Nrf2 signaling pathway

Journal Pre-proof Metformin promotes osteogenic differentiation and protects against oxidative stressinduced damage in periodontal ligament stem cells via activation of the Akt/Nrf2 signaling pathway Linglu Jia, Yixuan Xiong, Wenjing Zhang, Xiaoni Ma, Xin Xu PII:

S0014-4827(19)30596-8

DOI:

https://doi.org/10.1016/j.yexcr.2019.111717

Reference:

YEXCR 111717

To appear in:

Experimental Cell Research

Received Date: 10 August 2019 Revised Date:

4 November 2019

Accepted Date: 7 November 2019

Please cite this article as: L. Jia, Y. Xiong, W. Zhang, X. Ma, X. Xu, Metformin promotes osteogenic differentiation and protects against oxidative stress-induced damage in periodontal ligament stem cells via activation of the Akt/Nrf2 signaling pathway, Experimental Cell Research (2019), doi: https:// doi.org/10.1016/j.yexcr.2019.111717. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Metformin promotes osteogenic differentiation and protects against oxidative stress-induced damage in periodontal ligament stem cells via activation of the Akt/Nrf2 signaling pathway

Linglu Jia1,2, Yixuan Xiong1,2, Wenjing Zhang1,2, Xiaoni Ma1,2*, Xin Xu1,2*

1. School of Stomatology, Shandong University, Jinan, China 2. Shandong provincial key laboratory of oral tissue regeneration, Jinan, China

*

Corresponding author： ：

Xin Xu ([email protected]), No. 44-1, Wenhua Xi Road, Jinan, Shandong, 250012 P.R. China Tel./Fax: +86-531-88382923 Xiaoni Ma ([email protected]), No. 44-1, Wenhua Xi Road, Jinan, Shandong, 250012 P.R. China Tel./Fax: +86-531-88382923

Declarations of interest: none

Abstract Periodontal ligament stem cell (PDLSC)-based tissue engineering is an important method for regenerating lost bone in periodontitis. Maintaining or enhancing the osteogenic differentiation of PDLSCs, as well as enhancing the resistance of PDLSCs to oxidative stress, is necessary in this process. As a common hypoglycemic drug, metformin has been reported to have multiple effects on cell functions. This study found that low concentrations of metformin did not affect cell proliferation but did inhibit adipogenic differentiation and promote osteogenic differentiation of PDLSCs. This positive effect was associated with activation of Akt signaling by metformin. Moreover, applying metformin as either a pretreatment or co-treatment could reduce the amount of reactive oxygen species, enhance antioxidant capacity, and rescue the cell viability and osteogenic differentiation that were negatively affected by H2O2-induced oxidative stress in PDLSCs. In addition, metformin was found to activate the Nrf2 signaling pathway in PDLSCs, and knockdown of Nrf2 by siRNA impaired the protective effect of metformin. Taken together, these results indicate that metformin not only promotes osteogenic differentiation of PDLSCs, but also protects PDLSCs against oxidative stress-induced damage, suggesting that metformin could be potentially useful in promoting PDLSC-based bone regeneration in the treatment of periodontitis.

Key words Metformin; periodontal ligament stem cells; osteogenesis; oxidative stress

1 Introduction Periodontitis is a chronic inflammation of periodontal supporting tissues that is highly prevalent in adults [1]. The progressive inflammation could lead to the destruction and loss of the periodontal ligament, the cementum and the alveolar bone, which could ultimately result in tooth loss [1, 2]. Realizing the regeneration of destroyed tissue is the major goal of periodontal therapy, especially alveolar bone, which supports holding the teeth in the jaw [3]. Stem cell-based tissue engineering and therapy technology provide a new strategy for repairing periodontal supporting tissues [3]. Periodontal ligament stem cells (PDLSCs), which exist in the periodontal ligament and have strong self-renewal and multidifferentiation abilities, are regarded as potential “seed cells” for alveolar bone regeneration [4]. Several studies proved that the transplantation of in vitro expanded PDLSCs and scaffold materials to the defect could effectively promote alveolar bone regeneration in animal models [5-7]. The osteogenic differentiation ability of seed cells is essential for regeneration, so exploring methods to maintain or enhance this differentiation of PDLSCs is an important part of PDLSC-based therapy. Recently, an increasing number of studies have shown that the periodontal microenvironment of patients with periodontitis is in a state of oxidative stress, which is an imbalance between the production of reactive oxygen species (ROS) and the endogenous antioxidant system [8-10]. ROS are various oxidative substances with high oxidizability and reactive properties that are produced during the process of aerobic metabolism. Cells have an antioxidant system to combat with ROS, including some antioxidant enzymes such as the superoxide dismutase (SOD) enzyme family, heme oxygenase-1 (HO-1) and NADPH dehydrogenase quinone 1 (NQO1), as well as some nonenzyme substances, such as glutathione (GSH) [11]. In patients with periodontitis, local bacterial stimulation and a series of inflammatory reactions are reported to induce the excessive production of ROS, which could exceed the antioxidant capacity of cells and damage cell viability [12, 13]. For stem cells, large amounts of ROS are cytotoxic and could impair their self-renewal and multidifferentiation abilities, which is not conducive to their application in tissue regeneration [14, 15]. Therefore, PDLSC-based periodontal bone regeneration should incorporate antioxidant stress into treatment strategies, so exploring methods to protect PDLSCs from potential ROS damage is necessary. Metformin (1,1-dimethylbiguanide hydrochloride) is an oral hypoglycemic agent that is widely used for the treatment of type 2 diabetes mellitus [16]. Recently, some other functions of metformin have been

