Osteogenic protein-1 alleviates high glucose microenvironment-caused degenerative changes in nucleus pulposus cells

Abstract Increasing evidence has indicated a close relationship between diabetes mellitus (DM) and disc degeneration. As a potential therapeutic growth factor, osteogenic protein-1 (OP-1) has lots of protective effects on the healthy disc cell’s biology. The present study was aimed to investigate the effects of OP-1 on degenerative changes of nucleus pulposus (NP) cells in a high glucose culture. Rat NP cells were cultured in the baseline medium or the high glucose (0.2 M) culture medium. OP-1 was added into the high glucose culture medium to investigate whether its has some protective effects against degenerative changes of NP cells in the high glucose culture. NP cell apoptosis ratio, caspase-3/9 activity, expression of apoptosis-related molecules (Bcl-2, Bax, and caspase-3), matrix macromolecules (aggrecan and collagen II), and matrix remodeling enzymes (MMP-3, MMP-13, and ADAMTS-4), and immuno-staining of NP matrix proteins (aggrecan and collagen II) were evaluated. Compared with the baseline culture, high glucose culture significantly increased NP cell apoptosis ratio, caspase-3/9 activity, up-regulated expression of Bax, caspase-3, MMP-3, MMP-13 and ADAMTS-4, down-regulated expression of Bcl-2, aggrecan and collagen II, and decreased staining intensity of aggrecan and collagen II. However, the results of these parameters were partly reversed by the addition of OP-1 in the high glucose culture. OP-1 can alleviate high glucose microenvironment-induced degenerative changes of NP cells. The present study provides that OP-1 may be promising in retarding disc degeneration in DM patients.

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Resveratrol inhibits IL-1β-mediated nucleus pulposus cell apoptosis through regulating the PI3K/Akt pathway

Bioscience Reports ◽

10.1042/bsr20190043 ◽

2019 ◽

Vol 39 (3) ◽

Cited By ~ 9

Author(s):

Yanhai Jiang ◽

Zhijie Xie ◽

Jinying Yu ◽

Lianqiang Fu

Keyword(s):

Nucleus Pulposus ◽

Disc Degeneration ◽

Cell Apoptosis ◽

Inflammatory Cytokine ◽

Culture Medium ◽

Caspase 3 ◽

Signal Transduction Pathway ◽

Akt Pathway ◽

Protective Effects ◽

Apoptosis Ratio

Abstract Nucleus pulposus (NP) cell apoptosis is a classical cellular character during intervertebral disc degeneration (IDD). Previous studies have shown that inflammatory cytokine-induced NP cell apoptosis plays an important role in disc degeneration. The present study was aimed to investigate whether resveratrol can suppress IL-1β-mediated NP cell apoptosis and the potential signal transduction pathway. Experimental rat NP cells were treated with culture medium containing IL-1β (20 ng/ml) for 7 days. Control NP cells were cultured in the baseline medium. Resveratrol was added along with culture medium to investigate its effects. The inhibitor LY294002 was used to study the role of the PI3K/Akt pathway. NP cell apoptosis was reflected by the caspase-3 activity, cell apoptosis ratio, and expression of apoptosis-related molecules (Bcl-2, Bax, caspase-3, cleaved caspase-3, and cleaved PARP). Compared with the control NP cells, IL-1β significantly increased caspase-3 activity, NP cell apoptosis ratio and mRNA/protein expression of Bax, caspase-3, cleaved caspase-3 and cleaved PARP, but decreased mRNA expression of Bcl-2. However, resveratrol partly suppressed the effects of IL-1β on those cell apoptosis-related parameters. Further analysis showed that IL-1β significantly decreased activity of the PI3K/Akt pathway whereas resveratrol partly increased activity of the PI3K/Akt pathway in NP cells treated with IL-1β. Additionally, when the inhibitor LY294002 was added along with the resveratrol, its protective effects against IL-1β-induced NP cell apoptosis were attenuated. In conclusion, resveratrol suppresses IL-1β-mediated NP cell apoptosis through activating the PI3K/Akt pathway. Resveratrol may be an effective drug to attenuate inflammatory cytokine-induced disc degenerative changes.

