OBJECTIVE: To explore mechanisms of acid-sensing ion channel 1 (ASIC1) mediated lumbar nucleus pulposus cell apoptosis through extracellular-signalregulated protein kinase 5 (ERK5) signaling pathway and mitochondrial dysfunction pathway. METHODS: Totally 34 patients with degenerative lumbar disc herniation (LDH) admitted from January 2020 to December 2022 were collected as research objects, including 21 males and 13 females;aged from 29 to 52 years old with an average of (37.43±4.75) years old;22 patients with grade Ⅱ and 12 patients with grade Ⅳ, according to Pfirrmann grading criteria;15 patients with L_4,5 and 19 patients with L₅S₁. The expression of ASIC1 in nucleus pulposus of LDH patients was measured by immunohistochemical staining. Nucleus pulposus cells were cultured by primary culture method, identified by toluidine blue staining and immunohistochemical staining, and the expression of ASIC1 protein was located by immunofluorescence staining. According to the addition of siRNA-ASIC1, ASIC1 overexpression plasmid, and ERK5 inhibitors, the nucleus pulpocyte was divided into three groups, named as SIRNA-silenced group, overexpression group, and inhibitor group, with 3 patients in each group. Cells of each group were collected at 72 h after intervention, expression of ASIC1, ERK5, BCL-xL/BCL-2-associated Death promoter (Bad), B-cell lymphoma-2 associated X (Bax) and B-cell lymphoblast-2 gene (Bcl-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR);intracellular calcium ion levels were detected by calcium ion kit, mitochondrial membrane potential was detected by JC-1 kit, and apoptosis was observed by AV-PI kit. RESULTS: In LDH patients with grade Ⅳ, nucleus pulposus tissue removed during operation revealed poor elasticity, white color and poor ductility, and immunohistochemical results showed increased ASIC1 expression. There was no significant difference in mRNA relative expression of ASIC1 between siRNA silencing group (0.31±0.03) and inhibitor group (0.39±0.05) (P>0.05). The mRNA relative expression level of ERK5 in siRNA silencing group(0.32±0.05) was significantly higher than that in inhibitor group (0.15±0.04)(P<0.05), which suggested ERK5 was the downstream molecule of ASIC1. The mRNA relative expression levels of apoptosis promoting factor Bad and Bax in siRNA silencing group and inhibitor group were lower than those in overexpression group(P<0.05), the relative expression level of anti-apoptosis factor Bcl-2 mRNA was significantly increased (P<0.05). The calcium content in overexpression group was higher than that in siRNA silencing and inhibitor groups (P<0.05), the normal proportion of mitochondrial membrane potential in overexpression group was lower than that in siRNA silencing and inhibitor group (P<0.05), and the apoptosis rate in overexpression group was higher than that in siRNA silencing and inhibitor group (P<0.05). CONCLUSION After the activation of ASIC1 channel protein, calcium ions could enter the cells and act as a second messenger molecule to regulate apoptosis of nucleus pulposus cells by ERK5 signaling pathway and mitochondrial disorder pathway.
Humans ; Acid Sensing Ion Channels/physiology* ; Male ; Female ; Apoptosis ; Middle Aged ; Adult ; Signal Transduction ; Mitogen-Activated Protein Kinase 7/physiology* ; Mitochondrial Diseases/genetics* ; Nucleus Pulposus/metabolism* ; Intervertebral Disc Degeneration/metabolism* ; Mitochondria/metabolism* ; Intervertebral Disc Displacement/genetics*