gradually explored, such as antitumor, immunoregulatory and anti-inflammatory activities [17-19]. It is worth noting that metformin was reported to regulate the multidifferentiation abilities of several types of stem cells [20-22], while its effects on PDLSCs have not been fully elucidated. Moreover, several studies revealed that metformin could influence the intracellular ROS level of some cancer cells [23] or adult tissue cells [24], but whether metformin affects the oxidative stress condition of PDLSCs and the underlying mechanisms are not known. The serine/threonine kinase Akt (also known as protein kinase B or PKB) signaling cascades have been reported to play critical roles in regulating cellular functions [25]. Several membrane receptors can transmit signals to induce Akt phosphorylation, which leads to its activation and results in its regulation of a variety of downstream target proteins. Numerous studies have shown that Akt signaling could regulate the multidifferentiation of stem cells [26, 27]. Additionally, Akt was reported to promote the activation of Nrf2 [28, 29], which is an important transcription factor against oxidative stress in cells [30]. Activated Nrf2 can translocate from the cytoplasm into the nucleus and then bind the antioxidant response element (ARE) to promote the transcription of antioxidant enzymes, such as SOD, HO-1 and NQO1, and further promote the production of antioxidant substances, such as GSH [31]. Several studies reported that metformin could affect the activation of Akt or Nrf2 [32, 33], so we wondered whether metformin could regulate the biological behaviors of PDLSCs via these signaling pathways. This study explored the potential beneficial effects of metformin on PDLSC-based bone regeneration. First, the regulation of PDLSCs proliferation and multi-differentiation by metformin was analyzed. Next, the protective effects of metformin on PDLSCs against H2O2-induced oxidative stress and the underlying mechanisms associated with the AKt-Nrf2 signaling pathway were investigated. We hope that the results provide a theoretical basis for the application of metformin in PDLSC-based bone regeneration.

2 Material and methods 2.1 Cell isolation and culture The present study was approved by the Medical Ethical Committee of the School of Stomatology, Shandong University. Six healthy premolars that were extracted for orthodontic reasons from different donors (aged 16-24) were collected at the Stomatological Hospital of Shandong University. The periodontal ligament tissues from the middle third of the tooth root were scraped off and minced into small pieces and then digested in a solution of 3 mg/ml collagenase type I (Sigma-Aldrich, St. Louis, MO, USA)

and 4 mg/ml dispase (Sigma-Aldrich) at 37°C for 1 h . The cell suspension was plated into culture dishes containing complete culture medium in 5% CO2 at 37°C. The complete culture medium consisted of 90% α-MEM (BI, Beit Haemek, Israel) and 10% fetal bovine serum (FBS) (BI) and was replaced every 3 days. Cells were passaged after reaching 90% confluence, and cells at passages 3-6 were used for the experiments. 2.2 Phenotype analysis of PDLSCs by flow cytometry and colony formation assay The BD Stemflow™ hMSC Analysis kit (BD Biosciences, New Jersey, USA) was used to analyze the immunophenotype of hPDLSCs according to the instructions. In brief, the cells were trypsinized and washed with PBS and then incubated with fluorescent dye conjugated antibodies in the dark at 4°C for 30 min. The antibodies included CD90-FITC, CD44-PE, CD105-PerCP-Cy, CD73-APC and PE-negative cocktail (CD34PE, CD11b PE, CD19 PE, CD45 PE and HLA-DR PE). The respective isotype control was used as a systemic negative control. Flow cytometry was performed with a BD Accuri™ C6 flow cytometer. For colony formation assay, cells were seeded into 6 well culture-plate (200 cells per well) and cultured in the complete culture medium. 10 days later, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Solarbio, Beijing, China). Aggregates with more than 50 cells viewed under the microscope were regarded as a colony. 2.3 Multidifferentiation assay of PDLSCs To verify the multilineage differentiation capacity of PDLSCs, cells were cultured in osteogenic, adipogenic or chondrogenic medium for induction. The osteogenic medium contained complete culture medium supplemented with 10 nM dexamethasone (Solarbio), 10 mM β-glycerophosphate (Solarbio) and 50 mg/l ascorbic acid (Solarbio). After osteogenic induction for 21 d, cells were fixed with 4% paraformaldehyde and incubated with Alizarin Red solution (Sigma-Aldrich) to detect the formation of mineralized matrix. The adipogenic medium contained complete culture medium supplemented with 1 µM dexamethasone (Solarbio), 0.2 mM indomethacin (Solarbio), 0.01 g/l insulin (Solarbio) and 0.5 mM isobutyl-methylxanthine (Solarbio). After 28 d of adipogenic induction, cells were fixed with 4% paraformaldehyde and stained with the Oil Red O (Solarbio) to detect the formation of lipid droplets. For chondrogenic induction, cells were cultured in the chondrogenic medium (Cyagen, Santa Clara, CA, USA) using a particle culture method according to the manufacturer’s instructions. After 28 d,

PDLSCs were stained with the Alcian Blue solution (Cyagen) to detect the formation of cartilage. 2.4 Cell treatments To determine the effects of metformin on cell proliferation, PDLSCs were cultured in complete culture medium containing different concentrations of metformin (Solarbio) (0, 10, 100, 500, 1000, or 2500 µM), and cell viability was evaluated on days 1, 2, 3, 4 and 5. To evaluate the effects of metformin on osteogenic and adipogenic differentiation of PDLSCs, cells were cultured in osteogenic or adipogenic medium supplemented with different concentrations of metformin (0, 10, 100, 500, or 1000 µM). LY294002 (#9901, Cell Signaling Technology, CST, Danvers, MA, USA), an inhibitor of Akt signaling, was added into the medium according to the experimental design. To investigate the influence of oxidative stress on cell viability, PDLSCs were treated with complete culture medium containing different concentrations of H2O2 (0, 10, 50, 100, 500, 250, 300, 350, or 400 µM) for 24 h. Then, the cell viability was determined. To detect the protective effects of metformin against oxidative stress, PDLSCs or PDLSCs with Nrf2 knockdown were pretreated with metformin before with the H2O2 exposure, or they were cotreated with both metformin and H2O2 simultaneously. For pretreatment, PDLSCs were cultured in complete culture medium containing metformin (100, 500, or 1000 µM) for 24 h and then treated with H2O2 (250 µM) for another 24 h. For co-treatment, PDLSCs were cultured in the complete culture medium containing both metformin (100, 500, or 1000 µM) and H2O2 (250 µM) for 24 h. PDLSCs continuously cultured in complete culture medium without metformin or H2O2 were regarded as negative controls, and cells cultured in the medium with H2O2 (250 µM) for 24 h were regarded as positive controls. Then, the cells of each group were assayed for cell viability and osteogenic differentiation. 2.5 Cell viability assay The cell counting Kit-8 kit (CCK8) (Dojindo Laboratories, Kumamoto, Japan) was used to detect the cell viability based on the manufacturer’s instructions. Briefly, PDLSCs were seeded in 96-well plates (2000 cell/well) and received different treatments according to the experimental design. At the detection time, cells were incubated for 2 h with compete culture medium containing 10% CCK8 reagent, then the absorbance value at 450 nm was measured using a microplate reader (SPECTROstar Nano; BMG Labtech, Germany). The ratio of the absorbance value of each group to the control group was calculated when needed. 2.6 Osteogenic differentiation assay

