Inflammatory biomarkers in sera of patients with intervertebral disc degeneration

Luciano Miller Reis Rodrigues; Lilian Zerbinatti de Oliveira; Mariane de Barros Ribeiro da Silva; Camila de Melo Accardo; Adriana Braz Del Giglio; Maria Aparecida da Silva Pinhal

doi:10.31744/einstein_journal/2019AO4637

. 2019 Aug 27;17(4):eAO4637. doi: 10.31744/einstein_journal/2019AO4637

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Inflammatory biomarkers in sera of patients with intervertebral disc degeneration

Luciano Miller Reis Rodrigues ¹, Lilian Zerbinatti de Oliveira ², Mariane de Barros Ribeiro da Silva ², Camila de Melo Accardo ³, Adriana Braz Del Giglio ¹, Maria Aparecida da Silva Pinhal ^2,^✉

PMCID: PMC6711751 PMID: 31482941

ABSTRACT

Objective:

To evaluate intervertebral disc levels of inflammatory factor (interleukin 6) and proteinase activity (cathepsin B) in patients with a degenerative disease and serum levels of interleukin 6, serum cathepsin B activity and hyaluronic acid biomarkers.

Methods:

We conducted immunohistochemistry studies of intervertebral discs to analyze interleukin 6 and cathepsin B levels of patients with degenerative disease and spine fracture (Control Group) and to measure hyaluronic acid, interleukin 6 and cathepsin B activity from sera of intervertebral disc degeneration patients, fracture patients, and healthy individuals.

Results:

Interleukin 6 and cathepsin B seem to be related with physiopathology of intervertebral disc degeneration, since the levels of both were higher in discs of patients with intervertebral disc degeneration. Interleukin 6 and cathepsin B do not represent good biomarkers of degenerative intervertebral disc disease, since the level of such compounds is increased in the plasma of patients with fractures.

Conclusion:

Hyaluronic acid can be a biomarker for intervertebral disc degeneration, because hyaluronic acid levels were higher only in sera of patients with intervertebral disc degeneration.

Keywords: Biomarkers, Proteoglycans, Glycosaminoglycans, Intervertebral disc degeneration

INTRODUCTION

Intervertebral discs are composed of abundant cell matrix and low cell density. Two distinct regions in intervertebral discs can be characterize, i.e ., annulus fibrosus (AF) with high amounts of collagen and nucleus pulposus rich in proteoglycans.⁽¹⁾ The principal proteoglycans found in intervertebral discs are aggrecan noncovalently attached to hyaluronic acid (HA). These proteoglycans' function allows compressive loads to intervertebral discs.⁽²⁾ Intervertebral disc degeneration (IVD) is associated with the loss of extracellular matrix (ECM) molecules, leading to alterations in the biochemical and biomechanical properties of the tissue.⁽³^-⁵⁾

Enzymatic activity is believed to contribute to the degenerative process of IVD with increased collagen, proteoglycans and fibronectin fragmentation.⁽⁶⁾ Previous biochemical studies have shown the catabolism of these ECM molecules stimulated by several proteinases, such as metalloproteinases and collagenases.⁽⁷^,⁸⁾

Cathepsins are cysteine proteases, a family of matrix degrading enzymes. Although published literature on cathepsins associated with IVD is scarce, these proteases seem to play an important role in the catabolic process of disc degeneration. Studies have demonstrated that cathepsin B (CatB) concentration in the cartilage of osteoarthritis patients is significantly higher than the levels found in normal tissues.⁽⁹^,¹⁰⁾ Chu et al., suggested that CatB is released by synovial and inflammatory cells, and this releasing contributes to inflammation progression and cartilage destruction.⁽¹¹⁾

Inflammatory cytokines are key players in the pathogenesis of IVD because they promote ECM disruption. Interleukin 6 (IL-6) can up-regulate matrix metalloproteinases and disintegrin and metalloproteinase with thrombospondin motifs (ADAMT) expression.⁽¹²⁾ Increased circulating levels of IL-6 have been reported in rheumatoid arthritis and osteoarthritis patients, which turn this cytokine a possible biomarker for disc degeneration.⁽¹³^-¹⁵⁾

OBJECTIVE

To evaluate intervertebral disc levels of possible inflammatory factors (interleukin 6 and cathepsin B) in patients with a degenerative disease, and compared them with healthy subjects (control). In addition, this study aim to investigate whether serum levels of interleukin 6, serum cathepsin B activity, or hyaluronic acid biomarkers reflect on intervertebral disc degeneration tissue status among patients with the intervertebral degenerative disease, control patients and patients with fractures.