OBJECTIVE: To explore the potential causal relationship between intervertebral disc degeneration and certain autoimmune diseases. METHODS: Genome-wide association study (GWAS) data of 5 autoimmune diseases were obtained from large-scale GWAS databases. Data on internal vertebral disc degeneration (IVDD) were derived from the FinnGen consortium, which included 294, 770 controls and 41, 669 cases. A two-sample bidirectional Mendelian randomization (MR) analysis was performed to investigate the potential causal relationship between the 5 autoimmune diseases and IVDD. Multiple analytical methods were adopted, including MR methods such as inverse variance weighting(IVW), MR-Egger, weighted median, weighted mode, and simple mode. Cochran's Q test, leave-one-out analysis, and MR-Egger intercept test were conducted to assess heterogeneity, robustness, and pleiotropy. For the robustness of the results, MR-PRESSO was used to detect outliers, and MR analysis was re-conducted after removing the outliers. RESULTS: The MR analysis results showed that there might be a bidirectional causal relationship between ankylosing spondylitis(AS) and IVDD:AS on IVDD, OR=1.038, 95%CI (1.024, 1.053), P=0.000;and IVDD on AS, OR=2.117, 95%CI(1.065, 4.207), P=0.032. There might be a positive correlation between IVDD and rheumatoid arthritis(RA) as well as systemic lupus erythematosus(SLE):IVDD on RA, OR=1.184, 95%CI(1.071, 1.309), P=0.001;and IVDD on SLE, OR=1.678, 95%CI(1.187, 2.372), P=0.003. There was no significant correlation between ulcerative colitis(UC), autoimmune thyroiditis(ATD) and IVDD. After removing outliers by MR-PRESSO and re-conducting MR analysis, the results did not change qualitatively. Sensitivity analysis indicated that the results were robust to potential sources of bias. CONCLUSION AS and IVDD may be risk factors for each other, and IVDD may be a potential risk factor for RA and SLE. These findings provide a basis for guiding the prevention and combined diagnosis and treatment of IVDD, AS, RA, and SLE, while the specific underlying mechanisms still require further experimental basic research.
Humans ; Intervertebral Disc Degeneration/etiology* ; Mendelian Randomization Analysis ; Autoimmune Diseases/complications* ; Genome-Wide Association Study ; Spondylitis, Ankylosing/genetics* ; Arthritis, Rheumatoid/genetics*

This study investigated the mechanism by which kaempferol(KAE) affected intervertebral disc degeneration(IDD) through the p38 mitogen-activated protein kinase(p38 MAPK) signaling pathway. Rats were randomly divided into five groups: control group, model group, low-dose KAE group, medium-dose KAE group, and high-dose KAE group. An IDD model was established by needle puncture of the caudal intervertebral discs. Four weeks post-surgery, the rats were administered KAE via gavage for 8 consecutive weeks. Magnetic resonance imaging(MRI) was performed, and samples were collected. In vitro, an inflammation model of nucleus pulposus cells(NPCs) induced by tumor necrosis factor-alpha(TNF-α) was constructed. Anisomycin was used to activate the p38 MAPK signaling pathway. NPCs were divided into blank, model, KAE, agonist, and KAE + agonist groups. After 1 day of treatment, cell proliferation activity was assessed using the CCK-8. Protein expression levels were determined by Western blot, and mRNA expression was measured by real-time quantitative polymerase chain reaction. Cell apoptosis was detected by TUNEL staining, and immunofluorescence staining was used to detect type Ⅱ collagen and matrix metalloproteinase 3(MMP3). In vivo results indicated significant improvement in the degree of IDD in the treatment groups compared to the model group, with the medium-dose group showing more pronounced therapeutic effects than the low-and high-dose groups. In vitro results demonstrated that KAE treatment significantly enhanced NPC proliferation activity, down-regulated the expression levels of Bcl-2-associated X protein(Bax), interleukin-6(IL-6), interleukin-17A(IL-17A), MMP3, and a disintegrin and metalloproteinase with thrombospondin motifs 5, and inhibited the phosphorylation of p38 MAPK pathway-related proteins. Activation of the p38 MAPK signaling pathway by anisomycin reduced the therapeutic effects of KAE. The study concluded that KAE could improve the proliferation activity of degenerated NPCs, reduce inflammation levels, and slow the progression of IDD in rats, and the mechanism was likely related to the regulation of the p38 MAPK signaling pathway.
Animals ; p38 Mitogen-Activated Protein Kinases/genetics* ; Kaempferols/pharmacology* ; Intervertebral Disc Degeneration/genetics* ; Rats ; Rats, Sprague-Dawley ; Male ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Nucleus Pulposus/drug effects* ; Signal Transduction/drug effects* ; Humans ; MAP Kinase Signaling System/drug effects*

