A Combinatorial Relative Mass Value Evaluation of Endogenous Bioactive Proteins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Intervertebral Discs: Identification of Potential Target Proteins for Gene Therapeutic Approaches

Background/Aims: Nucleus pulposus cell (NPC) apoptosis is the main factor in intervertebral disc degeneration (IDD); thus, inhibiting the excessive apoptosis of nucleus pulposus cells may be a potential way to alleviate IDD. The effect of Hemeoxygenase-1 (HO-1) on human NPC apoptosis has never been reported. Our study aimed to investigate the effect and mechanism of HO-1 on apoptosis in human degenerative NPCs. Methods: Nucleus pulposus tissues were collected from patients with lumbar vertebral fracture (LVF) and IDD. The expression of HO-1 and P65 in intervertebral discs was determined using immunohistochemistry and western blot analysis. Apoptosis of human nucleus pulposus cells was quantified by flow cytometric analysis. A recombinant lentiviral vector overexpressing HO-1 and HO-1-siRNA was used to promote or silence the expression of HO-1 in nucleus pulposus cells. The NF-κB inhibitor PDTC was used to inhibit the NF-κB pathway. Results: Our study demonstrated that compared with normal samples, IDD samples showed down-regulation of HO-1 expression and up-regulation of P65 expression. Overexpression of HO-1 inhibited the increase in nucleus pulposus cell apoptosis after IL-1β treatment and simultaneously inhibited the expression of p-P65. Furthermore, after treatment with PDTC, the number of apoptotic cells was significantly decreased with or without overexpression of HO-1. Conclusion: HO-1 might play a significant role in IDD, and HO-1 protected degenerative human NPCs against apoptosis induced by IL-1β through the NF-κB pathway. These findings would aid in the development of novel therapeutic approaches for IDD treatment.

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Cytotoxic Effects of the Radiocontrast Agent Iotrolan and Anesthetic Agents Bupivacaine and Lidocaine in Three-Dimensional Cultures of Human Intervertebral Disc Nucleus Pulposus Cells: Identification of the Apoptotic Pathways

PLoS ONE ◽

10.1371/journal.pone.0092442 ◽

2014 ◽

Vol 9 (3) ◽

pp. e92442 ◽

Cited By ~ 13

Author(s):

Koji Iwasaki ◽

Hideki Sudo ◽

Katsuhisa Yamada ◽

Manabu Ito ◽

Norimasa Iwasaki

Keyword(s):

Intervertebral Disc ◽

Nucleus Pulposus ◽

Three Dimensional ◽

Nucleus Pulposus Cells ◽

Cytotoxic Effects ◽

Radiocontrast Agent ◽

Anesthetic Agents ◽

Apoptotic Pathways

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P34. Tissue-engineered Composite of Three Dimensional PLGA Scaffold Seeded with Nucleus Pulposus Cells for Intervertebral Disc Regeneration

The Spine Journal ◽

10.1016/j.spinee.2008.06.676 ◽

2008 ◽

Vol 8 (5) ◽

pp. 117S-119S ◽

Cited By ~ 1

Author(s):

Chao Zhang ◽

Ruan Dike ◽

Rongfeng Zhang ◽

Shenguo Wang ◽

Fei Yang

Keyword(s):

Intervertebral Disc ◽

Nucleus Pulposus ◽

Three Dimensional ◽

Plga Scaffold ◽

Nucleus Pulposus Cells ◽

Disc Regeneration ◽

Intervertebral Disc Regeneration

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Effect of Lentivirus-mediated GDF5 Transfection on Differentiation of Rabbit Nucleus Pulposus Mesenchymal Stem Cells