METHODS

Study population

This study was approved by the Ethics Committee on Research involving Human of the Faculdade de Medicina do ABC (approval number 262/2008). Patients who signed the Informed Consent statement were included. All spinal cord injuries were in the lumbar region. Disc degeneration were found in L4/L5 and L5/S1, and fractures were observed in L1/L2, L2/L3 and L3/L4, all in the lumbar region. We obtained serum samples and intervertebral disc specimens from 83 patients who underwent primary lumbar discectomy with acute low back pain associated with radicular pain for less than 2 weeks. Patients' blood samples were collected during the follow-up of those who underwent surgery. We also obtained serum samples from 33 healthy subjects, without any spinal injury or inflammatory conditions, and who were used as controls. In addition, we also obtained intervertebral disc specimens from six patients who underwent surgery because of accidental fracture of the spine, and required disc removal. These patients had no spinal injury or previous inflammatory conditions and serum samples were taken from them. This group of patients was required to provide non-degenerated disc tissue for the immunohistochemistry analysis. The individuals enrolled in the study did not present any co-morbidity (hypertension, diabetes mellitus, chronic kidney disease or cancer), since such co-morbidities might increase the incidence of disc degeneration.

Study design

It was a prospective study, and the subjects were selected from January 2015 to December 2017 at Hospital Mário Covas , in Santo André (SP), Brazil, and from the Orthopedic Surgery Department of the Faculdade de Medicina do ABC .

Study limitations

The increase of the number of samples may enhance the statistical differences. The controlling of immunohistochemical reactions could not be performed with intervertebral disc tissues of healthy individuals because this study is not recommended from the ethical point of view. Therefore, as control tissue sample, we used tissues from patients who were affected by spinal fracture. However, a limitation is that these patients can present an acute inflammation process, and their chronic inflammation process does not present the common characteristics of those patients who suffer from IVD disease.

Radiographic and clinical evaluation

All individuals were classified according to Pfirrmann's grading system for disc degeneration. A magnetic resonance imaging analysis was performed in all patients with a degenerative disc as well all spinal fracture patients (control). Only individuals classified as Pfirrmann grade III or IV, with one or two levels of degeneration, and nerve root compression at least in one level were included in the disc degeneration group, while Control Group was composed by individuals classified as Pfirrmann's grade I. Radiographies of the knee, shoulder, and hip of all individuals were also performed to assess the presence of degenerative changes in these joints. Exclusion criteria were defined as the presence of one or more of the following characteristics, systemic or inflammatory diseases, previous orthopedic surgery, ligament or muscular lesion, hypertension, hypercholesterolemia, diabetes, obesity (body mass index − BMI >30) or use of analgesic drugs during the preceding week.

Serum hyaluronic acid levels

Sera from patients and controls were assayed for HA by a non-competitive and non-isotopic fluoroassay. This method is based on the affinity of specific proteins extracted from bovine cartilage (globular HA-binding region of the aggrecan and link protein) for HA, that detects HA between 0.2 and 500 µ g/L. This method has highly specificity and sensitivity (<0.2 µ g/L), and gives intra- and interassay coefficients of variation of 2 and 5%, and 3 and 14% in sera from normal subjects.⁽¹⁶⁾

Serum interleukin 6 concentration

Serum levels of IL-6 were determined by the ELISA test using the kit Human IL-6 ELISA Ready-Set-Go!^® (eBioscience™ Inc, CA, USA) following the manufacturer's instructions.