OBJECTIVE: To investigate the expression and clinical significance of receptor interacting protein serine-threonine kinases 1 (RIPK1) in the nucleus pulposus of patients with lumbar disc herniation (LDH). METHODS: Nucleus pulposus tissue specimens of 40 patients with LDH patients underwent surgical treatment from January 2016 to January 2018 as the case group, and nucleus pulposus tissue specimens of 30 patients with lumbar spine fracture underwent surgical treatment at the same time as the control group. The expression of RIPK1 mRNA and protein of receptor interaction were detected by polymerase chain reaction (PCR) and Western blot, respectively. The expression of RIPK1 protein in the nucleus pulposus were detected by immunohistochemical staining. The concentrations of RIPK1 and tumor necrosis factor-α (TNF-α) in nucleus pulposus were detected by ELISA method. The relationship between the concentrations of RIPK1, TNF-α in nucleus pulposus and the Pearce grade of LDH patients was analyzed by one-way ANOVA. The correlation between RIPK1 and TNF-α was analyzed by Pearson. RESULTS: RIPK1 was weakly positively expressed in nucleus pulposus of control group, and RIPK1 protein was positively or strongly positively expressed in case group. The expression of RIPK1 mRNA in nucleus pulposus of case group was higher than that of control group ( CONCLUSION The expression levels of RIPK1 mRNA and protein in the intervertebral disc tissues of LDH patients are higher than those of normal intervertebral disc tissues, and increased with the increase of Pearce grade, which may be an important factor involved in LDH inflammatory disease.
Humans ; Intervertebral Disc/metabolism* ; Intervertebral Disc Degeneration ; Intervertebral Disc Displacement/genetics* ; Nucleus Pulposus ; Receptor-Interacting Protein Serine-Threonine Kinases/genetics* ; Tumor Necrosis Factor-alpha/metabolism*

Intervertebral disc degeneration (IDD) is one of major causes for intervertebral disc degenerative diseases, and patients with IDD usually suffer from serious low back pain. The current treatments for patients with IDD only relieve the clinical symptom rather than restore biological balance of IDD, leading to inadequate and unsatisfactory results. MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNA molecules, which regulate the gene expression at the post-transcription levels. Research evidences support the involvement of miRNAs in many biological processes, such as lipid metabolism, apoptosis, differentiation and organ development. Accumulating evidences indicate that the expressions of miRNAs change significantly in degenerative tissues. In addition, dysregulated miRNAs contribute to multiple pathological process of IDD, including proliferation and apoptosis of nucleus pulposus and extracellular matrix components, inflammatory response and cartilage endplates degeneration. In this review article, we summarize the expression profiles and roles of miRNAs in IDD, which may provide a novel strategy of biological therapy for the disease.
Apoptosis ; Extracellular Matrix ; pathology ; Gene Expression ; Gene Expression Profiling ; Humans ; Intervertebral Disc Degeneration ; genetics ; pathology ; MicroRNAs ; genetics

Neck shoulder pain or lumbocrural pain caused by intervertebral disc degeneration (IDD) could seriously affect the qualities life of patients. Current treatments mainly focus on alleviating pain and the symptoms of nerve compression, which could not radically stop the process of intervertebral disc degeneration, but conversely lead to high recurrence rate. In recent years, scholars have turned to study the biological treatment for repair and rebuild the intervertebral disc by biological molecular therapy, gene therapy, cell therapy and tissue engineering to solve the problem of intervertebral disc degeneration, while most of the above methods are still in animal experiments or in vitro experiments and the clinical application is still a long way to go.
Animals ; Biological Therapy ; Genetic Therapy ; Humans ; Intervertebral Disc ; metabolism ; Intervertebral Disc Degeneration ; genetics ; metabolism ; therapy

Degenerative disc disease is a multifaceted progressive irreversible condition and an inevitable part of aging, which has been found to be a contributing factor for low back pain and might cause radiculopathy, myelopathy, spinal stenosis, degenerative spondylolisthesis, and herniations. Its etiology is complex and multifactorial. Although genetics influence more dominant, the occupational and mechanical influences still persist as a major risk factor. This review emphasizes up-to-date knowledge regarding etiology of disc degeneration with special consideration on occupational, lifestyle factors, and genetic polymorphisms.
Humans ; Intervertebral Disc Degeneration ; etiology ; genetics ; Life Style ; Occupational Diseases ; etiology

