Dynamic Pressure Stimulation Upregulates Collagen II and Aggrecan in Nucleus Pulposus Cells Through Calcium Signaling

Abstract Background Low-intensity pulsed ultrasound (LIPUS) is a safe and noninvasive rehabilitative physical therapy with anti-inflammatory effects. The current study investigated the effect of LIPUS on the inflammation of nucleus pulposus (NP) cells and its underlying mechanism. Methods Human NP cells were acquired from lumbar disc herniation tissue samples and cultured for experiments. Human NP cells were treated with LPS and then exposed to LIPUS (15 mW/cm2, 30 mW/cm2 and 60 mW/cm2) for 20 min daily for 3 days to determine the appropriate intensity to inhibit the expression of the inflammatory factors TNF-α and IL-1β. The gene and protein expression of aggrecan, collagen II, MMP-3 and MMP-9 was measured by real‐time PCR and western blotting, respectively. The activity of the nuclear factor‐kappa B (NF‐κB) pathway was examined by western blotting and immunofluorescence. After pretreatment with the NF-κB inhibitor PDTC, the expression of TNF-α, IL-1β, MMP-3 and MMP-9 was measured by real‐time PCR. Results LIPUS at intensities of 15 mW/cm2, 30 mW/cm2 and 60 mW/cm2 inhibited LPS-induced NP cell expression of the inflammatory factors TNF-α and IL-1β, especially at 30 mW/cm2. LIPUS significantly upregulated the gene and protein expression of aggrecan and collagen II and downregulated the gene and protein expression of MMP-3 and MMP-9 in LPS-induced NP cells. The NF‐κB signaling pathway was inhibited by LIPUS through inhibiting the protein expression of p-P65 and the translocation of P65 into the nucleus in LPS-induced NP cells. In addition, LIPUS had similar effects as the NF-κB inhibitor PDTC by inhibiting the NF-κB signaling pathway, inflammation and catabolism in LPS-induced human degenerative nucleus pulposus cells. Conclusion LIPUS inhibited inflammation and catabolism through the NF‐κB pathway in human degenerative nucleus pulposus cells.

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CB2-mediated attenuation of nucleus pulposus degeneration via the amelioration of inflammation and oxidative stress in vivo and in vitro

Molecular Medicine ◽

10.1186/s10020-021-00351-x ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Xiaoqiang Cheng ◽

Jiayi Lin ◽

Zhanghuan Chen ◽

Yubo Mao ◽

Xiexin Wu ◽

...

Keyword(s):

Oxidative Stress ◽

Nucleus Pulposus ◽

Nucleus Pulposus Cell ◽

Immunocytochemical Staining ◽

Nucleus Pulposus Cells ◽

Collagen Ii ◽

Ivd Degeneration ◽

Oxide Synthase

Abstract Background Nucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity. Method In Vitro, H2O2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model. Result In vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H2O2) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3. Conclusions The experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H2O2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD.

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Anisodamine Maintains the Stability of Intervertebral Disc Tissue by Inhibiting the Senescence of Nucleus Pulposus Cells and Degradation of Extracellular Matrix via Interleukin-6/Janus Kinases/Signal Transducer and Activator of Transcription 3 Pathway

Frontiers in Pharmacology ◽

10.3389/fphar.2020.519172 ◽

2020 ◽

Vol 11 ◽

Author(s):

Ning Tang ◽

Yulei Dong ◽

Chong Chen ◽

Hong Zhao

Keyword(s):

Intervertebral Disc ◽

Nucleus Pulposus ◽

Nuclear Translocation ◽

Telomerase Activity ◽

Nucleus Pulposus Cells ◽

Stat3 Pathway ◽

Stat3 Phosphorylation ◽

Ecm Degradation ◽

Collagen Ii ◽

Induced Apoptosis

Objectives: Anisodamine (ANI) has been used to treat a variety of diseases. However, the study of ANI in intervertebral disc degeneration (IVDD) is unclear. This study investigated the effects of ANI on degenerative nucleus pulposus cells (NPCs) and IVDD rats, and its possible mechanisms.Methods: Human nucleus pulposus cells (HNPCs) were treated with IL-1β (20 ng/ml) to simulate IVDD, and an IVDD rat model was constructed. IL-1β-induced HNPCs were treated with different concentrations (10, 20, or 40 μM) of ANI, and IVDD rats were also treated with ANI (1 mg/kg).Results: ANI treatment significantly reduced the apoptosis, caspase-3 and SA-β-gal activities, and p53 and p21 proteins expression, while promoted telomerase activity and aggrecan and collagen II synthesis in IL-1β-induced HNPCs. Moreover, the introduction of ANI inhibited the expression of IL-6, phosphorylation of JAK and STAT3, and nuclear translocation of p-STAT3 in Degenerated HNPCs. Additionally, the application of ANI abolished the effects of IL-6 on apoptosis, SA-β-gal and telomerase activity, and the expression of p53, p21, aggrecan and collagen II proteins in degenerated HNPCs. Simultaneously, ANI treatment enhanced the effects of AG490 (inhibitor of JAK/STAT3 pathway) on IL-1β-induced apoptosis, senescence and ECM degradation in HNPCs. Furthermore, ANI treatment markedly inhibited the apoptosis and senescence in the nucleus pulposus of IVDD rats, while promoted the synthesis of aggrecan and collagen II. ANI treatment obviously inhibited JAK and STAT3 phosphorylation and inhibited nuclear translocation of p-STAT3 in IVDD rats.Conclusion: ANI inhibited the senescence and ECM degradation of NPCs by regulating the IL-6/JAK/STAT3 pathway to improve the function of NPCs in IVDD, which may provide new ideas for the treatment of IVDD.