PDLSCs were cultured in osteogenic medium, and their osteogenic differentiation abilities were evaluated through the alkaline phosphatase (ALP) activity measurement, ALP staining, Alizarin Red staining, western blot and qRT-PCR experiments. For determining the ALP activity, PDLSCs were analyzed after osteogenic induction for 7 and 14 d using an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. For ALP staining, PDLSCs were fixed with 4% paraformaldehyde and stained using an NBT/BCIP staining kit (Beyotime) on days 7 and 14. For Alizarin Red staining, PDLSCs were fixed with 4% paraformaldehyde and incubated with the Alizarin Red solution (Sigma-Aldrich) at 21 d. Then, the mineralized matrix was dissolved in 10% cetylpyridinium chloride (Solarbio), and the absorbance value at 562 nm was measured using a microplate reader. For western blot and qRT-PCR experiments, the expression of osteogenesis related genes, including ALP, type 1 collagen (COL1) or osteocalcin (OCN), was evaluated on day 7. 2.7 Adipogenic differentiation assay PDLSCs were cultured in adipogenic medium. After induction for 14 d, the expression of adipogenesis-related genes was detected through qRT-PCR; the analyzed genes included peroxisome proliferator-activated receptor γ (PPARγ), lipoprotein lipase (LPL) and CCAAT/enhancer binding protein Alpha (C/EBPα). On day 28, PDLSCs were fixed with 4% paraformaldehyde and stained with the Oil Red O (Solarbio). 2.8 Intracellular ROS and antioxidation assay PDLSCs were pretreated with metformin (100, 500, or 1000 µM) for 12 h before stimulation with H2O2 (250 µM) for 6 h, or they were cotreated with both metformin (100, 500, or 1000 µM) and H2O2 (250 µM) for 6 h. PDLSCs continuously cultured in the complete culture medium without metformin or H2O2 were regarded as negative controls, and cells cultured in the medium with H2O2 (250 µM) for 6 h were regarded as positive controls. Then, all the cell samples were assessed for ROS and antioxidant activity. To analyze the intracellular oxidative stress state, the level of intracellular ROS was assessed using the Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instruction. Briefly, cells were incubated with a 10 µM fluorescent probe, DCFH-DA, for 30 min in the dark, and then they were washed with PBS. Then, the cells were observed using a fluorescence microscope (Olympus IX73, Tokyo, Japan) In addition, cells incubated with DCFH-DA were collected and analyzed by flow cytometer (BD Accuri TM, USA).

To analyze the antioxidant capacity of cells, the intracellular SOD activities and GSH levels were measured. For SOD activity detection, a SOD assay kit (Nanjing Jiancheng Bioengineering Institute) was used. For GSH level detection, a GSH assay kit (Nanjing Jiancheng Bioengineering Institute) was used. All of these methods followed the manufacturer's instructions. 2.9 Protein extraction and western blot To extract the total proteins from cell samples, cells were washed with PBS and lysed in RIPA buffer (Solarbio) supplemented with 1% PMSF (Solarbio) and 1% phosphatase inhibitor (Bosterbio, Wuhan, China) on ice, and they were subsequently lysed by ultrasound. After centrifugation at 12000 rpm at 4 °C for 15 min, the supernatant containing the total protein was obtained. To extract the nuclear proteins, a Nuclear and Cytoplasmic Protein Extraction Kit (Bosterbio) was applied according to the manufacturer’s instructions. The concentration of each protein solution was measured by the BCA Protein Assay Kit (Solarbio). Then, the proteins from each group were separated by SDS–PAGE gel and transferred to polyvinylidene fluoride membranes. After being blocked with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C and further incubated with secondary antibodies for 1 h at room temperature. Finally, the signals on the membrane were visualized with the enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) under an ECL chemiluminescence detection system (Amersham Imager 600, USA). The band intensities were analyzed using ImageJ software, and the expression of target proteins was normalized to GADPH or Histone-H3 levels. The following antibodies were used:COL1A1 (#84336, CST), RUNX2 (ab23981, Abcam, Cambridge, MA, USA), Akt (pan) (#4691, CST), phospho-Akt (Ser473) (p-Akt) (#4060, CST), Nrf2 (ab62352, Abcam), Histone-H3 (17168-1-AP, Proteintech, Chicago, IN, USA) and GAPDH (HRP-60004, Proteintech). Histone H3 and GAPDH were used as internal controls. 2.10 RNA isolation and quantitative real-time PCR (qRT-PCR) analysis Total RNA was extracted using the RNAios Plus reagent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The total RNA was reverse transcribed to generate cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara), and the qRT-PCR reactions were performed in a 10 µl reaction volume with the TB Green PCR Core Kit (Takara) and the Roche LightCycler® 480II following the manufacturer’s instructions. Changes in gene expression were calculated by the 2 method. The primers used in this study are listed in Supplementary Table 1. 2.11 Nrf2 interfering

∆∆CT

Small interfering RNA (siRNA)-targeted Nrf2 (siNrf2) was designed and synthesized by GenePharma Corporation (Shanghai, China) and was transfected into PDLSCs with Micropoly-transfecterTM cell reagent (Micropoly, Jiangsu, China) to knockdown the expression of Nrf2. PDLSCs transfected with negative control siRNA (siNC) were regarded as the control group. qRT-PCR and western blot experiments were used to detect the knockdown efficiency. Then, the siNC PDLSCs and siNrf2 PDLSCs were analyzed in intracellular ROS assays, antioxidation assays and osteogenic differentiation assays according to the experimental design. 2.12 Statistical analysis All experiments were repeated at least three times, and all data are presented as the means ± the standard deviations. Student’s t test or one-way ANOVA was used to determine significant differences between groups. The statistical analyses were conducted by GraphPad software, and values of P < 0.05 were considered statistically significant.

