Low Concentration Flufenamic Acid Enhances Osteogenic Differentiation
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Liu et al. Stem Cell Research & Therapy (2019) 10:213 https://doi.org/10.1186/s13287-019-1321-y RESEARCH Open Access Low concentration flufenamic acid enhances osteogenic differentiation of mesenchymal stem cells and suppresses bone loss by inhibition of the NF-κB signaling pathway Xuenan Liu1, Zheng Li1, Hao Liu1, Yuan Zhu1, Dandan Xia2, Siyi Wang1, Ranli Gu1, Weiliang Wu3, Ping Zhang1*, Yunsong Liu1* and Yongsheng Zhou1 Abstract Background: As the representative of fenamic acids, an important group of NSAIDs, flufenamic acid (FFA) has been used for anti-inflammation and analgesia in the clinic. Recently, researches have focused on the role of some members of NSAIDs in promoting osteogenesis. However, little attention has been paid to the subgroup of fenamic acids, and it remains unclear whether FFA and other fenamic acids could regulate mesenchymal stem cells’ (MSCs) lineage commitment and bone regeneration. Methods: Here we treated two kinds of human MSCs with FFA at different concentrations in vitro and examined the effect of FFA on osteogenic differentiation of human MSCs. This was followed by heterotopic bone formation assay in nude mice. In addition, ovariectomized and aged mice were used as osteoporotic models to test the effect of FFA on osteoporosis. Besides, activators and inhibitor of nuclear factor-κB(NF-κB) signaling pathway and western blot were used to clarify the mechanism of the promoting effect of low concentration FFA on osteogenesis. Results: Our results indicated that low concentrations of FFA could significantly enhance osteogenic differentiation of human MSCs in vitro, as well as in vivo. In addition, FFA treatment suppressed bone loss in ovariectomized and aged mice. Mechanistically, FFA at low concentrations promoted osteogenesis differentiation of human MSCs by inhibition of the NF-κB signaling pathway. Conclusions: Collectively, our study suggested that low concentration FFA could be used in bone tissue engineering or osteoporosis by promoting osteogenic differentiation of human MSCs. Keywords: Flufenamic acid, Mesenchymal stem cells, Osteogenesis, Osteoporosis, Nuclear factor-κB Background in men [1], leading to low bone strength, caused by low As a common disease that can often be seen in the clinic, mineral density, architectural deterioration of the bone osteoporosis brings health and economic burdens to pa- structure, and susceptibility to fracture [2]. Previous studies tients and remains a clinical challenge. It is usually caused have shown that abnormal in osteogenic differentiation by estrogen deficiency in menopausal women or by aging capacity of bone marrow-derived mesenchymal stem cells (BMMSCs) contributes to these structural abnormalities * Correspondence: [email protected]; [email protected] [3–5], which is mainly caused by loss of bone homeostasis. 1Department of Prosthodontics, Peking University School and Hospital of Stomatology, National Laboratory for Digital and Material Technology of Non-steroidal anti-inflammatory drugs (NSAIDs) are Stomatology, Beijing Key Laboratory of Digital Stomatology, National Clinical commonly used in the clinic and can inhibit the synthe- Research Center for Oral Diseases, 22 Zhongguancun South Avenue, Beijing sis of prostaglandin by inhibiting cyclooxygenases 100081, People’s Republic of China Full list of author information is available at the end of the article (COXs) to achieve the anti-inflammatory and analgesia © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Stem Cell Research & Therapy (2019) 10:213 Page 2 of 14 effects [6–8]. However, the members of the NSAIDs are from Sigma-Aldrich (St. Louis, MO, USA) unless other- quite different in their chemical structures and other wise stated. This study was approved by the Institutional properties, which may cause different effects in addition Animal Care and Use Committee of the Peking Univer- to anti-inflammatory and analgesia effects. According to sity Health Science Center (LA2014233), and all experi- their chemical structure, NSAIDs can be divided into ments were performed in accordance with the approved several groups, for instance, salicylic acids, anilines, guidelines. acetic acids, oxicams, and fenamic acids [9]. Some mem- FBS, MEM, DMEM, and 100× penicillin and strepto- bers of NSAIDs have been proven to have complex ef- mycin mixture were purchased from Gibco (Grand Is- fects on the biological behaviors of mesenchymal stem land, NY, USA). Human BMMSCs and ASCs were cells (MSCs) and bone homoeostasis, such as aspirin cultured in proliferation medium (PM), consisting of [10], ibuprofen, and indomethacin [11]. However, little 10% (v/v) FBS, penicillin/streptomycin, and fresh MEM attention has been paid to the important group of (for hBMMSCs) or DMEM (for hASCs), with 