摘要
The FASEB JournalVolume 33, Issue 10 p. 11555-11566 ResearchFree Access BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration Jianle Wang, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, ChinaThese authors contributed equally to this work.Search for more papers by this authorJianing Hu, The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaThese authors contributed equally to this work.Search for more papers by this authorXimiao Chen, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Department of Orthopaedics, Affiliated Hospital of Guilin Medical College, Guilin, ChinaThese authors contributed equally to this work.Search for more papers by this authorChongan Huang, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorJialiang Lin, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorZhenxuan Shao, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorMingbao Gu, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorYaosen Wu, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorNaifeng Tian, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorWeiyang Gao, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorYifei Zhou, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorXiangyang Wang, Corresponding Author xiangyangwang@wmu.edu.cn Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, China Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: xiangyangwang@wmu.edu.cn Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: zhangxiaolei@wmu.edu.cnSearch for more papers by this authorXiaolei Zhang, Corresponding Author zhangxiaolei@wmu.edu.cn Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, China Chinese Orthopaedic Regenerative Medicine Society, Hangzhou, China Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: xiangyangwang@wmu.edu.cn Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: zhangxiaolei@wmu.edu.cnSearch for more papers by this author Jianle Wang, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, ChinaThese authors contributed equally to this work.Search for more papers by this authorJianing Hu, The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaThese authors contributed equally to this work.Search for more papers by this authorXimiao Chen, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Department of Orthopaedics, Affiliated Hospital of Guilin Medical College, Guilin, ChinaThese authors contributed equally to this work.Search for more papers by this authorChongan Huang, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorJialiang Lin, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorZhenxuan Shao, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorMingbao Gu, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorYaosen Wu, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorNaifeng Tian, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorWeiyang Gao, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorYifei Zhou, Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, ChinaSearch for more papers by this authorXiangyang Wang, Corresponding Author xiangyangwang@wmu.edu.cn Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, China Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: xiangyangwang@wmu.edu.cn Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: zhangxiaolei@wmu.edu.cnSearch for more papers by this authorXiaolei Zhang, Corresponding Author zhangxiaolei@wmu.edu.cn Department of Orthopaedics, The Second Affiliated Hospital–Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China The Second School of Medicine, Wenzhou Medical University, Wenzhou, China Chinese Orthopaedic Regenerative Medicine Society, Hangzhou, China Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: xiangyangwang@wmu.edu.cn Correspondence: Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 West Xueyuan Rd., Wenzhou, Zhejiang Province 325027, China. E-mail: zhangxiaolei@wmu.edu.cnSearch for more papers by this author First published: 22 July 2019 https://doi.org/10.1096/fj.201900703RCitations: 5 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat ABSTRACT Diabetes mellitus may lead to intervertebral disc degeneration (IVDD). Matrix metalloproteinase-13 (MMP-13) is one of the major catabolic factors in extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and contributes to diabetic IVDD. