High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis Lina Yang a, Jing Liu b, Qiusheng Shan c, Guannan Geng d, Ping Shao a, * a

Department of Orthodontics, The First Affiliated Hospital of Harbin Medical University, Harbin, 150081, PR China Department of Periodontology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150081, PR China c Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 7008525, Japan d Department of Endocrinology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150081, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 October 2019 Accepted 13 November 2019 Available online xxx

The inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone are well documented. However, a comprehensive study focused on the molecular mechanisms is still unknown. Recent studies have revealed that caspase-1 participates in the pathological processes of hepatic injury, cancers and diabetes related complications. However, the relationship between pyroptosis and proliferation and differentiation of osteoblasts has not been investigated. This study aimed to explore the possible pyroptosis participating in the inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone. The diabetes model was constructed both in vitro and in vivo to detect the expression of pyroptosis related factors. These results show that high glucose inhibits proliferation and differentiation of osteoblast in alveolar bone through pyroptosis pathway. Furthermore, caspase-1 inhibitor was co-administered with high glucose in ME3T3-E1 cells, which shows that caspase-1 inhibitor could repress effect of high glucose on the proliferation and differentiation of osteoblast. In conclusion, High glucose could activate the pyroptosis through the caspase-1/GSDMD/IL-1b pathway to inhibit the proliferation and differentiation of osteoblast in alveolar bone, which provides a theoretical basis for clinical treatment of alveolar bone disease in diabetic patients. © 2019 Elsevier Inc. All rights reserved.

Keywords: High glucose Osteoblast Pyroptosis Proliferation Differentiation

1. Introduction Diabetes Mellitus (DM) is an important risk factor for osteoporosis. The proliferation and differentiation of osteoblasts in alveolar bone of diabetic patients remains a great matter in dentists daily work, which could hinder many oral diseases treatments such as dental implants and oral surgery. Hyperglycaemia often displays in these patients and has negative effect on alveolar bone rebuilding [1]. The detail mechanism that high glucose inhibiting proliferation and differentiation of osteoblast are still unclear. However, the high levels of proinflammatory cytokines such as IL-1b in periodontal tissues under the condition of hyperglycemia are considered to be potentially important contributors for periodontal disease inhibiting the tissue formation. Meanwhile, the above-mentioned

* Corresponding author. Department of Orthodontics, The first affiliated Hospital of Harbin Medical University, Harbin City, Heilongjiang Province, China. E-mail address: [email protected] (P. Shao).

cytokines were also participated in pyroptosis which leads to the formation of apical periodontitis [2]. However, whether these factors also affect the other oral diseases is still unknown. Pyroptosis, also known as cell inflammatory necrosis, is mediated by caspase-1 or caspase-11 [3, 4]. Dissimilar other cell death processes, pyroptosis is characterized by swelling of the cell until the membrane dissolves leading to release of the cell substance and proinflammatory cytokines resulting in eventually cell death [5]. During pyroptosis, the activation of caspase-1 is then capable of converting the pro- IL-1b to bioactive IL-1b [6].Several latest studies demonstrated that Gasdermin D (GSDMD) is another serious component of the inflammasome cleaved by activated caspase-1, which is essential in pyroptosis because it can promote the secretion of matured IL-1b and damage the plasma membrane [7-9]. In addition, some studies also revealed that caspase-1 participates in the pathological processes of hepatic injury, cancers and diabetes related complications [10-12]. However, whether pyroptosis participates in the inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone has not been

https://doi.org/10.1016/j.bbrc.2019.11.080 0006-291X/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: L. Yang et al., High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.080

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investigated. In this study, the glucose and caspase-1 inhibitor were used to inhibit the proliferation and differentiation of osteoblasts and block pyroptosis pathway respectively to explore whether pyroptosis participates in proliferation and differentiation of osteoblast in diabetic patient alveolar bone, which will provide a theoretical basis for clinical treatment of alveolar bone disease in diabetic patients. 2. Materials and methods

which blocked in blocking solution (5% fat-free milk buffer dissolved in Tris-buffered saline -Tween-20) on a shaker for 2 h at room temperature. After the membrane was incubated respectively overnight at 4℃ with primary antibodies like p-AKT(1:1000, CST, MA, USA), b-catenin(1:1000, CST, MA, USA), caspase-1(1:1000, CST, MA, USA) and IL-1b(1:1000, CST, MA, USA), GSDMD(1:500, BIOSS, Beijing, China) and GAPDH(1:1000, ZSGB-BIO, Beijing, China). Then washing by TBST for 30 min, the membranes were incubated with HRB-labeled goat anti-mouse IgG or rabbit anti-IgG (1:1000, ZSGBBIO, Beijing, China) for 1 h. The bands were pictured using GelDox XR System (Bio-Rad, CA, USA) and quantified by Quantity One Software.