3 Results 3.1 Culture and identification of PDLSCs PDLSCs were successfully isolated, cultured and passaged. The cultured PDLSCs presented fibroblast-like morphology and were arranged in whirlpool formations (Supplementary Figure 1A). Colony formation assay (Supplementary Figure 1F) and CCK-8 assay (Supplementary Figure 1G) showed that PDLSCs owned strong colony-formation ability and proliferation activity. In the multidifferentiation assay, Alizarin Red–positive mineralized matrix (Supplementary Figure 1B), Oil Red O–positive lipid droplets (Supplementary Figure 1C) and Alcian Blue–positive cell aggregates (Supplementary Figure 1D) were observed after osteogenic, adipogenic, and chondrogenic induction, respectively, indicating that PDLSCs could differentiate into osteoblasts, adipocytes and chondrocytes. The immunophenotype assay showed that PDLSCs expressed MSC-specific surface markers (CD90, CD105, CD73 and CD44) and did not express hematopoietic and endothelial cell-specific markers (CD34, CD11b, CD19, CD45 and HLA-DR) (Supplementary Figure 1E). The above results indicated that the isolated PDLSCs exhibited the characteristics of mesenchymal stem cells. 3.2 Effects of metformin on the proliferation of PDLSCs PDLSCs were cultured in complete culture medium containing 0, 10, 50, 100, 500, 1000 or 2500 µM metformin for 5 d. The results of the CCK8 assay showed that low concentrations of metformin (≤1000 µM)

had no significant influence on the proliferation of PDLSCs, while high concentrations of metformin (2500 µM) slightly inhibited cell proliferation (Figure 1A). Thus, metformin was used at concentrations of less than 1000 µM in the following studies. 3.3 Metformin promoted the osteogenic differentiation of PDLSCs To study the influence of metformin on osteogenic differentiation of PDLSCs, cells were incubated in the osteogenic medium supplemented with 0, 10, 100, 500 or 1000 µM metformin for 21d. The results of the ALP activity assay and ALP staining assay showed that metformin at 10, 100, and 500 µM significantly increased the ALP activity of PDLSCs on days 7 and 14 (Figure 1B, C, D). The positive effects of 1000 µM metformin on ALP activity were smaller than what was observed for the other concentrations on day 7, while on day 14, the 1000 µM treatment led to a decrease in ALP activity (Figure 1B, C, D). Alizarin red staining and quantitative analysis proved that PDLSCs stimulated with metformin produced more mineralized matrix than the control group (Figure 1G, H). Among different concentrations, 100 µM metformin showed the greatest positive effects on the mineralized matrix formation (Figure 1G, H). In addition, metformin significantly improved the mRNA level of ALP, COL1, RUNX2 and OCN (Figure 1E) and the protein level of COL1 and RUNX2 (Figure 1F), and the concentrations of 10, 100 and 500 µM had a stronger positive effect than 1000 µM did. Taken together, these results showed that 10, 100 and 500 µM metformin promoted the osteogenic differentiation of PDLSCs. 3.4 Metformin inhibited the adipogenic differentiation of PDLSCs The osteogenic lineage and adipogenic lineage have the same origin and are considered to be two closely related directions in stem cell differentiation, so we also studied the influence of metformin on adipogenic differentiation of PDLSCs. The Oil Red O staining assay indicated that the number of lipid drops decreased when PDLSCs were incubated with metformin (Figure 1J). The results of qRT-PCR showed that the metformin treatment reduced the mRNA levels of PPARγ, LPL and C/EBPα in a dose-dependent manner (Figure 1I). These results indicated that metformin inhibited the adipogenic differentiation of PDLSCs. 3.5 Metformin promoted the osteogenic differentiation of PDLSCs through the Akt signaling pathway The 100 µM metformin treatment has the greatest positive effects on osteogenic differentiation based on the above results, so it was used to stimulate PDLSCs for 10 min, 30 min, 60 min and 120 min. As shown in Figure 2A, metformin significantly promoted the phosphorylation of Akt at 10 min, and the

degree of phosphorylation decreased with time until 120 min, although it was always more abundant than what was observed in the unstimulated group. This result indicated that metformin significantly activated Akt signaling in PDLSCs. In the following experiment, LY294002 was added to the medium to block with the effects of metformin on Akt. Western blot analysis showed that LY294002 inhibited the positive impact of metformin on Akt phosphorylation (Figure 2B). Then, the osteogenic differentiation abilities of PDLSCs cultured in the osteogenic medium with or without metformin or LY294002 were measured and compared. The results of the ALP activity assay and ALP staining showed that LY294002 reversed the positive effects of metformin on ALP activity (Figure 2C, D). Alizarin red staining and quantitative analysis indicated that there was less mineralized matrix in the metformin plus LY294002 group than in the metformin group (Figure 2E, F). In addition, LY294002 inhibited the expression of osteoblastic markers, including ALP, COL1, RUNX2 and OCN, which were induced by metformin (Figure 2G, H). Therefore, the inhibition of the Akt signaling pathway reversed the positive effects of metformin on PDLSCs osteogenic differentiation. 3.6 Metformin ameliorated H2O2-induced oxidative stress in PDLSCs H2O2 was used to induce oxidative stress in PDLSCs. The results from the CCK8 assays showed that cell viability decreased as the concentration of H2O2 increased (Figure 3A). When the concentration reached 250 µM, the cell viability decreased to approximately 75% of that of the control group, so treatment with 250 µM H2O2 was used to induce oxidative stress in subsequent experiments. The intracellular ROS level of PDLSCs was measured using the fluorescent probe DCFH-DA. Fluorescent microscope observations (Figure 3B) and flow cytometric analysis (Figure 3C, D) showed that the 250 µM H2O2-treated group exhibited a significant increase in the fluorescence intensity of PDLSCs compared with the negative control group, indicating that H2O2 caused an elevation in the intracellular ROS level in PDLSCs. However, the fluorescence intensity level was much lower in PDLSCs pretreated with 100, 500 or 1000 µM metformin prior to H2O2 stimulation than in those treated with H2O2 alone (Figure 3B, C, D), proving that metformin pretreatment effectively inhibited the increase in ROS induced by H2O2. In addition, with increasing concentrations, the inhibitory effect of metformin pretreatment on ROS generation became stronger. To further verify the protective effects of metformin, PDLSCs were cotreated with both metformin and H2O2. As shown in Figure 3B, C, D, the intracellular ROS level in PDLSCs treated with both metformin and H2O2 was lower than that observed in cells treated