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is implicated in chronic inflammation. Here, we report that the expression of BRD4 and MMP-13 was elevated in diabetic nucleus pulposus tissues as well as in advanced glycation end products (AGEs)-treated NPCs; also, the regulatory effect of BRD4 on MMP-13 was studied. We found that MMP-13 was regulated by MAPK and NF-κB signaling as well as autophagy in AGEs-treated NPCs. Next, we explored the role of BRD4 in regulation of MAPK, NF-κB signaling, and autophagy. The results showed that BRD4 is the upstream regulator of all of these 3 factors, and inhibition of BRD4 may suppress MAPK and NF-κB signaling while activating autophagy in AGEs-treated NPCs. Finally, we demonstrated that BRD4 inhibition may suppress MMP-13 expression in diabetic NPCs in vitro as well as in vivo; meanwhile, it may preserve ECM in diabetic rats. Our study demonstrates that inhibition of BRD4 may suppress MAPK and NF-κB signaling and activate autophagy to suppress MMP-13 expression in diabetic IVDD, and diabetic IVDD may be compromised by BRD4 inhibitors.—Wang, J., Hu, J., Chen, X., Huang, C., Lin, J., Shao, Z., Gu, M., Wu, Y., Tian, N., Gao, W., Zhou, Y., Wang, X., Zhang, X. BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration. FASEB J. 33, 11555–11566 (2019). www.fasebj.org ABBREVIATIONS 3-MA 3-methyladenine AGE advanced glycation end-product BRD4 bromodomain-containing protein 4 ECM extracellular matrix IVD intervertebral disc IVDD intervertebral disc degeneration LC3 microtubule-associated protein 1A/1B-light chain 3 MMP-13 matrix metalloproteinase-13 NP nucleus pulposus NPC nucleus pulposus cell PD PD98059 Rapa rapamycin SD Sprague-Dawley T2D type 2 diabetes WIPI2 WD repeat domain phosphoinositide interacting 2 Intervertebral disc degeneration (IVDD) is known to contribute to low back pain (1), which is the leading cause of disability around the world (2, 3). Studies from different groups including ours have shown that diabetes mellitus,# especially type 2 diabetes (T2D), is a major risk factor for IVDD (4–7). Animal study demonstrated that extracellular matrix (ECM) metabolism in nucleus pulposus (NP) was influenced by diabetes (8), and this phenomenon was further confirmed in human samples (9); however, how diabetes affects ECM metabolism in NP is still not clear. The ECM metabolism of nucleus pulposus cells (NPCs) is a dynamic process, which includes ECM anabolism and ECM catabolism. Matrix metalloproteinase-13 (MMP-13) was shown to be one of the major catabolic factors for IVDD (10–12). Studies demonstrated that MMP-13 was activated under diabetic conditions (13–15); however, how MMP-13 is regulated in diabetic IVDD remains elusive. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is required for maintaining chromatin stability (16). Also, it acts as an important regulator in multiple cellular processes, and its inhibitor (JQ1) draws much attention for therapeutic effects against tumor growth (17). Emerging studies have shown a close association between chronic inflammation and diabetes (18), whereas it has been recently discovered that BRD4 regulates inflammation (19, 20). Thus, we assume that BRD4 may be involved in diabetic IVDD, especially MMP-13 expression in NPCs. Inflammation-related signaling pathways such as MAPK [including p38, jun N-terminal kinase (JNK), and extracellular regulated protein kinase 1/2 (ERK1/2)] and NF-κB are reported to be activated by advanced glycation end products (AGEs), which are considered to be major pathogenic metabolites in diabetic conditions (21, 22). Growing evidence has demonstrated that activation of p38 and JNK signals may lead to MMP-13 expression enhancement (23–25), whereas NF-κB signaling was also reported to act as a crucial regulator in MMP-13 expression in NPCs (26). Meanwhile, our studies have found that autophagy may also be activated by AGEs and regulate MMP-13 in NPCs (27–29). These studies above suggest that MAPK, NF-κB signaling, and autophagy may regulate MMP-13 in IVDD, which may also be implicated in diabetic IVDD; however, the relationship between them and BRD4 in diabetic IVDD is still unknown. In the current study, we found: 1) the expression of BRD4 and MMP-13 were increased in diabetic NP tissues and AGEs-treated NPCs; 2) MAPK, NF-κB signaling pathways, and autophagy may regulate MMP-13 expression in AGEs-treated NPCs; 3) BRD4 inhibition, either by lentivirus or by BRD4 inhibitor JQ1, may suppress MAPK and NF-κB signaling pathways and activate autophagy in AGEs-treated NPCs; and 4) BRD4 inhibition suppressed diabetes-induced MMP-13 expression