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Overexpression of miR-150 Inhibits the NF-κB Signal Pathway in Intervertebral Disc Degeneration through Targeting P2X7

Cells Tissues Organs ◽

10.1159/000503281 ◽

2019 ◽

Vol 207 (3-4) ◽

pp. 165-176

Author(s):

Yan Zhang ◽

Yi-Shu Zhang ◽

Xiao-Juan Li ◽

Chao-Rong Huang ◽

Hui-Jin Yu ◽

...

Keyword(s):

Intervertebral Disc ◽

Nucleus Pulposus ◽

Disc Degeneration ◽

Intervertebral Disc Degeneration ◽

Signal Pathway ◽

Nucleus Pulposus Cells ◽

Tnf Α ◽

Collagen Ii ◽

Cox 2

Objective: To elaborate the mechanism of miR-150 in the regulation of the NF-κB signal pathway in intervertebral disc degeneration (IDD) by targeting P2X7. Methods: The degenerative and normal intervertebral disc tissues were collected to detect the expressions of miR-150 and P2X7. Nucleus pulposus cells were transfected and divided into different groups. Cell apoptosis was determined by flow cytometry and TUNEL staining. The expressions of IL-6, TNF-α, MMP-3, MMP-13, Cox-2, iNOS, collagen II and aggrecan, as well as NF-κB-associated proteins were measured by qRT-PCR and Western blotting. Furthermore, IDD rat models were established to validate the role of miR-150 in vivo.Results: miR-150 was down-regulated but P2X7 was up-regulated in the degenerative intravertebral disc tissues. The apoptosis of nucleus pulposus cells in the IL-1β-induced group with the transfection of miR-150 mimic and siP2X7 was significantly decreased, with reduced levels of IL-6, TNF-α, MMP-3, MMP-13, Cox-2 and iNOS, increased levels of collagen II and aggrecan, as well as decreased P2X7, p-p65/p65 and cleaved caspase-3. However, the above factors showed an opposite tendency after treatment with miR-150 inhibitor. Furthermore, the P2X7 siRNA transfection could reverse the effects caused by miR-150 inhibitor. Simultaneously, pcDNA P2X7 transfection also inhibited the function of miR-150 mimic in IL-1β-induced nucleus pulposus cells. In vivoexperiments further verified the protective role of miR-150 in IDD rats. Conclusion: miR-150 may alleviate the degeneration of the intervertebral disc partially since it could restrict the NF-κB pathway by targeting P2X7, and thereby inhibiting IL-1β-induced matrix catabolism, inflammatory responses and apoptosis of the nucleus pulposus cells.

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Quantitation of collagen I, collagen II and aggrecan mRNA and expression of the corresponding proteins in human nucleus pulposus cells in monolayer cultures

Cell and Tissue Research ◽

10.1007/s00441-005-1116-6 ◽

2005 ◽

Vol 321 (3) ◽

pp. 459-464 ◽

Cited By ~ 15

Author(s):

Adrijana Preradovic ◽

Guenther Kleinpeter ◽

Hans Feichtinger ◽

Ernest Balaun ◽

Walter Krugluger

Keyword(s):

Nucleus Pulposus ◽

Collagen I ◽

Nucleus Pulposus Cells ◽

Monolayer Cultures ◽

Collagen Ii

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miR-155 Inhibits Nucleus Pulposus Cells’ Degeneration through Targeting ERK 1/2

Disease Markers ◽

10.1155/2016/6984270 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Dongping Ye ◽

Libing Dai ◽

Yicun Yao ◽

Shengnan Qin ◽

Han Xie ◽

...

Keyword(s):

Intervertebral Disc ◽

Nucleus Pulposus ◽

Nucleus Pulposus Cell ◽

Microrna Expression ◽

Expression Level ◽

Nucleus Pulposus Cells ◽

Collagen Ii ◽

The Difference ◽

Cell Expression ◽

Normal Nucleus

We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2.

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