with H2O2 alone. This result proved that the presence of metformin inhibited the production of ROS induced by H2O2 and that higher concentrations of metformin had stronger effects. To explore the effect of metformin on the antioxidant capacity of PDLSCs, antioxidant markers including SOD activity and GSH concentration were detected. Compared with the negative control group, H2O2 incubation led to a significant decrease in SOD activity and GSH concentration (Figure 3E, F), suggesting that H2O2-induced oxidative stress impaired the antioxidant capacity of cells. However, the SOD activity and GSH concentration were much higher in the group pretreated with 100 or 1000 µM metformin before H2O2 incubation than in the H2O2-treated group (Figure 3E, F). Similarly, intracellular SOD activity and GSH concentration were increased in PDLSCs cotreated with metformin and H2O2 compared to the cells treated only with H2O2 (Figure 3E, F). The above results proved that both pretreatment and co-treatment with metformin could reverse the damage of antioxidant capacity caused by H2O2. 3.7 Metformin protected PDLSCs from oxidative stress-induced dysfunction Since oxidative stress may disrupt normal cellular function, we further assessed whether metformin could ameliorate oxidative stress-induced damage to proliferation and osteogenic differentiation in PDLSCs. The cell viability of PDLSCs decreased significantly after incubation with 250 µM H2O2 for 24 h compared with the negative control treatment (Figure 4A). However, cell viability increased after PDLSCs pretreatment with metformin for 24 h before exposure to H2O2 compared with the H2O2-treatment (Figure 4A). In addition, the cell viability of PDLSCs cotreated with metformin and H2O2 was also higher than that of the cells treated with H2O2 alone (Figure 4A), suggesting that metformin could partially reverse the damage to cell activity caused by H2O2. In the osteogenic differentiation assay, after treatment with 250 µM H2O2 for 24 h, PDLSCs were cultured in the osteogenic medium to detect their differentiation ability. The results of the ALP activity assay (Figure 4B) and ALP staining (Figure 4C) showed that H2O2 treatment decreased the ALP activity compared with the negative control treatment. Alizarin red staining (Figure 4D) and quantitative analysis (Figure 4E) showed that H2O2 treatment reduced the production of extracellular mineralized matrix after the 21 d osteogenic induction treatment. qRT-PCR showed that the H2O2-treated group had lower mRNA levels of osteogenic genes, such as ALP, COL1, RUNX2 and OCN, than the control group (Figure 4F). The protein level of COL1 and RUNX2 also decreased in the H2O2-treated group (Figure 4G). The above

results suggested that H2O2-induced oxidative stress impaired the osteogenic differentiation ability of PDLSCs. When PDLSCs were pretreated with metformin for 24 h before the H2O2 incubation or when they were incubated with H2O2 in the presence of metformin, the ALP activity (Figure 4B, C), the production of extracellular mineralized matrix (Figure 4D, E) and the expression of osteogenic genes (Figure 4F, G) were significantly improved. Thus, metformin pretreatment and co-treatment effectively rescued the osteogenic differentiation ability of PDLSCs that had been damaged by oxidative stress. 3.8 Metformin activated the Akt/Nrf2 signaling pathway under oxidative stress To explore the mechanism of the protective effects of metformin, the expression of p-Akt and Nrf2 were analyzed by western blot after PDLSCs were incubated with 100, 500 or 1000 µM metformin for 24 h. As shown in Figure 5A, metformin increased the phosphorylation of Akt and upregulated the protein level of Nrf2. We further extracted nuclear proteins from PDLSCs and found that metformin treatment increased the level of Nrf2 in the nucleus (Figure 5B). In addition, metformin promoted the expression of target genes of Nrf2, including NQO1 and HO-1 (Figure 5C). All these results indicated that metformin could activate the Akt/Nrf2 signaling pathway in PDLSCs. To further study the protective effects of metformin on cells under oxidative stress, we analyzed the Akt/Nrf2 signaling in the presence of H2O2. As shown in Figure 5D and E, H2O2 stimulation promoted the expression of Nrf2 and its downstream targets NQO1 and HO-1 in PDLSCs, indicating that H2O2-induced oxidative stress could activate Nrf2 signaling in PDLSCs. Moreover, the expression levels of p-Akt, Nrf2, NQO1 and HO-1 were further improved in the group pretreated with metformin before H2O2 stimulation or incubated with H2O2 and metformin simultaneously compared with the group treated with H2O2 alone (Figure 5D, E). These results proved that both metformin pretreatment and co-treatment could activate stronger Akt/Nrf2 signals under oxidative stress to enhance the antioxidant capacity of cells. 3.9 Nrf2 knockdown by siRNA reversed the protection effects of metformin on PDLSCs under oxidative stress siRNA was used to knockdown the expression of Nrf2. As shown in Figure 6A and B, the mRNA and protein levels of Nrf2 decreased significantly after siRNA transfection for 48 and 72 h. The expression of downstream targets of Nrf2, including NQO1 and HO-1, was also downregulated (Figure 6A). Then, the siNC PDLSCs and siNrf2 PDLSCs were used to analyze whether knocking down Nrf2 could reverse the protective effects of metformin under oxidative stress. In assaying intracellular ROS, the results of fluorescence microscope observations (Figure 6E) and