PURPOSE: To investigate the molecular responses of various genes and proteins related to disc degeneration upon treatment with cytokines that affect disc-cell proliferation and phenotype in living human intervertebral discs (IVDs). Responsiveness to these cytokines according to the degree of disc degeneration was also evaluated. MATERIALS AND METHODS: The disc specimens were classified into two groups: group 1 (6 patients) showed mild degeneration of IVDs and group 2 (6 patients) exhibited severe degeneration of IVDs. Gene expression was analyzed after treatment with four cytokines: recombinant human bone morphogenic protein (rhBMP-2), transforming growth factor-beta (TGF-beta), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). Molecular responses were assessed after exposure of cells from the IVD specimens to these cytokines via real-time polymerase chain reaction and immunofluorescence staining. RESULTS: mRNA gene expression was significantly greater for aggrecan, type I collagen, type II collagen, alkaline phosphatase, osteocalcin, and Sox9 in group 1 than mRNA gene expression in group 2, when the samples were not treated with cytokines. Analysis of mRNA levels for these molecules after morphogen treatment revealed significant increases in both groups, which were much higher in group 1 than in group 2. The average number of IVD cells that were immunofluorescence stained positive for alkaline phosphatase increased after treatment with rhBMP-2 and TGF-beta in group 1. CONCLUSION: The biologic responsiveness to treatment of rhBMP-2, TGF-beta, TNF-alpha, and IL-1beta in the degenerative living human IVD can be different according to the degree of degeneration of the IVD.
Adult ; Aggrecans/genetics/metabolism ; Alkaline Phosphatase/genetics/metabolism ; Biological Products/pharmacology/*therapeutic use ; Bone Morphogenetic Protein 2/pharmacology/therapeutic use ; Collagen Type I/genetics/metabolism ; Collagen Type II/genetics/metabolism ; Cytokines/*pharmacology/*therapeutic use ; Female ; Fluorescent Antibody Technique ; Gene Expression Regulation/drug effects ; Humans ; Interleukin-1/pharmacology/therapeutic use ; Intervertebral Disc/*drug effects/*pathology ; Intervertebral Disc Degeneration/*drug therapy/genetics/*pathology ; Male ; Middle Aged ; Osteocalcin/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/pharmacology/therapeutic use ; SOX9 Transcription Factor/genetics/metabolism ; Transforming Growth Factor beta/pharmacology/therapeutic use ; Tumor Necrosis Factor-alpha/pharmacology

Animals ; Extracellular Matrix Proteins ; antagonists & inhibitors ; chemistry ; genetics ; physiology ; Humans ; Intervertebral Disc Degeneration ; etiology ; therapy ; Repetitive Sequences, Amino Acid

Intervertebral disc (IVD) degeneration is implicated as a major cause of low back pain. The alternated phenotypes, reduced cell survival, decreased metabolic activity, loss of matrix production and dystrophic mineralization of nucleus pulposus (NP) cells may be key contributors to progressive IVD degeneration. IVD is the largest avascular structure in the body, characterized by low oxygen tension in vivo. Hypoxia-inducible factor (HIF) is a master transcription factor that is induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. This review summarizes relevant studies concerning the involvement of HIF in the regulation of biological behaviors of NP cells. We describe current data on the expression of HIF in NP cells and further discuss the various roles that HIF plays in the regulation of the phenotype, survival, metabolism, matrix production and dystrophic mineralization of NP cells. Here, we conclude that HIF may be a promising target for the prevention and treatment of IVD degeneration.
Animals ; Basic Helix-Loop-Helix Transcription Factors/genetics/*metabolism ; Cell Survival ; Extracellular Matrix/metabolism ; Humans ; Hypoxia-Inducible Factor 1/genetics/*metabolism ; Intervertebral Disc/*cytology/metabolism ; Intervertebral Disc Degeneration/*metabolism/*pathology