and ameliorated diabetic IVDD in vivo. To the best of our knowledge, this is the first study describing the role of BRD4 in diabetic IVDD and introducing BRD4 inhibition for diabetic IVDD therapy. MATERIALS AND METHODS Ethics statement All interventions, treatments, animal care [Sprague-Dawley (SD) rats; BKS mice], and primary cells (SD rats) extraction procedures were performed in strict accordance with the Animal Care and Use Committee of Wenzhou Medical University. Human NP tissue collection and experiments involved in human NP were approved by the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University Ethics Committee and following the guidelines of the Declaration of Helsinki (30). Reagents and antibodies The inhibitor (+)-JQ1 was purchased from Meilunbio (Dalian, China), and its purity was ≥98%. The glycoaldehyde-AGEs modified bull serum albumin (BSA) (AGEs-BSA) was purchased from Biovision (Milpitas, CA, USA). The primary antibodies of BRD4, IκBα, p-p65, p65, WD repeat domain phosphoinositide interacting 2 (WIPI2), p62, and β-actin were acquired from Abcam (Cambrige, MA, USA). The p-p38, p38, p-JNK, JNK, p-ERK, and ERK antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA). DAPI was purchased from Beyotime (Shanghai, China). Alexa-Fluor-488–tagged and Alexa-Fluor-594–tagged second antibodies were from Abcam. The reagents for cell culture were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Human NP collection To study the relationship between BRD4 and diabetes, 7 NP tissues from patients with IVDD (Table 1) were collected to detect BRD4 level according to the Pfirrmann grading scale (31). In our study, there are no other complications except diabetes, such as hypertension, etc., related to the IVDD in patients whose NP tissues were collected in surgery. The collected NPCs were cultured to 70% in the first passage and lysed for Western blot subsequently. Table 1. Information of patients involved in this study No. Age Sex Piffirmann grade FBG (mM) HbA1c (%) Diabetes mellitus 1 52 F IV 4.5 4.8 No 2 46 F IV 3.8 5.9 No 3 43 M IV 5.9 5.2 No 4 56 F IV 7.5 8.7 Yes 5 51 M IV 7.9 10.8 Yes 6 49 F IV 16.1 13.2 Yes 7 55 M IV 9.6 9.1 Yes Range of normal values: FBG: 3.9–6.1 mM; HbA1c: 4–6%. F, female; M, male. Rat nucleus pulposus cells isolation and culture The gel-like NP tissues were collected from the tails of 3–4-wk-old SD rats (60 female rats and 60 male rats; totally 120 rats). The NP tissues were digested in 0.2% type II collagenase (MilliporeSigma, Burlington, MA, USA) for 4 h at 37°C. After washing with PBS, the digested tissues were transferred to DMEM/F12 (Thermo Fisher Scientific) with 15% fetal bovine serum (Thermo Fisher Scientific) and antibiotics (1% streptomycin/penicillin) in the incubator at 5% CO2 at 37°C. When confluent, the cells were passaged after trypsinizing with 0.25% Trypsin-EDTA (Thermo Fisher Scientific) and replanted into 10-cm culture plates at the appropriate density. We used the first 3 passages cells in our experiments. NPCs from 90 rats were used to be performed Western blots, and the remaining NPCs were used for further experiments such as immunofluorescence, western blotting, and transmission electron microscopy. Experimental design In our study, we used primary NPCs extracted from SD rats. In order to simulate the normal condition and the diabetic condition in vitro, NPCs in a serum-free medium were cultured with BSA or AGEs-BSA at concentration of 3 µM for 24 h or different time intervals (0, 12, 24, and 48 h), with or without agonists or inhibitors of autophagy and inhibitors of MAPK and NF-κB signaling pathways. All these reagents, including p38 inhibitor SB202190 (10 µM), JNK inhibitor SP600125 (10 µM), ERK1/2 inhibitor PD98059 (PD) (10 µM), NF-κB inhibitor BAY-11-7082 (20 µM), autophagy agonist rapamycin (Rapa) (500 nM), and autophagy inhibitor 3-methyladenine (3-MA; 5 mM) were added into culture medium before treatment with BSA or AGEs-BSA. To determine the effects of BRD4 inhibition in different signaling pathways, NPCs were treated with or without BRD4 knockdown or JQ1 at 200 nM. In in vivo studies we performed experiments by using BKS wild-type and BKS db/db mice (30 female mice and 30 male mice; totally 60 BSK mice). We used a total of 60 BKS mice. To evaluate the effects of BRD4 inhibition in BKS db/db mice, JQ1 dissolved in DMSO solution was injected intraperitoneally daily at a dose of 50 mg/kg for 4-wk period. General experimental animal