flow cytometric analysis (Figure 6F, G) showed that the level of ROS in siNrf2 PDLSCs was much higher than it was in siNC PDLSCs, even though both were pretreated or cotreated with metformin under oxidative stress. At the same time, the SOD activity and GSH concentration were much lower in siNrf2 PDLSCs than in siNC PDLSCs when they were pretreated or cotreated with metformin (Figure 6C, D). These results proved that knocking down Nrf2 inhibited the antioxidant stress effect of metformin on PDLSCs. We further examined the protective effect of metformin on oxidative stress-induced dysfunction of PDLSCs when Nrf2 was knocked down. In analyzing cell viability, the results of the CCK8 assay showed that the rescue effect of metformin on the H2O2-induced reduction in cell viability was diminished by the knockdown of Nrf2 (Figure 6H). In the osteogenic differentiation assay, stimulation with H2O2 for 24 h led to a decrease in the osteogenic differentiation ability of siNC PDLSCs, while pretreatment with metformin before H2O2, or co-treatment with metformin and H2O2 rescued osteogenesis of cells. However, lower ALP activity (Figure 6I, J) and lower expression levels of osteogenesis markers (Figure 6K, L) were observed in the siNrf2 plus metformin group compared with the siNC plus metformin group, indicating that the protection effects of both pretreatment or co-treatment with metformin on PDLSCs was diminished when Nrf2 was knocked down.

4 Discussion In PDLSC-based bone tissue engineering, it is important to maintain or promote the osteogenic differentiation of PDLSCs and enhance the resistance of PDLSCs to adverse environments. The present study proved that low concentrations of metformin (less than 1000 µM) did not affect cell proliferation but promoted the osteogenic differentiation of PDLSCs. In addition, when PDLSCs were exposed to H2O2-induced oxidative stress, metformin could reduce ROS levels, enhance antioxidant capacity, and rescue cell viability and osteogenic differentiation of PDLSCs that were impaired by oxidative stress. Therefore, metformin could potentially be useful in the application of PDLSCs in alveolar bone regeneration. In proliferation assays, we found that metformin had a concentration-dependent effect on the proliferation of PDLSCs, because low concentrations of metformin (less than 1000 µM) did not significantly affect proliferation, but the cell viability was inhibited when the concentration reached 2500 µM. In fact, previous studies on other types of stem cells also found that a high concentration of metformin

(usually more than 1000 µM) significantly inhibited cell viability [34], and metformin was even used as an antitumor agent to inhibit cancer stem cell proliferation [35]. However, the influence of a low concentration of metformin (less than 1000 µM) on the proliferative activity of stem cells from different sources was reported to be different: metformin was reported to promote the proliferation of bone marrow stem cells (BMSCs) [36] and iPSC-derived neural stem cells [37], and it could inhibit that of human chorionic villous mesenchymal stem cells [38], while it had no significant effects on the proliferation of dental pulp stem cells [39] and human umbilical cord mesenchymal stem cells [40]. Thus, the effect and mechanism of metformin on cell proliferation is complicated, and different types of stem cells may respond differently to metformin, which requires further study. The differentiation potential is closely related to PDLSC-based bone regeneration, and the present study showed that 10-1000 µM metformin had a positive effect on the osteogenic differentiation of PDLSCs in vitro. At the same time, metformin inhibited the adipogenic differentiation of PDLSCs, which is thought to have a balanced relationship with osteogenic differentiation [41]. Our results are in line with some previous studies, which reported that metformin could promote the osteogenic differentiation of some mesenchymal stem cells and osteoblastic cell lines [42, 43]. It is worth noting that the optimum concentration of metformin for different cell types was reported to be quite different, suggesting that exploring the appropriate concentration of metformin is of great significance to its application in tissue engineering. On the premise of not inhibiting cell proliferation, the present study showed that 100 µM metformin had a more prominent positive effect on the osteogenic differentiation of PDLSCs, which provides guidance for the application of metformin in PDLSC-based bone regeneration. Of course, additional in vivo experiments are needed to verify the positive influence of metformin on PDLSCs osteogenic differentiation, which is our future research direction. To date, the underlying mechanism of metformin's effect on osteogenic differentiation of stem cells or osteoblastic cell lines remains unclear. Metformin was reported to activate LKB1/AMPK signaling in iPS-derived mesenchymal stem cells [44], inhibit GSK3β and activate Wnt/β-catenin signaling in BMSCs [45], improve the expression of SIRT6 in mouse preosteoblasts [46] and promote the transactivation of Runx2 via the USF-1/ SHP cascade in preosteoblasts [42] to promote osteogenesis. The results of the above studies suggest that the role of metformin in regulating osteogenic differentiation involves a variety of molecular mechanisms and signaling pathways, and it may vary depending on the source of stem cells. The present study found that metformin significantly promoted the activation of the Akt signaling pathway

in PDLSCs, which has been reported to improve the osteogenesis of PDLSCs [47]. When using LY294002 to interfere with Akt signals, the positive effects of metformin on osteogenic differentiation of PDLSCs were counteracted. These results prove that the Akt signaling pathway mediates the positive effects of metformin on the osteogenic differentiation of PDLSCs, at least in part. This result is similar to some previous studies that proved that metformin could activate the Akt signaling cascade to exert protective effects in neural cells [32] and cardiomyocytes[48]; however, the data are in conflict with other studies, which supported that high concentrations of metformin (usually more than 5 mM) inhibited the Akt signaling pathway in several types of cancer cells [49-51]. These conflicting observations may be the result of differences in cell types, culture conditions, or concentrations of metformin. The present study showed that treating PDLSCs with H2O2 led to a significant decrease in cell viability and osteogenesis, so enhancing the resistance of PDLSCs to oxidative stress is another important issue in PDLSC transplantation therapy for periodontitis. Here, we proved that metformin (100-1000 µM) pretreatment or co-treatment reduced intracellular ROS, increased antioxidant capacity, and alleviated the decrease in cell activity and osteogenic differentiation ability caused by H2O2, indicating that metformin could protect PDLSCs from oxidative stress-induced damage to cellular function. In fact, previous studies have reported that metformin could influence cellular ROS levels: some scholars have supported that metformin could reduce ROS in cardiomyocytes [24], enterocytes [52] and neural cells [53], while others have reported that metformin treatment increased ROS levels in several types of cancer cells [54, 55]. These inconsistent conclusions reflect the complexity of metformin's action, and attention should be paid to the timing and concentration of metformin in its application. Nrf2 has been reported to play important roles in responding to oxidative stress in cells [30]. Our study showed that incubating PDLSCs with metformin increased the expression of Nrf2, promoted Nrf2 entry into the nucleus, and further increased the expression of downstream target genes of Nrf2, indicating that metformin can activate the Nrf2 signaling pathway in PDLSCs. In addition, the protective effect of metformin against oxidative stress was eliminated when Nrf2 was knocked down by siRNA. Considering that Akt signaling could activate Nrf2 [28, 29], we support that the antioxidative stress effect of metformin in PDLSCs is through activation of the Akt/Nrf2 signaling pathway (Figure 7). It is worth noting that some other mechanisms that act on the antioxidative stress effect of metformin have been reported, such as the activation of AMPK signaling by metformin [52]. Whether these mechanisms participate in the effects of metformin on PDLSCs requires further investigation.