2.1. Cell culture 2.5. Immunofluorescence staining Mouse pre-osteoblast ME3T3-E1 cell line was purchased from Sabakang Biology (Shanghai, China). The cells were maintained in high-glucose DMEM (GE Healthcare HyClone, Logan, UT, USA) supplemented with 10%FBS (BI, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin solution (Beyotime, Biotechnology, Jiangsu, China) at 37℃ in 5% CO2 atmosphere prior to drug treatment. 2.2. CCK8 assay The effect of high glucose with different concentrations on the proliferation of ME3T3-E1 was analyzed by CCK8 assay. The ME3T3-E1 cells was added into a 96-well plate at a density of 104/ 200 mL and incubated overnight (37℃, 5% CO2). After 24 h, add different concentrations of high glucose (25 mM, 50 mM, 100 mM) and the mixture contained caspase-1 inhibitor and different concentrations of high glucose, which incubated for 24 h, 48 h, 72 h respectively. The medium of each well was replaced by 100 mL medium contained CCK8 solution (10 mL) and incubated for another 4 h at 37
C in dark. The absorbance of each well was measured by enzyme-linked immunosorbent assay reader at 450 nm. 2.3. Animal models and treatment The study was approved by the ethics committee of Harbin Medical University, and animal protocols were in accordance with the guidelines of the Animals (Scientific Procedures) Act 1986. Male C57BL/6 mice weighing 18-20 g were purchased from the Animal Experimental Center of the Second Affiliated Hospital of Harbin Medical University (Harbin, China). All mice were fed adaptively with routine diet for one week (temperature 22±1℃; 12 h light and dark cycle). The mice were randomly divided into three groups: control group (control), diabetic group (DM) and diabetes-caspase1 inhibitor group (DMþCaspase-1I). Citric acid buffer containing streptozotocin (STZ, Sigma, St. Louis, MO) 50 mg/kg/day (PH¼4.6) was injected intraperitoneally for 5 consecutive days. After one week, the blood glucose level in the tail vein was measured by Contour Glucose Meter (Roche, Germany) and the diabetic mice model was successfully established when the blood glucose level reached more than 16.7 mmol/L. The DMþCaspase1 I group treated with 0.1 mg/kg Caspase-1 inhibitor AC-YVAD-CMK (Cayman Chemical, MI, USA) through intraperitoneal injection every day. All the mice were fed for 12 weeks (n ¼ 5 in each group). 2.4. Protein extraction and western blot Alveolar bones and cells were collected and proteins were extracted using RIPA buffer (Beyotime, Jiang Su, China) containing 1Protease Inhibitor. Protein concentration was determined by BCA Protein Assay Kit (Beyotime, Jiang Su, China). The protein sample was separated by 12% acrylamide gel electrophoresis (SDS-PAGE) based on the manufacturer’s instructions and then electrotransferred to the nitrocellulose membrane (Millipore, MA, USA) ,