arrangements are shown in Fig. 1. Figure 1Open in figure viewer Schematic of experimental animal arrangement in this study. IHC, immunohistochemistry; IF, immunofluorescence; S&O, safranin O-fast green staining; WB, Western blots. Lentivirus transfection When reaching 30–50% confluence, rat NPCs were transfected with lentivirus (GeneChem, Shanghai, China) at multiplicity of infection of 100. The culture medium was changed after 12 h of transfection, when more than 95% of cells were still alive. Three days later, all transfected cells were passaged for further experiments. The level of BRD4 was quantified by Western blot analysis. Transmission electron microscopy After being fixed in 2.5% glutaraldehyde overnight, rat NPCs were fixed in 2% osmium tetroxide for 1 h and stained with 2% uranyl acetate for 1 h. Before embedding into araldite and cut into semithin sections, these samples were dehydrated in an ascending series of acetone. Then, semithin sections were stained with toluidine blue to locate cells before being observed with a transmission electron microscope (Hitachi, Tokyo, Japan) in 80 nm. Western blot assay NPCs were lysed in ice-cold RIPA with 1 mM PMSF (Beyotime). Protein concentrations of samples were measured by the BCA protein assay kit (Beyotime). Proteins of NPCs were separated on 7.5–12.5% SDS-PAGE and were transferred to PVDF membrane (MilliporeSigma) followed by blocking with 5% nonfat milk. After that, the bands were probed with primary antibodies specific to BRD4 (1:1000), WIPI2 (1:1000), microtubule-associated protein 1A/1B-light chain 3 (LC3; 1:1000), p62 (1:1000), p-p38 (1:1000), p38 (1:1000), p-JNK (1:1000), JNK (1:1000), p-ERK (1:1000), ERK (1:1000), p-p65 (1:500), p65 (1:1000), IκBα (1:1000), MMP-13 (1:1000), and β-actin (1:1000) overnight at 4°C, before incubated with goat anti-rabbit IgG H&L horseradish peroxidase (HRP) secondary antibody. Last, after incubation with Super ECL Reagent (Wanleibio, Shenyang, China) for 60 s, bands were quantified using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA) to determine the intensity. Immunofluorescence analysis After being incubated with 0.5% Triton-X for 15 min, cells were blocked by 10% goat serum for 30 min at 37°C. Primary antibodies against BRD4 (1:100) and LC3 (1:100) were applied to the incubation of samples at 4°C overnight. Then, the slides were incubated with FITC- or tetramethyl rhodamine isothiocyanate–conjugated second antibodies for 1 h and labeled with DAPI for 5 min. Lastly, slides were observed in a fluorescence microscope (Olympus, Tokyo, Japan). Immunohistochemical analysis After sections embedded in paraffin were deparaffinized, rehydrated endogenous peroxidase was blocked by 3% hydrogen peroxide. Then, the sections were incubated with 0.4% pepsin (Sangon Biotech, Shanghai, China) in 5 mM HCl at 37°C for 30 min for antigen retrieval. The sections were incubated with 10% goat blocking serum for 30 min at room temperature, then with primary antibody against MMP-13 (1:100) overnight at 4°C, and finally with HRP-conjugated secondary antibody. Rinsed sections were counterstained with hematoxylin. Bright-field images were acquired by using light microscopy (Olympus). Histopathologic analysis BKS mice were executed with an intraperitoneal lethal dose injection of pentobarbital, and tails were harvested. After being fixed in formaldehyde and decalcified, the specimens were dehydrated and embedded in paraffin. The tissues were cut into 5-µm sections. Slides of each joint were stained with safranin O-fast green and alcian blue staining. Bright-field images were acquired using light microscopy. Statistical analysis All the experiments were performed at ≥3 times. The results were expressed as means ± sd. Raw statistical analysis was processed by SPSS statistical software v.20.0 (IBM SPSS, Chicago, IL, USA). Data were analyzed by 1-way ANOVA followed by the Tukey's test for comparison between control and treatment groups. Nonparametric data (Pfirrmann MRI grade scores) were analyzed by the Kruskal-Wallis H test. Differences were suggested to be statistically significant when P < 0.05. RESULTS BRD4 level increases with higher MMP-13 level in diabetic IVDD in human NP To investigate the BRD4 and MMP-13 expression in relatively healthy humans and diabetic human NPs, we collected human NP tissues from patients undergoing intervertebral discs (IVDs) surgery and divided them into nondiabetes group and diabetes group. As