Cathepsin B activity

CatB activity was measured spectrofluorimetrically using the fluorogenic substrate Z-FR-MCA (Sigma, MO, USA) at 37°C. The fluorescence intensity was monitored in a microplate reader, with excitation and emission wavelengths set at 365 and 420nm. The assay was performed by preincubating the serum samples with the enzyme activator dithiothreitol (DTT) 2mM for 20 minutes and, then, by adding the irreversible inhibitor E-64, azocasein (Sigma, MO, USA), as previously described in study conducted by Almeida et al.⁽⁹⁾

Immunohistochemical staining

Representative lumbar intervertebral degenerative disc regions were chosen based on the results of a hematoxylineosin staining study of the corresponding tissue sections. Three-micrometer-thick sections of formalin-fixed paraffin-embedded tissues were deparaffinized and rehydrated. The primary antibodies for IL-6 (Santa Cruz Biotechnology, CA, USA) and CatB (Calbiochem, USA) were diluted 1:350 and incubated overnight. A secondary biotinylated antibody (LSAB^®, DakoCytomation^®, Glostrup, Denmark) was applied for 30 minutes, and the slides were subsequently incubated with a peroxidase-labeled streptavidin complex (LSAB^®, DakoCytomation^®, Glostrup, Denmark) for additional 30 minutes. Sections were developed using 3,3'-diaminobenzidine (DAB) as the chromogen for 1 minute and, subsequently, counterstained with hematoxylin.

Digital quantification

The slides were analyzed using a TS100 Nikon Eclipse^® light microscope to identify areas that best represented the immunostaining of IL-6 and CatB (hot spots). In each case, quantification of the degree of immunostaining was performed using a digital computer-assisted image analysis method. Photomicrographs (640×480 pixels) were obtained from non-coincident consecutive fields for each case at a magnification of 400x using a 4300 Nikon Coolpix^® digital camera adjusted to the same parameters. The obtained images were analyzed using the image processing and analysis system ImageLab^® (Softium Informática^®, São Paulo, Brazil) adjusted to the micrometer scale. The images were performed at the same magnification fold (400x). The technique described by Matos et al.,⁽¹⁷⁾ was used to obtain the index of positive percentage of labeled cells (IP), the index of immunostaining intensity expression (ItE) and the index of expression (IE).

Statistical analysis

The measures of the central tendency and value dispersion of the samples and the statistical tests for comparison between them were based on the data distribution type. The distributions were determined to be parametric by Kolmogorov-Smirnov testing. Each continuous variable value of the study was expressed by means and standard deviations. Absolute and relative frequencies were used for categorization. The analisys of variance (ANOVA) and Student's t test were used to compare the averages of the sample populations. The χ² test was conducted to compared percentages. The significance level adopted was based on the chance of the occurrence of a type I error lower than 5% (p<0.05). For all analyses we used the Statistical Package for Social Science (SPSS), version 19.0 (SPSS Inc.; Illinois, USA).

RESULTS

Table 1 shows the comparison of general characteristics (age, gender, smoking habits or manual labor) of patients with IVD degeneration, spine fracture and non-affected individuals (Control Group). No significant difference was seen among studied groups.

Table 1. General characteristics of the study population.

	Controls	Disc degeneration	Fracture	p value
Number of patients	33	83	6
Age	36.48±7.83	36.41±9.80	33.83±5.12	0.794
Gender (female/male)	18/15	32/51	2/4	0.260
Smokers	5 (15.2)	20 (24.1)	1 (16.7)	0.511
Manual laborers	6 (18.2)	31 (37.3)	2 (33.3)	0.136

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Results expressed as n, mean±standard deviation or n (%).