ME3T3-E1 cells treated with different concentrations of glucose were stained with DAPI (Beyotime, Shanghai, China) for 30 min and treated with 4% paraformaldehyde buffer at room temperature for 20 min. Followed by penetrated buffer (1% BSA and 0.1% Triton-X) for 2 h and blocked by 5% BSA buffer for 1 h at room temperature. Then cells were treated with the primary antibodies of Caspase-1, GSDMD, IL-1b, p-AKT and b-catenin (1: 100) overnight at 4 ℃ respectively. The cells were washed with PBS for three times and incubated with secondary antibody for 1 h. The results were captured using a fluorescence microscope. 2.6. HE staining and immunohistochemistry Osteoblast morphology and internal protein expression were detected by HE and immunohistochemistry respectively. The alveolar bone was decalcified by EDTA for 2 weeks when tissue was fixed in 4% paraformaldehyde for 48 h and embedded in paraffin and cut into 4 mm sections which were used for HE and immunohistochemical staining. The tissue slices were incubated with caspase-1, GSDMD, IL-1b, p-AKT and b-catenin (1:200) overnight respectively. After that, the sections were washed with PBS for 3 times followed by the treatment with secondary antibodies at room temperature for 1 h. The sections were finally stained with diaminobenzidine and the results were captured by fluorescence microscopy. 2.7. Statistical analysis The statistical analysis was conducted by GraphPad Prism 5.0 software (San Diego, CA, USA). All data are shown as mean ± standard error of mean (mean ± SEM). The comparison of data between more than two groups were conducted by one-way ANOVA. The comparison of data between two groups were analyzed by the t-test. Values of at least P<0.05 were considered statistically significant. 3. Results 3.1. High glucose inhibited the proliferation of osteoblast through activating the pyroptosis pathway The inhibition of high glucose on the proliferation of osteoblast ME3T3-E1 line was analyzed by CCK8 assay (Fig. 1 A). The results showed that the 25 mM high glucose induced significant reduction in viability of ME3T3-E1 cells at 48 h, which were afterwards employed as the concentration and time point for the subsequent experiment. Furthermore, the expression levels of apoptosisrelated proteins caspase-1 and GSDDM were detected by immunofluorescence (Fig. 1 B, C) and Western blot assays (Fig. 1 D, E). The results showed that high glucose could promote the expression of above-mentioned proteins and activate pyroptosis pathway.

Please cite this article as: L. Yang et al., High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.080

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Fig. 1. High glucose inhibits the proliferation of osteoblasts through activating the pyroptosis pathway. (A) The CCK8 assay was used to detect the effects of high glucose on ME3T3-E1 at 24, 48 and 72 h respectively. (B, C) The immunofluorescence staining of caspase-1 and GSDMD in ME3T3-E1 cells; microphotographs are representative of three individual experiments. (D, E) the expression level of caspase1 and GSDMD was examined by western Blot, GAPDH served as an internal control. **p < 0.01 versus Control, ***p < 0.001 versus Control; n ¼ 3.

3.2. High glucose inhibited the proliferation and differentiation of osteoblasts through IL-1 b /AKT/ b-catenin pathway Since cells viability was significantly reduced under a light microscope after administration of high glucose and the pyroptosis

pathway was activated. The IL-1b/p-AKT/b-catenin pathway plays an important role in regulating the proliferation and differentiation of osteoblasts. Therefore, the expression level of above-mentioned proteins was examined by immunofluorescence (Fig. 2 A, B, C) and western blot (Fig. 2 D, E, F). The results indicated that high glucose

Please cite this article as: L. Yang et al., High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.080

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promotes the expression level of IL-1b and inhibits the expression of p-AKT and b-catenin. all of these data demonstrated that high glucose could inhibit the proliferation and differentiation of osteoblast by IL-1b/p-AKT/b-catenin pathway.

3.3. Caspase-1 inhibitor could alleviate the inhibition of high glucose on the proliferation and differentiation of osteoblasts The caspase-1 inhibitor was used to block the pyroptosis

Fig. 2. High glucose inhibited the proliferation and differentiation of osteoblasts through IL-1 b/AKT/b-catenin pathway (A, B and C) The immunofluorescence staining of IL-1 b, p-AKT andb-catenin in ME3T3-E1 cells; microphotographs are representative of three individual experiments. (D, E and F) Relative expression of caspase-1 and IL-1b protein in ME3T3-E1 were detected by Western blot, GAPDH served as an internal control. **p < 0.01 versus Control, ***p < 0.001 versus Control; n ¼ 3.