10.21203/rs.3.rs-40034/v1 ◽

2020 ◽

Author(s):

kun zhu ◽

Rui Zhao ◽

Yuchen Ye ◽

Gang Xu ◽

Changchun Zhang

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Nucleus Pulposus ◽

Lentiviral Vector ◽

Intervertebral Discs ◽

Degenerative Disease ◽

The Other ◽

Control Group ◽

Nucleus Pulposus Cells ◽

Qrt Pcr

Abstract Background: Disc degenerative disease is a common senile degenerative disease, which seriously affects the quality of life of patients.The purpose of this study is to observe the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs), and to determine the effect of growth differentiation factor 5(GDF5) on the differentiation of rabbit NPMSCs by lentivirus transfection.Methods: In vitro culture model of rabbit NPMSCs was established and NPMSCs cells were identified by flow cytometry (FCM)and quantitative real-time PCR(qRT-PCR). Then NPMSCs were divided into three groups: lentiviral vector carrying GDF5 was used to transfect NPMSCs, to determine the transfection rate, which was recorded as transfection group, and the NPMSCs transfected with ordinary lentiviral vector was recorded as control group, NPMSCs without processing was recorded as normal group. FCM, qRT-PCR and Western Blot(WB) were used to detected the change of NPMSCs.Results: The transfected NPMSCs by GDF5 became longer and narrower, and the cell density decreased,and the positive rate of GDF5 in the transfected group was significantly higher than that in the other two groups (P<0.05). The mRNA expression of KRT8, KRT18, KRT19 in the transfected group was significantly higher than the other two groups(P<0.05),the result of WB were the same to qRT-PCR. Conclusions: GDF5 can induce the differentiation of NPMSCs and repair degenerative intervertebral discs. Lentiviral vector carrying GDF5 can be integrated into the chromosome genome of NPMSCs and promote differentiation of NPMSCs into nucleus pulposus cells(NPCs).

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Propionibacterium acnes Accelerates Intervertebral Disc Degeneration by Inducing Pyroptosis of Nucleus Pulposus Cells via the ROS-NLRP3 Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/4657014 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Guoqing Tang ◽

Xiaoguang Han ◽

Zhijie Lin ◽

Hongbin Qian ◽

Bing Chen ◽

...

Keyword(s):

Nucleus Pulposus ◽

Propionibacterium Acnes ◽

Strong Association ◽

Intervertebral Discs ◽

Inflammatory Factors ◽

Death Process ◽

Dependent Manner ◽

Nucleus Pulposus Cells ◽

Alternative Treatment Strategy ◽

Ivd Degeneration

Our previous study verified the occurrence of Propionibacterium acnes (P. acnes), a low-virulence anaerobic bacterium, latently residing in degenerated intervertebral discs (IVDs), and the infection had a strong association with IVD degeneration. We explored whether P. acnes induces nucleus pulposus cell (NPC) pyroptosis, a more dangerous cell death process than apoptosis, and accelerates IVD degeneration via the pyroptotic products interleukin- (IL-) 1β and IL-18. After coculturing with P. acnes, human NPCs showed significant upregulation of NOD-like receptor 3 (NLRP3), cleaved IL-1β, cleaved caspase-1, and cleaved gasdermin D in response to the overexpression of IL-1β and IL-18 in a time- and dose-dependent manner. In addition, the gene expression of inflammatory factors and catabolic enzymes significantly increased in normal NPCs when cocultured with pyroptotic NPCs in a transwell system, and the adverse effects were inhibited when NPC pyroptosis was suppressed. Furthermore, inoculation of P. acnes into the IVDs of rats caused significant pyroptosis of NPCs and remarkable IVD degeneration. Finally, coculture of NPCs with P. acnes induced the overexpression of reactive oxygen species (ROS) and NLRP3, while inhibition of both factors reduced NPC pyroptosis. Therefore, P. acnes induces NPC pyroptosis via the ROS-NLRP3 signaling pathway, and the pyroptotic NPCs cause an IVD degeneration cascade. Targeting the P. acnes-induced pyroptosis of NPCs may become an alternative treatment strategy for IVD degeneration in the future.