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Osteogenic protein-1 attenuates nucleus pulposus cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture

Bioscience Reports ◽

10.1042/bsr20181708 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 3

Author(s):

Yan Yang ◽

Xiyang Wang ◽

Zheng Liu ◽

Xiao Xiao ◽

Wenkai Hu ◽

...

Keyword(s):

Protein Expression ◽

Nucleus Pulposus ◽

Cell Apoptosis ◽

Caspase 3 ◽

Mtor Pathway ◽

Protective Effects ◽

Fk 506 ◽

Osteogenic Protein ◽

Apoptosis Ratio

Background: Previous studies have indicated that osteogenic protein-1 has protective effects on the biological functions of intervertebral disc cells. Hyperosmolarity is an important physicochemical factor within the disc nucleus pulposus (NP) region, which obviously promotes NP cell apoptosis. Objective: To study the effects of osteogenic protein-1 (OP-1) on NP cell apoptosis induced by hyperosmolarity and the potential signaling transduction pathway. Methods: Rat NP cells were cultured in a hyperosmotic medium with or without OP-1 addition for 7 days. Inhibitor 294002 and inhibitor FK-506 were used to investigate the role of the PI3K/Akt/mTOR pathway in this process. NP cell apoptosis were evaluated by cell apoptosis ratio, activity of caspase-3/9 and gene/protein expression of apoptosis-related molecules (Bax, Bcl-2, caspase-3/cleaved caspase-3 and cleaved PARP). Results: OP-1 addition obviously decreased cell apoptosis ratio and caspase-3/9 activity, down-regulated gene/protein expression of pro-apoptosis molecules (Bax, caspase-3/cleaved casepase-3 and cleaved PARP), up-regulated gene/protein expression of anti-apoptosis molecule (Bcl-2) in a hyperosmotic culture. Moreover, OP-1 addition significantly increased protein expression of p-Akt and p-mTOR. Further analysis showed that addition of LY294002 and FK-506 partly attenuated these protective effects of OP-1 against NP cell apoptosis and activation of the PI3K/Akt/mTOR pathway in a hyperosmotic culture. Conclusion: OP-1 can attenuate NP cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture. The present study sheds a new light on the protective role of OP-1 in regulating disc cell biology and provides some theoretical basis for the application of OP-1 in retarding/regenerating disc degeneration.

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Osteogenic protein-1 inhibits nucleus pulposus cell apoptosis through regulating the NF-κB/ROS pathway in an inflammation environment

Bioscience Reports ◽

10.1042/bsr20181530 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 5

Author(s):

Wei Yu ◽

Jiabin Fu ◽

Yan Liu ◽

Yuchi Wu ◽

Dianming Jiang

Keyword(s):