shown in Fig. 2, our results showed that the levels of BRD4 and MMP-13 were elevated in NP of patients with diabetes compared with patients without diabetes, leading to our further experiments to explore BRD4 and MMP-3 levels in different species. Figure 2Open in figure viewer Expression of BRD4 and MMP-13 in diabetic NP tissues in human. A) The BRD4 level of NP from patients with or without diabeties (n = 5). B) BRD4/β-actin of control. C) MMP-13/β-acton of control. All experiments were performed as means ± sd of 3 times in duplicate. *P < 0.05, **P < 0.01. BRD4 level increases with higher MMP-13 expression in murine diabetic IVDD In order to verify the results in Fig. 2, we did further experiments in murine. Immunofluorescence assay for BRD4 and immunohistochemistry for MMP-13 in BKS mice IVDs sections from different groups were performed. As shown in Fig. 3A–D, db/db mice displayed higher fluorescence intensity of BRD4 and higher absorbance of MMP-13 in IVDs compared with wild-type mice. AGEs-BSA, which is considered as a potential biomarker for T2D (21), was used to establish diabetic IVDD in vitro. Our data showed that AGEs-BSA promoted BRD4 level with increasing content of MMP-13 in rat NPCs, indicating the relevance between BRD4 and MMP-13 (Fig. 3E–G). Therefore, based on the similar results of in vitro and in vivo experiments, we assume that BRD4 might be involved in regulation of MMP-13 in diabetic conditions. Figure 3Open in figure viewer Expression of BRD4 and MMP-13 in diabetic NP tissues and AGEs-teated NPCs in murine. A, B) Representative immunofluorescence images with details for BRD4 of mice IVDs from wild-type group and db/db group. Samples performed according to our protocal without primary antibody were shown as negative control group (n = 5). Scale bar, 1000 µm. C, D) Representative immunohistochemistry images with details for MMP-13 of mice IVDs from wild-type group and db/db group. Samples performed according to our protocal without primary antibody were shown as negative control group. Brown: MMP-13; Blue: nucleus (n = 5). Scale bar, 100 µm. E–G) Representative Western blots and quantitative data for BRD4 and MMP-13 in rat NPCs treated with AGEs-BSA (3 µM) for increasing time periods (0, 12, 24, and 48 h) (n = 5). All experiments were performed as means ± sd of 3 times in duplicates. *P < 0.05, **P < 0.01. MMP-13 is regulated by MAPK, NF-κB signaling pathways, and autophagy in diabetic NPCs As previously described, MMP-13 could be regulated by MAPK, NF-κB signaling pathways and autophagy; however, it has not been proven in diabetic NPCs (32). Therefore. we aimed to confirm it in AGEs-treated NPCs. As shown in Fig. 4A, B, we found that p38 inhibitor SB203580 and JNK inhibitor SP600125 but not ERK1/2 inhibitor PD down-regulated the level of MMP-13 in NPCs treated with AGEs-BSA, indicating that AGEs-BSA induced MMP-13 activation via p38 and JNK MAPK signaling pathways. Bay-11-7082 is a specific inhibitor for IκB phosphorylation used to suppress activation of NF-κB signaling pathway. Our results showed that inhibition of NF-κB by Bay-11-7082 blocked MMP-13 level augment in NPCs treated with AGEs-BSA, suggesting that NF-κB aggravates MMP-13 expression in NPCs under the AGEs-BSA induced diabetic conditions (Fig. 4C, D). Figure 4Open in figure viewer MAPK, NF-κB, and autophagy are implicated in the mechanism of BRD4 on MMPs in AGEs-BSA–treated NPCs. Reagent: p38 inhibitor SB202190, JNK inhibitor SP600125 (SP), ERK1/2 inhibitor PD, NF-κB inhibitor BAY-11-7082, autophagy agonist Rapa, and autophagy inhibitor 3-MA. After pretreatment with reagents of different signaling pathways, rat NPCs were incubated with or without BSA (3 µM) or AGEs-BSA (3 µM) for 48 h. A, B) The effects of MAPK on MMP-13 in AGEs-BSA–treated NPCs (n = 5). C, D) Effects of NF-κB on MMP-13 in AGEs-BSA–treated NPCs (n = 5). E, F) The role of autophagy in regulation of MMP-13 in AGEs-BSA–treated NPCs (n = 5). All experiments were performed as means ± sd of 3 times in duplicate. *P < 0.05, **P< 0.01. Autophagy inducer (Rapa) and inhibitor (3-MA) were used to investigate the correlation of autophagy with MMP-13 expression. As shown in Fig. 4E, F, autophagy activation by Rapa repressed the MMP-13 level, whereas 3-MA aggravated MMP-13 level enhancement in AGEs-BSA–treated NPCs, implying that autophagy is capable of depressing MMP-13 activation in NPCs treated with AGEs-BSA. Together, these results demonstrate that MMP-13 is re