Please cite this article as: L. Yang et al., High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.080

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pathway to exam the role and function of pyroptosis in the inhibition of high glucose on the proliferation and differentiation of osteoblast. CCK-8 assay results showed that the cell viability was significantly increased after adding the caspase-1 inhibitor

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especially at 48h (Fig. 3 A). Furthermore, the expression level of pyroptosis related proteins and p-AKT/b-catenin were examined by immunofluorescence (Fig. 3 B) and western blot (Fig. 3 C, D, E, F, G). The results showed that the expression of caspase-1, GSDMD and

Fig. 3. Caspase-1 inhibitor can alleviate the inhibition of high glucose on the proliferation and differentiation of osteoblasts. (A) The CCK8 assay was used to detect the effects of high glucose and AC-YVAD-CMK on ME3T3-E1 at 24, 48 and 72 h respectively. (B) The immunofluorescence staining of caspase-1, GSDMD, IL-1b, p-AKT and b-catenin in different groups; microphotographs are representative of three individual experiments. (C, D, E, F and G) Relative expression of caspase-1, GSDMD, IL-1b, p-AKT and b-catenin in different groups were detected by Western blot. GAPDH served as an internal control, ***p < 0.001 versus Control; ##p < 0.01 vs. AC-YVAD-CMK 100 mM, ###p < 0.001 vs. AC-YVAD-CMK 100 mM; n ¼ 3.

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IL-1b protein increased, while the expression of p-AKT and b-catenin decreased. all of these results indicated that caspase-1 inhibitor can reverse the inhibition of high glucose on the proliferation and differentiation of osteogenesis via the pyroptosis pathway.

3.4. The proliferation and differentiation of alveolar osteoblasts were inhibited due to the activation of pyroptosis pathway in diabetic mice We further confirmed that high glucose could inhibit

Fig. 4. The proliferation and differentiation of alveolar osteoblasts were inhibited due to the activation of pyroptosis pathway in diabetic mice. (A) Hematoxylineosin (HE) staining were performed to detection of the number of alveolar bone osteoblasts. Scale bar, 20 mm. (B) Immunohistochemistry analysis was performed to detect the expression caspase-1 and GSDMD; microphotographs are representative of three individual experiments. (C) Immunohistochemistry analysis was performed to detect the expression of IL-1b、 p-AKT and b-catenin; microphotographs are representative of three individual experiments. (D) Fasting blood glucose levels measured at 12 weeks after STZ injection in the different groups. (E,F, G, H and I) Western blot was conducted to determine the protein expression of caspase-1, GSDMD IL-1b, p-AKT and b-catenin. ***p < 0.001 versus Control; ##p < 0.01 vs. AC-YVAD-CMK 100 mM ###p < 0.001 vs. AC-YVAD-CMK 100 mM; n ¼ 5.

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proliferation and differentiation of osteoblasts via activating pyroptosis pathway in vivo. Firstly, the model of diabetic mice was established and the blood glucose was measured after 12 weeks. The results showed that the blood glucose in the diabetic group was significantly higher than that in the control group (Fig. 4 D). Secondary, the number of osteoblasts was measured by HE staining which showed that the number of diabetic group bone cells decreased compared with the control group and the number of osteoblasts was increased after the administration of the AC-YVADCMK (Fig. 4 A). Next, the expression of pyroptosis associated proteins and proliferation and differentiation of osteoblast protein was detected by immunohistochemistry, which indicated that the expression level of Caspase-1, GSDMD and IL-1b were significantly increased in the diabetic group compared with the control group, while p-AKT and b-catenin were significantly decreased. The expression level of above-mentioned protein was reversed after administrating AC-YVAD-CMK (Fig. 4 B, C). Western Blot is consistent with the expression of immunohistochemistry (Fig. 4 E, F, G, H, I). All of these data suggested that the activation of the pyroptosis pathway of osteoblasts in alveolar bone leads to the inhibition of proliferation and differentiation of osteoblasts in the state of diabetes. 4. Discussion The mechanism that high glucose inhibiting proliferation of cell has been extensively studied [13,14]. However, the detail mechanism that high glucose inhibiting the proliferation and differentiation of osteoblast is still unclear. In this study, we found that high glucose could inhibit the proliferation and differentiation of alveolar bone osteoblasts via activate pyroptosis pathway, which can be reversed by caspase-1 inhibitors both in vivo and in vitro. In agreement, another experiment has also demonstrated high glucose suppressed proliferation of human retinal endothelial cells by activating pyroptosis pathway [13]. Ac-YVAD-CMK, as caspase-1 inhibitor, could inhibit the activation of caspase-1, which results in the alleviation of bone necrosis in diabetic mice models [15]. All of these findings demonstrated that the inhibition of diabetes on proliferation and differentiation of osteoblast in alveolar bone was closely correlated with activated pyroptosis pathway. creasing evidence has shown that the activation of pyroptosis plays a crucial role in diseases, including diabetic cardiomyopathy, hepatitis and breast cancer [12,16,17]. Pyroptosis is a type of inflammatory caspase-mediated necrotic cell death process, which become more and more important [18]. Caspase-1 lead to the conversion of IL-1b precursors into mature IL-1b in pyroptosis pathway [4]. Simultaneously, caspase-1 cleaves the amino- terminal and carboxyl-terminal linkers of GSDMD facilitating bind of the released amino-terminal fragment to the cell membrane to form oligomeric asymptotic pores [7,19,20]. In this study, we also discover the high expression level of IL-1b in alveolar bone of diabetic mice. The inferior expression of IL-1b was observed accompanied with addition proliferation and differentiation of osteoblast under Ac-YVAD-CMK condition. These findings suggested that IL-1b production caused by caspase-1 plays a key role of in suppression of proliferation and differentiation of osteoblast diabetic alveolar bone. Several studies also found that IL-1b plays an important role in many bone diseases such as high expression levels of IL-1b under estrogen shortage exacerbate osteoporosis by inhibiting osteogenic differentiation [21]. IL-1b expression has been found positively associated with the development of bone cancer pain in bone metastasis [22]. In periodontal diseases, the upregualted IL-1b protein could lead to the loss of alveolar bone and the damage of periodontal tissue in obesity-associated metabolic disorders rats [23]. Some research also confirmed that alveolar

