Resveratrol Suppresses Annulus Fibrosus Cell Apoptosis through Regulating Oxidative Stress Reaction in an Inflammatory Environment

During disc degeneration, the increase of inflammatory cytokines and decrease of disc cell density are two prominent features. Enhanced inflammatory reaction contributes to disc annulus fibrosus (AF) cell apoptosis. In this study, we investigated whether resveratrol can suppress AF cell apoptosis in an inflammatory environment. Rat disc AF cells were cultured in medium with or without tumor necrosis factor-α (TNF-α). Resveratrol was added along with the culture medium supplemented with TNF-α. Caspase-3 activity, cell apoptosis ratio, expression of apoptosis-associated molecules (Bcl-2, Bax, caspase-3, cleaved PARP, and cleaved caspase-3), reactive oxygen species (ROS) content, and the total superoxide dismutase (SOD) activity were measured. Our results showed that TNF-α significantly increased caspase-3 activity and AF cell apoptosis ratio and upregulated gene/protein expression of Bax, caspase-3, cleaved caspase-3, and cleaved PARP, whereas it downregulated the expression of Bcl-2. Moreover, TNF-α significantly increased ROS content but decreased the total SOD activity. Further analysis demonstrated that resveratrol partly attenuated the effects of TNF-α on AF cell apoptosis-associated parameters, decreased ROS content, and increased the total SOD activity in the AF cells treated with TNF-α. In conclusion, resveratrol attenuates inflammatory cytokine TNF-α-induced AF cell apoptosis through regulating oxidative stress reaction in vitro. This study sheds a new light on the protective role of resveratrol in alleviating disc degeneration.

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Responses of apoptosis and matrix metabolism of annulus fibrosus cells to different magnitudes of mechanical tension in vitro

Bioscience Reports ◽

10.1042/bsr20182375 ◽

2019 ◽

Vol 39 (2) ◽

Cited By ~ 1

Author(s):

Yanhai Jiang ◽

Lianqiang Fu ◽

Yeliang Song

Keyword(s):

Protein Expression ◽

Cell Apoptosis ◽

Annulus Fibrosus ◽

Caspase 3 ◽

Mechanical Load ◽

Control Group ◽

Mechanical Tension ◽

Matrix Metabolism ◽

Apoptosis Ratio

Abstract Background: Annulus fibrosus (AF) is important to confine disc nucleus pulposus (NP) tissue during mechanical load experience. However, the knowledge on AF cell biology under mechanical load is much limited compared with disc NP. Objective: The present study aimed to investigate responses of apoptosis and matrix metabolism of AF cells to different magnitudes of mechanical tension in vitro. Methods: Rat AF cells were subjected to different magnitudes (5, 10, and 20% elongations at a frequency of 1.0 Hz for 6 h per day) of mechanical tension for 7 days. Control AF cells were cultured without mechanical tension. Cell apoptosis ratio, caspase-3 activity, gene/protein expression of apoptosis-related molecules (Bcl-2, Bax, caspase-3/cleaved caspase-3 and cleaved PARP), matrix macromolecules (aggrecan and collagen I) and matrix metabolism-related enzymes (TIMP-1, TIMP-3, MMP-3, and ADAMTS-4) were analyzed. Results: Compared with 5% tension group and control group, 10 and 20% tension groups significantly increased apoptosis ratio, caspase-3 activity, up-regulated gene/protein expression of Bax, caspase-3/cleaved caspase-3, cleaved PARP, MMP-3, and ADAMTS-4, whereas down-regulated gene/protein expression of Bcl-2, aggrecan, collagen I, TIMP-1, and TIMP-3. No significant difference was found in these parameters apart from Bcl-2 expression between the control group and 5% tension group. Conclusion: High mechanical tension promotes AF cell apoptosis and suppresses AF matrix synthesis compared with low mechanical tension. The present study indirectly indicates how mechanical overload induces disc degeneration through affecting AF biology.

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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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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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Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

January 2018 - Pain Physician ◽

10.36076/ppj/2019.22.155 ◽

2019 ◽

Vol 2 (22.2) ◽

pp. 155-164

Author(s):

Liang Zhang

Keyword(s):

Methylene Blue ◽

Cell Apoptosis ◽

Annulus Fibrosus ◽

Caspase 3 ◽

In Vitro Study ◽

Collagen Type I ◽

Type I ◽

Dependent Manner ◽

Concentration Dependent Manner

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood. Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro. Study Design: This study used an experimental design. Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University. Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 μg/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor β1 [TGF-β1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting. Results: Our results indicated that MB reduced cell viability in a concentration- and timedependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells. Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols. Key words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine

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Salvianolic Acid B Protects Intervertebral Discs from Oxidative Stress-Induced Degeneration via Activation of the JAK2/STAT3 Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6672978 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Shouqian Dai ◽

Ting Liang ◽

Xiu Shi ◽

Zongping Luo ◽

Huilin Yang

Keyword(s):

Oxidative Stress ◽

Cell Proliferation ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Caspase 3 ◽