Protein Expression ◽

Disc Degeneration ◽

Cell Apoptosis ◽

Caspase 3 ◽

Protective Effects ◽

Transduction Pathway ◽

Signaling Transduction Pathway ◽

Tnf Α ◽

Osteogenic Protein ◽

Apoptosis Ratio

Background: Intervertebral disc degeneration is a pathological process that involves an inflammation response. As a classical cellular feature, several studies have demonstrated that inflammation can promote nucleus pulposus (NP) cell apoptosis. Therefore, attenuation of NP cell apoptosis may be a potential way to retard disc degeneration. Objective: The present study was aimed to investigate the protective effects of osteogenic protein-1 (OP-1) against NP cell apoptosis in an inflammation environment, and the potential signaling transduction pathway. Methods: Rat NP cells were cultured in medium with or without inflammatory cytokine tumor necrosis factor (TNF)-α for 6 days. The exogenous TNF-α was added into the medium to investigate its protective effects. NP cell apoptosis was evaluated by cell apoptosis ratio, caspase-3 activity, gene/protein expression of apoptosis-related molecules (Bcl-2, Bax, and caspase-3). Additionally, the intracellular reactive oxygen species (ROS) content and activity of the NF-κB pathway were also analyzed. Results: Compared with the control NP cells, TNF-α significantly increased cell apoptosis ratio, caspase-3 activity, gene/protein expression of Bcl-2, Bax and caspase-3, ROS content, and activity of the NF-κB pathway. However, OP-1 partly attenuated these effects in NP cells treated with TNF-α. Conclusion: OP-1 is effective in attenuating TNF-α-caused NP cell apoptosis, and the ROS/NF-κB pathway may be the potential signaling transduction pathway. The present study indicates that OP-1 may be helpful to inhibit inflammation-mediated disc degeneration.

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High-glucose environment accelerates annulus fibrosus cell apoptosis by regulating endoplasmic reticulum stress

Bioscience Reports ◽

10.1042/bsr20200262 ◽

2020 ◽

Vol 40 (7) ◽

Author(s):

Lianglong Pang ◽

Keshi Yang ◽

Zhi Zhang

Keyword(s):

Endoplasmic Reticulum ◽

Protein Expression ◽

Er Stress ◽

Disc Degeneration ◽

High Glucose ◽

Cell Apoptosis ◽

Annulus Fibrosus ◽

Culture Medium ◽

Caspase 3 ◽

Apoptosis Ratio

Abstract Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. However, how DM affects annulus fibrosus (AF) biology remains unclear. The present study was aimed to investigate the effects and mechanism of high glucose on AF cell biology. Rat AF cells were cultured in baseline medium and culture medium with 0.2 M glucose. The inhibitor 4-PBA was added along with the high glucose culture medium to study the role of endoplasmic reticulum (ER) stress in this process. Compared with the control cells, high glucose significantly increased cell apoptosis ratio and caspase-3/9 activity, up-regulated mRNA/protein expression of Bax and caspase-3/cleaved caspase-3, but down-regulated mRNA/protein expression of Bcl-2. Moreover, high glucose increased mRNA and protein expression of CHOP, ATF-6 and GRP78. However, once ER stress was inhibited by the inhibitor 4-PBA in the high glucose group, cell apoptosis ratio and caspase-3/9 activity were decreased, mRNA/protein expression of Bax and caspase-3/cleaved caspase-3 was down-regulated, but mRNA/protein expression of Bcl-2 was up-regulated. In conclusion, high glucose condition can promote AF cell apoptosis through inducing ER stress. The present study helps us understand the mechanism of disc degeneration in DM patients.

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Osteogenic protein-1 attenuates apoptosis and enhances matrix synthesis of nucleus pulposus cells under high magnitude compression though inhibiting the p38 MAPK pathway

Bioscience Reports ◽

10.1042/bsr20180018 ◽

2018 ◽

Vol 38 (2) ◽

Cited By ~ 3

Author(s):

Haolin Fang ◽

Xianzhou Li ◽

Haiming Shen ◽

Buwei Sun ◽

Haijun Teng ◽

...

Keyword(s):