5% CO2 fenamic acids. In particular, the effect of fenamic acids atmosphere at 37 °C. The OM comprised fresh DMEM on MSC fate has not been reported. or MEM containing 10 nM dexamethasone, 10 mM β- A variety of transcription and signaling factors partici- glycerophosphate, 0.2 mM L-ascorbic acid, 10% (v/v) pate in lineage commitment of MSCs toward osteogenic FBS, and penicillin/streptomycin. TNF-α was purchased cells, including the nuclear factor kappa B (NF-κB) signal- from R&D Systems (Minneapolis, MN, USA), and ing pathway. As a regulator of immune and inflammatory BAY117082 was purchased from Selleck (Houston, TX, signals [12, 13], the NF-κB signaling pathway plays an im- USA). Cells at the fourth to sixth passage were used for portant role in regulating the differentiation and function the in vitro experiments. of osteocytes and in bone metabolism [14–18]. Activation of this pathway can prevent osteogenic differentiation of Preparation of concentrated FFA solution MSCs [14] and inhibit osteoblastic SMAD activation [18] Flufenamic acid was first dissolved in DMSO to obtain a − and the synthesis of matrix protein in bone [19], as well as concentrated solution at 200 mmol L 1, to test the effect suppressing postnatal bone formation in vivo [15]. Be- of FFA at different concentrations on the differentiation sides, several genes participate in bone metabolism of hMSCs, and to choose the optimal concentration for through the NF-κB signaling pathway [20, 21]. However, osteogenic differentiation of hMSCs in vitro. whether fenamic acids can affect the functions of MSCs through the NF-κB signaling pathway remains unknown. Cell proliferation assay In the present study, we aimed to explore the in vitro Human BMMSCs or ASCs were seeded in 12-well plates and in vivo effects of FFA, a representative of the at 2 × 104 per well. After that, the cells were cultured in fenamic acids of NSAIDs, on the osteogenic differenti- PM, or PM with 25, 50, 100, and 200 μM FFA, respect- ation of hMSCs and its potential mechanism. The in ively. Three wells of cells in the same treatment method vivo effects of FFA in an osteoporotic animal model were tested daily from day 1 to day 7. The cells were were also involved. Our results demonstrated that low counted using a Cell Counting Kit-8 (CCK8, Dojindo La- concentration FFA promoted osteogenic differentiation boratories, Kumamoto, Japan), according to the manu- of hMSCs in vitro and the effect declined with the con- facturer’s instructions and growth curves of the cells centration increasing. The best concentration for osteo- were obtained according to the cell number. genesis promoting was 50 μM. Fifty micromolar FFA could also promote osteogenic differentiation of hMSCs Alkaline phosphatase staining in vivo. Besides, 50 μM FFA suppressed bone loss in Human BMMSCs or ASCs were seeded in 6-well plates OVX mice and aged mice. Mechanistically, FFA pro- at the same initial density. After 7 days of culture in PM, moted osteogenesis through inhibition of NF-κB signal- OM, or OM with different concentrations of FFA, the ing pathway. Overall, our study provided valuable clues cells were washed with PBS, fixed in 95% cold ethanol, for the potential use of FFA to treat bone metabolic dis- and washed with PBS again. An alkaline phosphatase ease and in bone tissue engineering. (ALP) Staining Kit (CWBIO, Beijing, China) was used for staining, according to the manufacturer’s instruc- Methods tions. The cells were then gently washed with distilled Culture and osteogenic induction of hMSCs water, and images were obtained using a scanner. Primary human BMMSCs and human adipose-derived mesenchymal stem cells (hASCs) were purchased from Quantification of ALP activity ScienCell Company (San Diego, CA, USA). All cell- Human BMMSCs or ASCs were seeded in 6-well plates based in vitro studies were repeated three times using at the same initial density. After 7 days of culture in PM, MSCs from three donors. All materials were purchased OM, or OM with different concentrations of FFA, the Liu et al. Stem Cell Research & Therapy (2019) 10:213 Page 3 of 14 cells were gently washed with PBS first. Then, the cells TGGT-3′; IL8,(forward)5′-ACCACACTGCGCCAACA were collected and centrifuged at 13362×g for 30 min at CAG-3′ and (reverse) 5′-TGCACCCA GTTTTCCT 4 °C after being lysed with 1% Triton X-100 on ice. A TGGGG-3′;andICAM1,(forward)5′-TGCACCCAGT BCA protein assay kit (Pierce Thermo Scientific, Wal- TTTCCTTGGG G-3′ and (reverse) 5′-GGCGCCGG tham, MA, USA) was used to measure the total protein AAAGCTGTAGAT-3′. concentration, according to the manufacturer’s instruc- tions. An ALP assay kit (Nanjing Jiancheng Bioengineer- ing Institute, Nanjing, China) was used to measure the Western blot ALP activity, and the ALP activity was normalized to the First, hBMMSCs were washed with cold PBS softly for total protein content of each sample.