Intervertebral Discs ◽

Salvianolic Acid B ◽

Stat3 Signaling ◽

Stat3 Signaling Pathway

Objective. To evaluate the influence of salvianolic acid B (SAB), an antioxidant derived from Danshen, on intervertebral disc degeneration (IDD) and its possible molecular mechanisms. Methods. Sixty adult rats were randomly grouped (control, IDD, and SAB IDD groups). IDD was induced using needle puncture. The rats received daily administration of SAB (20 mg/kg) in the SAB IDD group while the other two groups received only distilled water. The extent of IDD was evaluated using MRI after 3 and 6 weeks and histology after 6 weeks. Oxidative stress was assessed using the ELISA method. In in vitro experiments, nucleus pulposus cells (NPCs) were treated with H2O2 (100 μM) or SAB+H2O2, and levels of oxidative stress were measured. Cell apoptosis was assessed by flow cytometry, expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins. Cell proliferation rate was assessed by EdU analysis. Pathway involvement was determined by Western blotting while the influence of the pathway on NPCs was explored using the pathway inhibitor AG490. Results. The data demonstrate that SAB attenuated injury-induced IDD and oxidative stress, caused by activation of the JAK2/STAT3 signaling pathway in vivo. Oxidative stress induced by H2O2 was reversed by SAB in vitro. SAB reduced the increased cell apoptosis, cleaved caspase-3 expression, and caspase-3 activity induced by H2O2. Reduced cell proliferation and decreased Bcl-2/Bax ratio induced by H2O2 were rescued by SAB. Additionally, the JAK2/STAT3 pathway was activated by SAB, while AG490 counteracted this effect. Conclusion. The results suggest that SAB protects intervertebral discs from oxidative stress-induced degeneration by enhancing proliferation and attenuating apoptosis via activation of the JAK2/STAT3 signaling pathway.

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Testosterone aggravates cerebral vascular injury by reducing plasma HDL levels

Open Life Sciences ◽

10.1515/biol-2020-0107 ◽

2020 ◽

Vol 15 (1) ◽

pp. 1042-1048

Author(s):

Tao Jin ◽

Lu Wang ◽

Dongbo Li ◽

Tao Yang ◽

Yuefei Zhou

Keyword(s):

Oxidative Stress ◽

Soybean Oil ◽

Vascular Injury ◽

Cerebral Artery ◽

Cell Apoptosis ◽

Density Lipoprotein ◽

Tnf Α ◽

And Oxidative Stress ◽

Cerebral Vascular

AbstractTestosterone is often used to improve the physiological function. But increased testosterone levels affect blood lipids and cause inflammation and oxidative stress, which are risk factors for vascular diseases. This study aimed at investigating the effects of testosterone on cerebral vascular injury using an established intracranial aneurysm (IA) model. Sixteen-week-old female C57Bl/6 mice were subcutaneously infused with testosterone propionate (TP; 5 mg/kg day) or plain soybean oil (controls) for 6 weeks. After 2 weeks of treatment, mice were given angiotensin II-elastase for another 4 weeks. The results showed that TP significantly increased cell apoptosis and reactive oxygen species production in cerebral artery, together with increases in plasma tumor necrosis factor-α (TNF-α) levels and in urinary 8-isoprostane levels. Plasma assays showed that 2 weeks after TP or soybean oil administration, the high-density lipoprotein (HDL) level was higher in the TP group than in controls. In vitro studies showed that testosterone increased TNF-α and monocyte chemotactic protein-1 mRNA and protein expression levels in RAW 264.7 macrophages. In summary, by reducing the HDL level, TP aggravates cerebral artery injury by increasing cell apoptosis, inflammation, and oxidative stress.

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Rehmanniae Decoction Improve Brain Function in Vascular Dementia Rats Through Phosphoinositide 3-Kinase (PI3K)/AKT Signaling Pathway

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2286 ◽

2020 ◽

Vol 10 (4) ◽

pp. 538-544

Author(s):

Peng Sang ◽

Jiahui Zhao ◽

Shun Wang ◽

Hui Yang ◽

Huijuan Shi

Keyword(s):

Oxidative Stress ◽

Learning And Memory ◽

Cell Apoptosis ◽

Caspase 3 ◽

Sham Group ◽

Akt Signaling ◽

Sod Activity ◽

Memory Ability ◽

Group I ◽

Mda Content

Objective: Studies have shown that Rehmanniae Decoction (Reh) has therapeutic effect on vascular dementia (VD). PI3K/AKT signaling regulates oxidative stress damage and cell apoptosis. Our study intends torehmanniae decoction's effect on the neural function in VD mice. Methods: The mice were divided into Sham group, VD group, low dose Reh+ VD group and high dose Reh+ VD group. Water maze test was used to assess learning and memory ability. The activity of caspase-3, the content of MDA and the activity of SOD enzyme in hippocampus were detected. In vitro , HT22 cells were divided into control group, I–R group, I–R+ 2% Reh serum, I–R+ 4% Reh serum. Flow cytometry was used to detect the intracellular content of ROS and cell apoptosis. Results: Compared with sham group, the learning and memory ability of mice in VD group was significantly decreased. p-AKT level and SOD activity in the hippocampus was decreased, the Caspase-3 activity and MDA content was significantly increased. After treatment of Reh, the learning and memory ability of VD model mice was significantly improved, p-AKT protein expression and SOD activity were up-regulated, and Caspase-3 activity and MDA content were reduced. Conclusion: Rehmanniae decoction alleviates the oxidative stress and inhibits cell apoptosis to improve the function of brain by regulating PI3K/AKT pathway.