Immunohistochemistry analysis of intervertebral disc tissue

The protein expression of IL-6 in degenerative disc tissues and in discs of patients who had suffered accidental spine fracture was obtained by digital quantification of immunohistochemistry reactions. Immunoreactivity for IL-6 was more intense in the AF and nucleus pulposus (NP) of degenerative discs than in the spine fracture ( Figure 1A ). Annulus fibrosus from degenerative discs presented more intense immunoreactivity for IL-6 in the ECM compared with the labeling in fibroblasts. Strong immunoreactivity for IL-6 was also present in the chondrocytes of NP from degenerative discs, whilst no labeling can be observed in the ECM ( Figure 1A ). The IE of IL-6 was significantly higher in the degenerative discs compared with the fracture IVD ( Figure 1B ). The expression of CatB was also investigated in the degenerative intervertebral discs and spine fracture using immunohistochemistry. The test demonstrated a low-intensity CatB labeling in the AF and NP of fracture discs ( Figure 2A ), whereas IVD with degeneration presented intense immunoreactivity ( Figure 2A ). Intense CatB labeling both in chondrocytes and ECM was observed in the NP from degenerative discs ( Figure 2A ). Therefore, protein intensity of CatB was significantly higher in the degenerative IVD compared to the fracture discs ( Figure 2B ).

* p<0.05. AF: annulus fibrosus; NP: nucleus pulposus; IE: index expression.

Circulating levels of interleukin-6, cathepsin B and hyaluronic acid

Circulating levels of IL-6 in disc degeneration and fracture patients were statistically higher compared to the Control Group (p<0.001), as demonstrated in figure 3A . There was no significant difference comparing IL-6 levels between IVD and fracture patients ( Figure 3A ). Measurement of CatB activity in sera of patients with IVD, intervertebral disc fracture and non-affected individuals (control), demonstrated that there was no significant difference among groups ( Figure 3B ). However, serum levels of HA were statistically higher in patients with IVD (p<0.001) compared with Control Group ( Figure 4 ), whilst, no significant difference was observed in the circulating HA levels between fracture patients and Control Group ( Figure 4 ).

DISCUSSION

CatB is a protease which is believed to have an important role in the degradation of cartilage ECM proteins, such as aggrecan and collagen.⁽¹⁰^,¹²^,¹⁸⁾ Enhanced expression of CatB has been reported in chondrocytes of osteoarthritis patients.⁽¹⁹⁾ In addition, intervertebral degenerated cells has been believed to be responsible of secretion pro-inflammatory cytokines, including IL-6.⁽²⁰⁾ The persistent proinflammatory activity of IL-6 favors mononuclear cell accumulation at the site of injury, angio-proliferation and antiapoptotic functions of T cells.⁽²¹^-²³⁾

Data suggest that both IL-6 and CatB are close related to physiopathology of IVD. In our study, CatB protein expression was significantly increased in degenerative disc tissues compared with fracture discs. Intense immunoreactivity for CatB was found in the ECM of degenerative disc specimens, suggesting that the acidic microenvironment is likely to cause inflammatory process favored by CatB secretion. Also, IL-6 protein levels were higher compared with control intervertebral discs.

No significant difference was found concerning CatB activity and IL-6 among groups when sera were analyzed. Circulating IL-6 and CatB concentrations in patients with spine fracture were also significantly higher compared with the Control Group, showing that increase of IL-6 and CatB in serum is not specific to IVD. The increased serum concentration of IL-6 and CatB in fracture patients may be due to the acute inflammation caused by the fracture, especially if we consider that peripheral blood was collected just after trauma.

Quantitative analyses of serum HA are useful in the diagnosis of several inflammatory diseases.⁽¹⁶^,²⁴^,²⁵⁾ Extracellular matrix degradation and turnover result in the release of HA and HA fragments into the systemic circulation.⁽²⁶⁾ Different from patients who underwent anti-inflammatory treatment, low levels of HA were found in the circulation.⁽²³^,²⁶⁾

We may suggest that increased levels of HA might indicate a degeneration process. However, other studies are necessary to confirm this conclusion, considering that our study represent preliminary results.

Degradation of ECM components on the IVD process is modulated by several proteolytic enzymes of which matrix metalloproteinases (MMPs) and aggrecanases play a critical role in the activation of IL-6, IL-8, cyclo-oxygenase-2 (COX2), MMP1/13 and Toll Like Receptors (TLR2).⁽²⁷^,²⁸⁾

Several strategies aiming the regeneration of the intervertebral disc have been used lately. These strategies, with promising results, involve cellular therapies, as well as biomaterials such as the HA scaffold.⁽²⁸^,²⁹⁾

Our results may contribute to further studies and may promote the use of these target molecules for the IVD process.