Nucleus Pulposus ◽

P38 Mapk ◽

Disc Degeneration ◽

Cell Apoptosis ◽

Caspase 3 ◽

Mapk Pathway ◽

High Magnitude ◽

P38 Mapk Pathway ◽

Regulated Expression ◽

Osteogenic Protein

Disc degeneration is correlated with mechanical load. Osteogenic protein-1 (OP-1) is potential to regenerate degenerative disc. To investigate whether OP-1 can protect against high magitude compression-induced nucleus pulposus (NP) cell apoptosis and NP matrix catabolism, and its potential mechanism; porcine discs were cultured in a bioreactor and compressed at a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days. OP-1 was added along with the culture medium to investigate the protective effects of OP-1. NP cell apoptosis and matrix biosynthesis were evaluated. Additionally, activity of the p38 MAPK pathway is also analyzed. Compared with the control group, high magnitude compression significantly promoted NP cell apoptosis and decreased NP matrix biosynthesis, reflected by the increase in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity, the up-regulated expression of Bax and caspase-3 mRNA and down-regulated expression of Bcl-2 mRNA, and the decreased Alcian Blue staining intensity and expression of matrix proteins (aggrecan and collagen II). However, OP-1 addition partly attenuated the effects of high magnitude compression on NP cell apoptosis and NP matrix biosynthesis. Further analysis showed that inhibition of the p38 MAPK pathway partly participated in this process. OP-1 can attenuate high magnitude compression-induced NP cell apoptosis and promoted NP matrix biosynthesis, and inhibition of the p38 MAPK pathway may participate in this regulatory process. The present study provides that OP-1 may be efficient in retarding mechanical overloading-exacerbated disc degeneration.

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Resveratrol attenuates high glucose-induced nucleus pulposus cell apoptosis and senescence through activating the ROS-mediated PI3K/Akt pathway

Bioscience Reports ◽

10.1042/bsr20171454 ◽

2018 ◽

Vol 38 (2) ◽

Cited By ~ 18

Author(s):

Wenping Wang ◽

Pei Li ◽

Jiagang Xu ◽

Xiangkun Wu ◽

Zhiliang Guo ◽

...

Keyword(s):

Nucleus Pulposus ◽

High Glucose ◽

Cell Apoptosis ◽

Culture Medium ◽

Nucleus Pulposus Cell ◽

Akt Pathway ◽

Control Group ◽

Ros Generation ◽

Protective Effects ◽

Potential Mechanism

Background: Diabetes mellitus is closely correlated with disc degeneration. Nucleus pulposus (NP) cell apoptosis and senescence are typical cellular features within the degenerative disc. Resveratrol is a newly identified phytoalexin that has protective effects on cartilaginous tissue. Objective: To investigate the whether resveratrol can protect against high glucose-induced NP cell apoptosis and senescence, and the potential mechanism in this process. Methods: Rat NP cells were cultured in either 10% FBS culture medium (control group) or 10% FBS with a high glucose concentration (0.2 M, experiment group) for 3 days. Resveratrol or the combination of resveratrol and LY294002 was added into the culture medium of experiment group to investigate the effects of resveratrol and the PI3K/Akt pathway. Results: High glucose significantly promoted NP cell apoptosis and NP cell senescence compared with the control group. Resveratrol exhibited protective effects against high glucose-induced NP cell apoptosis and senescence. Further analysis showed that resveratrol suppressed reactive oxygen species (ROS) generation and increased the activity of the PI3K/Akt pathway under the high glucose condition. However, the LY294002 had no significant effects on ROS content in the resveratrol-treated high glucose group. Conclusion: Resveratrol can attenuate high glucose-induced NP cell apoptosis and senescence, and the activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

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High glucose promotes annulus fibrosus cell apoptosis through activating the JNK and p38 MAPK pathways

Bioscience Reports ◽

10.1042/bsr20190853 ◽

2019 ◽

Vol 39 (7) ◽

Cited By ~ 6

Author(s):

Lizhen Shan ◽

Di Yang ◽

Danjie Zhu ◽

Fabo Feng ◽

Xiaolin Li

Keyword(s):

