viability was evaluated using Software, Inc.) was used to determine protein band density. 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium Each experiment was performed in triplicate and repeated 5-carboxanilide (XTT, Sigma-Aldrich; Merck KGaA) method three times. in accordance with a previous report (10). Each experiment was performed in triplicate and repeated four times. Calcium accumulation. Calcium Colorimetric Assay kits (cat. no. MAK022; Sigma-Aldrich; Merck KGaA) were Flow cytometry. Cells at a density 5x103 cells/ml were seeded used to measure calcium deposition (expressed as µg/mg in a 96-well plate, then treated with H2O2 (100 µM) or isop- cellular protein) according to the manufacturer's instructions. soralen (1 µM) + H2O2 (100 µM) for 12, 24 and 48 h. Cell Each experiment was performed in triplicate and repeated apoptotic rate was measured by flow cytometry according to the three times. manufacturer's instructions. In brief, OB-6 osteoblastic cells were collected and fixed in 70% ethanol for 30 min at room ROS production. Intracellular ROS production was measured temperature. Cells were stained with Annexin V‑fluorescein using a CellROX™ Green Reagent (cat. no. C10444; isothiocyanate/propidium iodide (50 µg/ml) using an Invitrogen; Thermo Fisher Scientific, Inc.) according to the Annexin V-FITC Apoptosis Detection kit (Roche Molecular manufacturer's guidelines. ROS levels were measured with Diagnostics) prior to analysis using a FACScanVantage™ SE a FACSCalibur™ flow cytometer (BD Biosciences) with (BD Biosciences, San Jose, CA, USA). Each experiment was the excitation source at 488 nm and emission at 525 nm. performed in triplicate and repeated four times and data were Data were analyzed using the FloJo software version 10.1.1 analyzed using the FloJo software version 10.1.1 (FlowJo, (FlowJo LLC). Each experiment was performed in triplicate LLC). and repeated three times. Reverse transcription‑quantitative polymerase chain Mitochondrial respiratory chain complex I activity. Enzymatic reaction (RT‑qPCR). RT-qPCR was used to determine OCN activities of complex I were determined spectrophotometri- and RUNX2 mRNA expression. Total cellular RNA was cally as described in a previous study (23). Mitochondrial isolated using TRIzol reagent (Invitrogen; Thermo Fisher respiratory chain complex I activity was calculated as the Scientific) and cDNA synthesis was performed using TaKaRa rotenone-sensitive

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