Notably, the periodontal microenvironment of patients with periodontitis is quite complex. In addition to oxidative stress that studied in this paper, the local bacterial stimulation and inflammatory factors, such as tumor necrosis factor-α, Interleukin-6 and Interleukin-8, could also cause damage to seed cells in the regeneration process [56]. Whether the application of metformin could protect PDLSC from these stimulations requires further experiments in vitro and in vivo to verify. Several scholars proved that metformin could inhibit inflammatory response in some adult cells [19, 57, 58], which provides clues for other potential benefits of metformin in PDLSC-based bone regeneration in periodontitis. Based on the positive and protective effects of metformin on PDLSCs, we propose a potential application strategy of metformin in PDLSC-based alveolar bone regeneration for periodontitis. First, metformin can be used to pretreat PDLSCs before transplantation to enhance the antioxidant stress ability of cells. Second, metformin can be further applied after transplantation to promote the osteogenic differentiation and protect cells against antioxidant stress injury. Application of scaffold materials that continuously release metformin may be a good choice, and the optimal duration and concentration of drug release need to be determined by further experiments. Anyway, more in vivo experiments are needed to verify the positive influence of metformin on PDLSC-based tissue regeneration, which is our future research direction.

5 Conclusion The present study showed that low concentrations of metformin promoted osteogenic differentiation and inhibited adipogenic differentiation of PDLSCs without affecting cell proliferation in vitro. Moreover, metformin reduced the production of ROS stimulated by H2O2, enhanced the antioxidant capacity, and alleviated the damage of oxidative stress on proliferation and osteogenic differentiation in PDLSCs. These beneficial effects of metformin were partly mediated by activation of the Akt/Nrf2 signaling pathway. The application of metformin could facilitate the PDLSC-based alveolar bone regeneration in the treatment of periodontitis.

Acknowledgements This work was supported by the Construction Engineering Special Fund of Taishan Scholars (grant number ts201511106), Natural Science Foundation of Shandong Province (grant number ZR2017MH031), Shandong medical and health science and technology development plan (grant number 2017WS112) and the Youth scientific research funds of School of Stomatology, Shandong University（grant number

2018QNJJ01）.

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Figure 1. Effects of Metformin on proliferation and multi-differentiation of PDLSCs A, cell viability of PDLSCs treated with 0, 10, 100, 500, 1000 or 2500 µM metformin for 5 d. B-H, PDLSCs were cultured in the osteogenic medium supplemented with 0, 10, 100, 500 or 1000 µM metformin, then ALP activity assay on days 7 and 14 (B), ALP staining on days 7 and 14 (C, D), the mRNA levels assay of

ALP, COL1, RUNX2 and OCN on day 7 (E), the protein levels assay of COL1 and RUNX2 on day 7 (F), Alizarin Red staining and quantitative analysis of Alizarin Red-positive mineralized matrix on day 21 (G,H) were performed. I, J, PDLSCs were cultured in the adipogenic medium supplemented with 0, 10, 100 or 1000 µM metformin, then the mRNA levels assay of PPARγ, LPL and C/EBPα on day 14 (I) and Oil Red O staining on day 28 (J) were performed. The histograms in F represent the relative expression levels of proteins normalized to GAPDH. *p<0.05 vs negative control (0 µM metformin), **p<0.01 vs negative control, *** p<0.001 vs negative control.

Figure 2 Metformin promoted the osteogenic differentiation of PDLSCs through the Akt signaling pathway A, the phosphorylation of Akt in PDLSCs stimulated with 100 µM metformin for 120 min was analyzed via western blot. B, the phosphorylation of Akt in PDLSCs stimulated with 100 µM metformin or 100 µM metformin plus LY294002 for 12 h was analyzed via western blot. C-H, PDLSCs were cultured in the osteogenic medium supplemented with metformin or metformin plus LY294002, and then ALP activity assay on day 7 (C), ALP staining on day 7 (D), Alizarin Red staining and quantitative analysis on day 21 (E, F), the mRNA levels assay of ALP, COL1, RUNX2 and OCN on day 7 (G), and the protein levels assay of COL1 and RUNX2 on day 7 were performed. The histograms in A, B and H represent the relative expression levels of proteins normalized to GAPDH. *p<0.05 vs negative control (metformin-, LY294002-), **p<0.01 vs negative control, #p<0.05 vs positive control (metformin+, LY294002-), ##p<0.01 vs positive control.

Figure 3 Metformin reduced ROS level and increased antioxidant capacity of PDLSCs A, cell viability assay of PDLSCs stimulated with H2O2 for 24 h. B-D, PDLSCs were pretreated with metformin for 12h before with H2O2 for 6h, or cotreated with both metformin and H2O2 for 6h, then the cells were labeled with DCFH. B, representative DCFH staining images of PDLSCs. C, flow cytometry assay of DCFH-stained PDLSCs. D, quantitative analysis of flow cytometry results. E, F, PDLSCs were pretreated with metformin for 12h before with H2O2 for 6h, or cotreated with both metformin and H2O2 for 6h, then the SOD activity (E) and GSH level (F) of PDLSCs were analyzed. *p<0.05 vs negative control (H2O2-, metformin-), **p<0.01 vs negative control, *** p<0.001 vs negative control, #p<0.05 vs positive control (H2O2+, metformin-), ##p<0.01 vs positive control.