CONCLUSION

Serum hyaluronic acid levels are significantly higher in patients with intervertebral disc degeneration than in non-affected patients, and those who had spine fracture. Data confirm that interleukin 6 and cathepsin B are possible related to the physiopathology of intervertebral disc degeneration, however they do not represent a biomarker in serum.

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Einstein (Sao Paulo). 2019 Aug 27;17(4):eAO4637. [Article in Portuguese]

Biomarcadores inflamatórios no plasma de pacientes com degeneração do disco intervertebral

RESUMO

Objetivo:

Avaliar os níveis de fatores inflamatórios nos discos intervertebrais (interleucina 6) e proteinase (catepsina B) em pacientes com doença degenerativa de disco intervertebral, além de verificar os níveis séricos de interleucina 6, ácido hialurônico e atividade sérica da catepsina B.

Métodos:

Foi realizado exame imuno-histoquímica dos discos intervertebrais de pacientes com doença degenerativa e fratura da coluna (Grupo Controle) e análise do plasma de pacientes com doença degenerativa de disco intervertebral. Como controle, foram utilizados plasma de pacientes com fraturas, além de indivíduos saudáveis.

Resultados:

Interleucina 6 e catepsina B sugerem relação com a fisiopatologia da doença degenerativa de disco intervertebral, uma vez que os níveis de ambos foram maiores nos discos de pacientes com doença degenerativa de disco intervertebral. Interleucina 6 e catepsina B não representam bons biomarcadores da doença degenerativa do disco intervertebral, já que também encontram níveis aumentados em plasma de pacientes com fratura.

Conclusão:

O ácido hialurônico é um possível biomarcador de doença degenerativa de disco intervertebral, porque os níveis de ácido hialurônico foram maiores apenas em plasma de pacientes com doença degenerativa de disco intervertebral.

Descritores: Biomarcadores, Proteoglicanas, Glicosaminoglicanos, Degeneração do disco intervertebral

INTRODUÇÃO

Os discos intervertebrais são compostos por matriz celular abundante e baixa densidade celular. Há possibilidade de caracterização de duas regiões distintas nos discos intervertebrais: anel fibroso, com altas quantidades de colágeno, e núcleo pulposo (NP), rico em proteoglicanos.⁽¹⁾Os principais proteoglicanos encontrados nos discos intervertebrais são agrecan aderidos de forma não covalente ao ácido hialurônico (AH). Sua função é permitir cargas compressivas nos discos intervertebrais.⁽²⁾

A degeneração do disco intervertebral (DDI) é associada à perda de moléculas da matriz extracelular (MEC), e a alterações nas propriedades bioquímicas e biomecânicas do tecido.⁽³^-⁵⁾

É aceito que a atividade enzimática contribui para o processo degenerativo da DIV com aumento do colágeno, proteoglicanos e fragmentação da fibronectina.⁽⁶⁾Estudos bioquímicos prévios evidenciaram o catabolismo dessas moléculas de MEC por várias proteinases, como metaloproteinases e colagenases.⁽⁷^,⁸⁾

As catepsinas são proteases de cisteína, uma família de enzimas que degradam a matriz e, embora a literatura sobre elas na DDI seja escassa, essas proteases possivelmente desempem papel importante no processo catabólico da degeneração discal. Estudos demonstraram que a concentração de catepsina B (CatB) na cartilagem de pacientes com osteoartrite é significativamente maior do que no tecido normal.⁽⁹^,¹⁰⁾Chu et al., sugeriram que a CatB é liberada pelas células sinoviais e inflamatórias, contribuindo para a progressão da inflamação e a destruição da cartilagem.⁽¹¹⁾

As citocinas inflamatórias são elementos-chave na patogênese da DDI, promovendo a ruptura do MEC. A interleucina 6 (IL-6) pode regular positivamente as metaloproteinases da matriz e a expressão de A disintegrin and metalloproteinase with thrombospondin motifs (ADAMT).⁽¹²⁾Os níveis circulantes aumentados de IL-6 foram encontrados em pacientes com artrite reumatoide e osteoartrite, tornando essa citocina um possível biomarcador para degeneração discal.⁽¹³^-¹⁵⁾