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bone in periodontal tissues of nondiabetic periodontitis patients damage less compared with uncontrolled type-2 diabetic patients with periodontitis [24]. Simultaneously, we also detected the increasing expression level of GSDMD in the alveolar bone of diabetic mice accompanied with inhibition of proliferation and differentiation of osteoblast. The relevant literature also demonstrated that the high expression of GSDMD would result in osteomyelitis, while has not been reported in osteoblasts under alveolar bone of diabetes [25]. The repair of implant-bone and bone defects was promoted by proliferation and differentiation of osteoblasts. Many protein factors such as p-AKT and b-catenin are involved in the process of proliferation and differentiation of osteoblasts [26]. In our study， we discovered that the expression levels of p-AKT and b-catenin were decreased in the alveolar bone of diabetic mice compared with the normal controls. However, the expression levels of above factors in diabetic mice were elevated in the treatment of AC-YVADCMK. AKT is a downstream serineethreonine kinase that transmits survival signals from growth factors closely connected with the differentiation of osteoblast [27,28]. Simultaneously, b-catenin, as transcriptional co-activator for the enhancer factor, could activate gene transcription in differentiation of bone [29]. These studies have shown that the AKT and b-catenin expression is positively associated with the proliferation and differentiation of osteoblast. According to related reports, the expression of AKT and b-catenin was inhibited by IL-1b, which lead to the inhibition of osteogenesis [26,30]. These published messages implicated that the inhibition of proliferation and differentiation of osteoblast may be correlated with the impeded AKT and b-catenin expression caused by overexpressed IL-1b. The molecular mechanisms IL-1b regulated proliferation and differentiation of osteoblast is still incomplete elucidated in diabetes alveolar bone. In summary, we confirmed that high glucose inhibits proliferation and differentiation of osteoblasts activated via pyroptosis, which is reversed by caspase-1 inhibitor. However, the mechanism that high glucose regulating the pyroptosis pathway need to be further studied. Therefore, this study will provide a theoretical basis for further research on diabetic periodontal disease and clinical treatment. Author contribution PS designs the experiment and provides the financial fund; LY and GG conduct the experiment; QS writes the manuscript; JL conducts the data analysis. Declaration of competing interest The authors declare no potential conflicts of interest in this manuscript. Acknowledgements This work was supported by Harbin Science and Technology Innovation Talent Fund (No. 2016RQQYJ232). The author thanks Yanru Liang for her help in article modification. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.080. References [1] J.S. Colombo, D. Balani, A.J. Sloan, S.J. Crean, J. Okazaki, R.J. Waddington,

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Please cite this article as: L. Yang et al., High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.080