P38 Mapk ◽

High Glucose ◽

Cell Apoptosis ◽

Annulus Fibrosus ◽

Caspase 3 ◽

Mapk Pathway ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Jnk Pathway ◽

Regulated Expression

Abstract Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. A high glucose niche-mediated disc cell apoptosis is an implicate causative factor for the spine degenerative diseases related with DM. However, the effects of a high glucose niche on disc annulus fibrosus (AF) cell apoptosis and the potential signaling transduction pathway is unclear. The present study is to investigate the effects of high glucose on disc AF cell apoptosis and the role of two potential signaling pathways in this process. Rat AF cells were cultured in baseline medium or medium with different concentrations (0.1 and 0.2 M) of glucose for 3 days. Flow cytometry was used to assess the degree of apoptosis. Activity of caspase 3/9 was evaluated by chemical kit. Expression of pro-apoptotic and anti-apoptotic molecules was analyzed by real-time polymerase chain reaction and Western blot. In addition, activity of the C-Jun NH2-terminal kinases (JNK) pathway and p38 mitogen-activated protein kinase (MAPK) pathway was evaluated by Western blot. Compared with the control group, high glucose culture increased cell apoptosis ratio and caspase-3/9 activity, up-regulated expression of bax, caspase-3, cleaved caspase-3 and cleaved PARP, and down-regulated expression of bcl-2 in a glucose concentration-dependent manner. Additionally, high glucose culture increased expression of the p-JNK and p-p38 MAPK in a concentration-dependent manner. Further results showed that inhibition of the JNK or p38 MAPK pathway attenuated the effects of high glucose on AF cell apoptosis. Together, high glucose promoted disc AF cell apoptosis through regulating the JNK pathway and p38 MAPK pathway in a glucose concentration-dependent manner.

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Melatonin Suppresses Apoptosis of Nucleus Pulposus Cells through Inhibiting Autophagy via the PI3K/Akt Pathway in a High-Glucose Culture

BioMed Research International ◽

10.1155/2021/4604258 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Jian Li ◽

Chengqiang Wang ◽

Lixin Xue ◽

Fan Zhang ◽

Jianqiang Liu

Keyword(s):

Protein Expression ◽

Nucleus Pulposus ◽

Disc Degeneration ◽

High Glucose ◽

Cell Apoptosis ◽

Caspase 3 ◽

Akt Pathway ◽

Protective Effects ◽

Nucleus Pulposus Cells

Diabetes mellitus- (DM-) associated hyperglycemia promotes apoptosis of disc nucleus pulposus (NP) cells, which is a contributor to intervertebral disc degeneration (IDD). Melatonin is able to protect against cell apoptosis. However, its effects on apoptosis of NP cell in a high-glucose culture remain unclear. The purpose of the present study was to investigate the effects and molecular mechanism of melatonin on NP cell apoptosis in a high-glucose culture. NP cells were cultured in the baseline medium supplemented with a high-glucose concentration (0.2 M) for 3 days. The control cells were only cultured in the baseline medium. Additionally, the pharmaceutical inhibitor LY294002 was added along with the culture medium to investigate the possible role of the PI3K/Akt pathway. Apoptosis, autophagy, and activity of the PI3K/Akt pathway of NP cells among these groups were evaluated. Compared with the control NP cells, high glucose significantly increased cell apoptosis ratio and caspase-3/caspase-9 activity and decreased mRNA expression of Bcl-2, whereas it increased mRNA or protein expression of Bax, caspase-3, cleaved caspase-3, cleaved PARP, and autophagy-related molecules (Atg3, Atg5, Beclin-1, and LC3-II) and decreased protein expression of p-Akt compared with the control cells. Additionally, melatonin partly inhibited the effects of high glucose on those parameters of cell apoptosis, autophagy, and activation of PI3K/Akt. In conclusion, melatonin attenuates apoptosis of NP cells through inhibiting the excessive autophagy via the PI3K/Akt pathway in a high-glucose culture. This study provides new theoretical basis of the protective effects of melatonin against disc degeneration in a DM patient.