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Acetylcholine ameliorated TNF-α-induced primary trophoblast malfunction via muscarinic receptors†

Biology of Reproduction ◽

10.1093/biolre/ioaa158 ◽

2020 ◽

Vol 103 (6) ◽

pp. 1238-1248

Author(s):

Zheng Wang ◽

Adoulaye Issotina Zibrila ◽

Shuhua Liu ◽

Gongxiao Zhao ◽

Yubei Li ◽

...

Keyword(s):

Oxidative Stress ◽

Muscarinic Receptors ◽

Caspase 3 ◽

Migration And Invasion ◽

Vagal Activity ◽

Trophoblast Cells ◽

Human Trophoblast ◽

Tnf Α

Abstract Oxidative stress and apoptosis of trophoblasts are involved in preeclampsia (PE). Numerous studies have shown that acetylcholine (ACh), the principal vagal neurotransmitter, plays a crucial role in attenuating oxidative stress, inflammation, and apoptosis in a variety of human diseases. However, the role of ACh in PE management remains unclear. Here, we aimed to determine the effects of ACh on TNF-α-treated human primary trophoblast cells. Western blotting, CCK-8, DHE, TUNEL immunofluorescence staining, transwell assays, and wound-healing assays were performed to evaluate the role of ACh in vitro. We found that both TNF-α expression and the apoptotic index were higher in placentas from preeclamptic women than in normal placentas. TNF-α enhanced oxidative stress and increased the number of TUNEL-positive nuclei, Bax/Bcl-2 ratio, and the cleaved caspase-3/caspase-3 ratio while decreasing cell viability in primary human trophoblast cells. TNF-α promoted cell migration and invasion. PDTC, a selective NF-κB inhibitor, significantly blunted TNF-α-induced effects. ACh treatment attenuated oxidative stress and apoptosis while further promoting migration and invasion of TNF-α-treated primary trophoblast cells. The effects of ACh could be reversed by the muscarinic receptor antagonist atropine. Overall, our findings indicate that ACh significantly ameliorates TNF-α-induced oxidative stress and apoptosis of human primary trophoblast cells via muscarinic receptors. This is the first time that the improvement of vagal activity served as a therapeutic strategy for PE-like trophoblasts, suggesting its potential value in clinical practice.

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Transforming growth factor-β1-regulated Fas/FasL pathway activation suppresses nucleus pulposus cell apoptosis in an inflammatory environment

Bioscience Reports ◽

10.1042/bsr20191726 ◽

2020 ◽

Vol 40 (2) ◽

Cited By ~ 2

Author(s):

Jingjing Xie ◽

Bo Li ◽

Bing Yao ◽

Pingchao Zhang ◽

Lixin Wang ◽

...

Keyword(s):

Growth Factor ◽

Nucleus Pulposus ◽

Disc Degeneration ◽

Cell Apoptosis ◽

Caspase 3 ◽

Transforming Growth Factor ◽

Tnf Α ◽

Gene And Protein Expression ◽

Tgf Β1

Abstract Background: During disc degeneration, inflammatory cytokine tumor necrosis factor (TNF)-α is correlated with nucleus pulposus (NP) cell apoptosis. Transforming growth factor (TGF)-β1 has the potential to regenerate degenerative disc. Objective: To investigate the protective role of TGF-β1 against TNF-α-mediated NP cell apoptosis and the underlying mechanism. Methods: Rat NP cells were treated with TNF-α (100 ng/ml) for 48 h. TGF-β1 was added into the culture medium to investigate its protective effects against TNF-α-induced NP cell apoptosis. Exogenous FasL was used to investigate the potential role of the Fas/FasL pathway in this process. Flow cytometry assay was used to analyze NP cell apoptosis. Real-time PCR and Western blotting were used to analyze gene and protein expression of apoptosis-related molecules. Results: In TNF-α-treated NP cells, TGF-β1 significantly decreased NP cell apoptosis, declined caspase-3 and -8 activity, and decreased expression of Bax and caspase-3 (cleaved-caspase-3) but increased expression of Bcl-2. However, exogenous FasL partly reversed these effects of TGF-β1 in NP cells treated with TNF-α. Additionally, expression of Fas and FasL in TNF-α-treated NP cells partly decreased by TGF-β1, whereas exogenous FasL increased expression of Fas and FasL in NP cells treated with TGF-β1 and TNF-α. Conclusion: TGF-β1 helps to inhibit TNF-α-induced NP cell apoptosis and the Fas/FasL pathway may be involved in this process. The present study suggests that TGF-β1 may be effective to retard inflammation-mediated disc degeneration.

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