Figure 4 Metformin protected PDLSCs from oxidative stress-induced dysfunction A, cell viability assay of PDLSCs which were pretreated with metformin for 24h before with H2O2 for 24h or cotreated with both metformin and H2O2 for 24h. B-E, PDLSCs were pretreated with metformin for 24h before with H2O2 for 24h, or cotreated with both metformin and H2O2 for 24h, then the cells were cultured in the osteogenic medium. B, ALP activity assay on day 7. C, ALP staining on day 7. D, E, Alizarin Red staining and quantitative analysis on day 21. F, G, PDLSCs were pretreated with 1000 µM metformin for

24h before with 250 µM H2O2 for 24h, or cotreated with both 1000 µM metformin and 250 µM H2O2 for 24h, then the cells were cultured in the osteogenic medium. F, the mRNA levels assay of ALP, COL1, RUNX2 and OCN on day 7. G the protein levels assay of COL1 and RUNX2 on day 7 (Pre: pretreatment, Co: co-treatment). The histograms represent the relative expression levels of proteins normalized to GAPDH. *p<0.05 vs negative control (H2O2-, metformin-), **p<0.01 vs negative control, #p<0.05 vs positive control (H2O2+, metformin-), ##p<0.01 vs positive control. NC refers to negative control (H2O2-, metformin-).

Figure 5 Metformin activated the Akt/Nrf2 signaling pathway under oxidative stress A, the protein level of p-Akt and Nrf2 in PDLSCs stimulated with 100, 500 or 1000 µM metformin for 24 h. B, the protein level of Nrf2 in nucleus of PDLSCs stimulated with 1000 µM metformin for 6 h. C, the mRNA levels of Nrf2 and its downstream targets, including NQO1 and HO-1, in PDLSCs stimulated with 1000 µM metformin for 24 h. D, the expression of p-Akt and Nrf2 in PDLSCs pretreated with 1000 µM metformin for 24h before with 250 µM H2O2 for 24h, or cotreated with 1000 µM metformin and 250 µM H2O2 for 24h (Pre: pretreatment, Co: co-treatment). E, the mRNA levels of Nrf2, NQO1 and HO-1 in PDLSCs pretreated with 1000 µM metformin for 24h before with 250 µM H2O2 for 24h, or cotreated with 1000 µM metformin and 250 µM H2O2 for 24h. The histograms in A, B and D represent the relative expression levels of proteins normalized to GAPDH or Histone H3. *p<0.05 vs negative control (H2O2-, metformin-), **p<0.01 vs negative control, ***p<0.001 vs negative control, #p<0.05 vs positive control (H2O2+, metformin-), ##p<0.01 vs positive control. NC refers to negative control (H2O2-, metformin-).

Figure 6 Nrf2 knockdown reversed the protection effects of metformin on PDLSCs under oxidative stress A, the mRNA levels of Nrf2 and its downstream targets, including NQO1 and HO-1, in PDLSCs transfected with siNrf2 and siNC for 48h. B, the protein level of Nrf2 in PDLSCs transfected with siNrf2 and siNC for 72 h. The histograms represent the relative expression levels of proteins normalized to

GAPDH. C, D, siNC PDLSCs and siNrf2 PDLSCs were pretreated with metformin for 12h before with H2O2 for 6h or cotreated with metformin and H2O2 for 6h, then the SOD activity (C) and GSH level (D) of PDLSCs were analyzed. E-G, siNC PDLSCs and siNrf2 PDLSCs were pretreated with metformin for 12h before with H2O2 for 6h or cotreated with metformin and H2O2 for 6h, then the cells were labeled with DCFH. E, representative DCFH staining images of PDLSCs. F, flow cytometry assay of DCFH-stained PDLSCs. G, quantitative analysis of flow cytometry results. H, cell viability assay of siNC PDLSCs and siNrf2 PDLSCs which were pretreated with metformin for 24h before with H2O2 for 24 or cotreated with metformin and H2O2 for 24h. I-L, siNC PDLSCs and siNrf2 PDLSCs were pretreated with 1000 µM metformin for 24h before with 250 µM H2O2 for 24h or cotreated with 1000 µM metformin and 250 µM H2O2 for 24h, then the cells were cultured in the osteogenic medium. I, ALP staining on day 7. J, ALP activity assay on day 7. K, the protein level assay of COL1 and RUNX2 on day 7(Pre: pretreatment, Co: co-treatment). L, the mRNA level assay of ALP, COL1 RUNX2 and OCN on day 7. The histograms in B and K represent the relative expression levels of proteins normalized to GAPDH. *p<0.05 vs negative control (H2O2-, metformin-), **p<0.01 vs negative control, ***p<0.001 vs negative control, #p<0.05 vs positive control (H2O2+, metformin-), ##p<0.01 vs positive control, &p<0.05, &&<0.01.

Figure 7 A model illustrating metformin promotes osteogenic differentiation and protects against oxidative stress via activation of the Akt/Nrf2 signaling pathway in PDLSCs Stimulation with metformin leads to the activation of Akt signaling, further promotes the osteogenic differentiation of PDLSCs. Moreover, metformin protects PDLSCs against H2O2-induced oxidative stress, partly through increasing the expression and nuclear localization of Nrf2, which combines with antioxidant response element (ARE) to promote the expression of antioxidant enzymes or substances such as NQO1, HO-1, SOD and GSH.

Highlights

1. Metformin promotes the osteogenic differentiation of PDLSCs. 2. Metformin protects PDLSCs against oxidative stress. 3. Metformin activates the Akt/Nrf2 signaling pathway in PDLSCs.

Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, Metformin promotes osteogenic differentiation and protects against oxidative stress-induced damage in periodontal ligament stem cells via activating Akt/Nrf2 signaling pathway