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Protective Effect of Polygonatum sibiricum Polysaccharides on Apoptosis, Inflammation, and Oxidative Stress in Nucleus Pulposus Cells of Rats with the Degeneration of the Intervertebral Disc

International Journal of Polymer Science ◽

10.1155/2019/8925807 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Zhaohui Zhai ◽

Zhaoxin Li ◽

Zhonglei Ji ◽

Xiaosheng Lu

Keyword(s):

Oxidative Stress ◽

Intervertebral Disc ◽

Nucleus Pulposus ◽

Disc Degeneration ◽

Rat Model ◽

Intervertebral Disc Degeneration ◽

Intervertebral Discs ◽

New Drugs ◽

Nucleus Pulposus Cells ◽

And Oxidative Stress

Objective. Polygonatum sibiricum polysaccharide (PSP) has antioxidant activity, immune enhancement, and other biological properties. However, the effect of PSP on intervertebral disc degeneration has not been reported. In this study, we mainly investigated the effect of PSP on the apoptosis, inflammation, and oxidative stress of nucleus pulposus cells (NPCs) during the process of intervertebral disc degeneration. Methods. A rat NPC model induced by H2O2 was constructed. The CCK8 method was used to measure the effects of PSP on the apoptosis of rat NPCs induced by H2O2. The effects on the activity of SOD and content of MDA were also determined. The rat model of intervertebral disc degeneration was treated with PSP for 1 month, and the mRNA expression levels of IL-1β, COX2, iNOS, Col2α1, Col10α1, and MMP3 were measured by qPCR in the tissue of intervertebral disc. NPCs from the degenerated intervertebral discs were separated, and the cell viability was measured by the CCK8 method. The contents of SOD and MDA in NPCs were determined as well. Results. PSP significantly reduced the apoptosis of NPCs induced by H2O2, significantly increased the SOD content, and decreased the content of MDA in H2O2-induced NPCs. The expression level of IL-1β, COX2, and iNOS in the rat model with intervertebral disc degeneration was significantly downregulated after 1 month of PSP treatment. PSP treatment increased the expression of Col2α1 type and significantly decreased the expression of Col10α1 type collagen and MMP3 in rats with disc degeneration. PSP treatment significantly reduced NPC apoptosis and increased its SOD content and reduced MDA content, which is consistent with the results from cell-level experiments. Conclusion. PSP can effectively reduce the apoptosis, inflammation, and oxidative stress of H2O2-induced NPCs in rats with intervertebral disc degeneration and mitigate the progression of intervertebral disc degeneration, which has the potential to be developed as new drugs for the treatment of intervertebral disc degeneration.

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Intervertebral Disc Morphology in Cervical Spine Biomechanics

10.1115/imece1999-0464 ◽

1999 ◽

Author(s):

Srirangam Kumaresan ◽

Frank A. Pintar ◽

Narayan Yoganandan ◽

Phaladone J. Khouphongsy ◽

Joseph F. Cusick

Keyword(s):

Cervical Spine ◽

Intervertebral Disc ◽

Nucleus Pulposus ◽

Annulus Fibrosus ◽

Sagittal Plane ◽

Three Dimensional ◽

Intervertebral Discs ◽

Old Adult ◽

Staining Methods ◽

Histological Images

Abstract Although qualitative descriptions of degenerative changes in the intervertebral disc components have been reported, methods to quantify these changes are lacking. A methodology was developed in this study to quantify the three-dimensional geometrical variations of the annulus fibrosus and nucleus pulposus. Fresh isolated intervertebral discs with adjacent vertebral bodies of skeletally mature young and old adult primates were sectioned sequentially, and different staining methods were used to distinguish the annulus and nucleus. Histological images were examined using light microscopy and exported to a computer to trace the boundaries of the annulus fibrosus and nucleus pulposus. Dorsal to ventral depth, medial to lateral width, and caudal to cranial height measurements of the nucleus pulposus and its relative location to the annulus pulposus were obtained. In the young adult, the nucleus was translucent with scattered notochordal cells. In the older adult, the nucleus appeared as a dense region of amorphous, irregular collagen material. A higher geometrical variation of nucleus due to degeneration was noted in the sagittal plane compared to coronal plane. Determination of the three-dimensional geometrical variations and histology analyses will assist mathematical modelers to better define the disc to study the biomechanics of the cervical spine.

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