RESUMO
Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω)-propyl-L-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum_4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5 h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.
Assuntos
Degeneração do Disco Intervertebral/enzimologia , Disco Intervertebral/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/patologia , Imageamento por Ressonância Magnética , Masculino , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos Wistar , Região SacrococcígeaRESUMO
Lumbar intervertebral disc degeneration (IDD) is a common clinical pathology and has become a focus for research in recent years. Matrix metalloproteinases (MMPs) are enzymes responsible for the degradation of almost all extracellular matrix proteins (ECM). The over-expression of MMPs or tissue inhibitors of metalloproteinases (TIMPs) may disrupt the dynamic balance of the ECM. Therefore, in the current study, the expression levels of MMP-1 and TIMP-1 in lumbar IDD patients were evaluated in an attempt to elucidate their role in IDD pathogenesis and progression. In total, 60 IDD patients were recruited as the experimental group, along with 20 cases of lumbar vertebral injury without disc degeneration as the control group. Preoperative venous blood samples were collected, and intervertebral disc tissues were collected from the lesion during surgery. Serum and tissue levels of MMP-1 and TIMP-1 were quantified by enzyme-linked immunosorbent assay and immunohistochemical staining, respectively. Serum and tissue MMP-1 levels in IDD patients were significantly higher than those in the control group (P < 0.05). Additionally, sub-group analysis revealed that severe IDD patients had higher MMP-1 levels compared with mild or moderate IDD patients (P < 0.05). However, there were no significant differences in TIMP- 1 levels in either the serum or tissues of IDD patients compared to patients in the control group (P > 0.05). These results demonstrate that MMP-1 expression is increased in IDD, with higher expression observed in more severe cases, whereas TIMP-1 expression was similarly expressed in both normal and degenerated discs.
Assuntos
Degeneração do Disco Intervertebral/enzimologia , Metaloproteinase 1 da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Adulto , Idoso , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/patologia , Região Lombossacral/patologia , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade , Radiografia , Inibidor Tecidual de Metaloproteinase-1/genética , Adulto JovemRESUMO
STUDY DESIGN: This is a quantitative study of heparanase isoforms expression in degenerative and nondegenerative intervertebral discs (IVDs). OBJECTIVE: To quantify the expression of both heparanase isoforms (HPSE1 and HPSE2) in IVD tissues as classified by different degeneration grades using the Pfirrmann grading system, and to correlate the expression with the loss of extracellular matrix molecules observed in patients with the disease. SUMMARY OF BACKGROUND DATA: The loss of proteoglycans as observed in IVD degeneration may occur due to the enhanced expression of matrix degrading enzymes, such as heparanase. However, the heparanase function in IVD degeneration remains unclear. METHODS: This study comprised 53 surgical samples of degenerative discs obtained from patients with lumbar disc degeneration and 12 control samples collected from healthy individuals without any degenerative lumbar disc alterations who had accidental spine fractures.All patients underwent magnetic resonance imaging based on the Pfirrmann grading system for disc degeneration. Only the specimens that were classified according to magnetic resonance imaging evaluations as Pfirrmann grades I, II, III, and IV were analyzed.The tissue sections of the disc samples were subject to immunohistochemical staining with antibodies against the heparanase isoforms and to quantitative real time PCR to amplify heparanase isoforms cDNA. Protein and mRNA expressions were quantified. Analysis of variance and Student t test were used to compare the means of the study populations. RESULTS: The data demonstrated a gradual increase in both the heparanase isoform protein expression and disc degeneration progression. Besides, mRNA expression of both heparanase isoforms were significantly higher in degenerative than nondegenerative IVDs. CONCLUSION: The overexpression of HPSE1 and HPSE2 in the intervertebral degenerated discs suggests a role for these factors in mediating extracellular matrix remodeling in degenerative discs during disease development.
Assuntos
Glucuronidase/metabolismo , Degeneração do Disco Intervertebral/enzimologia , Disco Intervertebral/enzimologia , Vértebras Lombares , Adulto , Análise de Variância , Matriz Extracelular/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Glucuronidase/genética , Humanos , Imuno-Histoquímica , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/classificação , Degeneração do Disco Intervertebral/metabolismo , Isoenzimas/metabolismo , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.
Assuntos
Matriz Extracelular/metabolismo , Glucuronidase/metabolismo , Degeneração do Disco Intervertebral/enzimologia , Deslocamento do Disco Intervertebral/enzimologia , Adolescente , Adulto , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica , Isoenzimas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.