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Resveratrol attenuates mechanical compression-induced nucleus pulposus cell apoptosis through regulating the ERK1/2 signaling pathway in a disc organ culture

Bioscience Reports ◽

10.1042/bsr20171703 ◽

2018 ◽

Vol 38 (2) ◽

Cited By ~ 8

Author(s):

Zhiwen Zhang ◽

Feng Wen ◽

Chengjian He ◽

Jun Yu

Keyword(s):

Organ Culture ◽

Nucleus Pulposus ◽

Cell Apoptosis ◽

Caspase 3 ◽

Dependent Manner ◽

Protective Effects ◽

Mechanical Compression ◽

High Magnitude ◽

Regulated Expression ◽

Dose Dependent

Background: Nucleus pulposus (NP) cell apoptosis is a typical feature within the degenerative disc. High magnitude compression significantly promotes NP cell apoptosis. Several studies have indicated that resveratrol has protective effects on disc cell’s normal biology. Objective: The present study aims to investigate whether resveratrol can attenuate mechanical overloading-induced NP cell apoptosis in a disc organ culture. Methods: Isolated porcine discs were cultured in culture chambers of a mechanically active perfusion bioreactor and subjected to a relatively high magnitude compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days. Different concentrations (50 and 100 μM) of resveratrol were added into the culture medium to observe the protective effects of resveratrol against NP cell apoptosis under mechanical compression. The noncompressed discs were used as controls. Results: Similar with the previous studies, this high magnitude compression significantly promoted NP cell apoptosis, reflected by the increased number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining-positive NP cells and enzyme (caspase-9 and caspase-3) activity, the up-regulated expression of proapoptotic molecules (Bax and caspase-3/cleaved caspase-3), and down-regulated expression of antiapoptotic molecule (Bcl-2). However, resveratrol partly attenuated NP cell apoptosis under this high magnitude compression in a dose-dependent manner. Additionally, though the ERK1/2 pathway was significantly activated in the mechanical compression group, resveratrol partly attenuated activation of the ERK1/2 pathway under mechanical compression in a dose-dependent manner. Conclusion: Resveratrol attenuates mechanical overloading-induced NP cell apoptosis in a dose-dependent manner, and inhibiting activation of the ERK1/2 pathway may be one potential mechanism behind this regulatory process.

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Liraglutide inhibits the apoptosis of human nucleus pulposus cells induced by high glucose through PI3K/Akt/caspase-3 signaling pathway

Bioscience Reports ◽

10.1042/bsr20190109 ◽

2019 ◽

Vol 39 (8) ◽

Cited By ~ 3

Author(s):

Yao Ming-yan ◽

Zhang Jing ◽

Guo Shu-qin ◽

Bai Xiao-liang ◽

Li Zhi-hong ◽

...

Keyword(s):

Signaling Pathway ◽

Nucleus Pulposus ◽

High Glucose ◽

Caspase 3 ◽

Maximum Effect ◽

Ros Generation ◽

Protective Effects ◽

Small Interfering Rnas ◽

Nucleus Pulposus Cells ◽

Pi3k Inhibitor Ly294002

Abstract Diabetes mellitus (DM) is a potential etiology of disc degeneration. Glucagon-like peptide-1 (GLP-1) is currently regarded as a powerful treatment option for type 2 diabetes. Apart from the beneficial effects on glycaemic control, GLP-1 has been reported to exert functions in a variety of tissues on modulation of cell proliferation, differentiation, and apoptosis. However, little is known regarding the effects of GLP-1 on nucleus pulposus cells (NPCs). In the present study, we investigated the effects of liraglutide (LIR), a long-lasting GLP-1 analogue, on apoptosis of human NPCs and the underlying mechanisms involved. We confirmed the presence of GLP-1 receptor (GLP-1R) in NPCs. Our data demonstrated that liraglutide inhibited the apoptosis of NPCs induced by high glucose (HG), as detected by Annexin V/Propidium Iodide (PI) and ELISA assays. Moreover, liraglutide down-regulated caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further analysis suggested that liraglutide suppressed reactive oxygen species (ROS) generation and stimulated the phosphorylation of Akt under HG condition. Pretreatment of cells with the Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY) and small interfering RNAs (siRNAs) GLP-1R abrogated the liraglutide-induced activation of Akt and the protective effects on NPCs’ apoptosis. In conclusion, liraglutide could directly protect NPCs against HG-induced apoptosis by inhibiting oxidative stress and activate the PI3K/Akt/caspase-3 signaling pathway via GLP-1R.

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