OBJETIVO

Avaliar os níveis de disco intervertebral de possíveis fatores inflamatórios (interleucina 6 e catepsina B) em pacientes com doença degenerativa comparados a indivíduos saudáveis (controle) e investigar se os níveis séricos de interleucina 6, a atividade sérica de catepsina B ou os biomarcadores de ácido hialurônico refletem no status do tecido com degeneração do disco intervertebral entre pacientes com degeneração do disco intervertebral, controles e com fratura.

METÓDOS

População do estudo

O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa com seres humanos da Faculdade de Medicina do ABC (parecer 262/2008 do Comitê de Ética em Pesquisa). Os pacientes foram incluídos após assinatura do Termo de Consentimento. Todas as lesões na medula espinhal estavam localizadas na região lombar. O nível de degeneração discal foi em L4/L5 e L5/S1, e o nível de fratura foi L1/L2, L2/L3 e L3/L4, sendo todos na coluna lombar. Foram obtidas amostras de plasma e dos discos intervertebrais de 83 pacientes submetidos à discectomia lombar primária, com dor lombar aguda associada à dor radicular por menos de 2 semanas. Amostras de sangue dos pacientes foram coletadas durante o acompanhamento dos pacientes que necessitaram de cirurgia. Além disso, também obtivemos amostras de plasma de 33 indivíduos saudáveis, sem qualquer lesão medular ou condição inflamatória, que compuseram o Grupo Controle. Além disso, espécimes do disco intervertebral também foram obtidos de seis pacientes submetidos à cirurgia por fratura acidental da coluna vertebral, o que exigiu a remoção do disco. Tais pacientes não apresentavam lesão medular e nem condições inflamatórias prévias e forneceram amostras de plasma. Este grupo de pacientes foi obrigado a fornecer tecido discal não degenerado para a análise imuno-histoquímica. Os sujeitos inscritos no estudo não apresentaram qualquer comorbilidade (hipertensão, diabetes mellitus , doença renal crônica ou cancro), uma vez que é sabido que tais comorbidades poderiam aumentar a incidência de degeneração discal.

Desenho do estudo

Estudo prospectivo em que os sujeitos foram selecionados no período de janeiro de 2015 a dezembro de 2017 no Hospital Mário Covas, em Santo André (SP), e no Departamento de Cirurgia Ortopédica da Faculdade de Medicina do ABC.

Limitação do estudo

O aumento do número de amostras pode beneficiar as diferenças estatísticas. O controle das reações imuno-histoquímicas não pode ser realizado com os tecidos do disco intervertebral de indivíduos saudáveis, pois não é recomendado do ponto de vista ético. Assim, como amostra de tecido controle, foram utilizados pacientes afetados por fratura da coluna vertebral. No entanto, esses pacientes são conhecidos por apresentarem processo agudo de inflamação, mas não por terem um processo de inflamação crônica, que é uma característica comum em pacientes que sofrem de doença DDI.

Avaliação radiográfica e clínica

Todos os indivíduos foram classificados de acordo com o sistema de classificação de Pfirrmann para degeneração discal. A análise por ressonância magnética foi realizada em todos os pacientes com um disco degenerativo, bem como com todos aqueles apresentando fratura da coluna vertebral (controle). Apenas indivíduos classificados como Pfirrmann grau III ou IV, com 1 ou 2 níveis de degeneração, e compressão de raiz nervosa em pelo menos um nível, foram incluídos no grupo de degeneração discal, enquanto o Grupo Controle foi composto por indivíduos classificados como Pfirrmann grau I. As radiografias de joelho, ombro e quadril de todos os indivíduos também foram conduzidas para avaliar a presença de alterações degenerativas nessas articulações. Os critérios de exclusão foram definidos também com presença de uma ou mais das seguintes características: doenças sistêmicas ou inflamatórias, cirurgia ortopédica prévia, lesão ligamentar ou muscular, hipertensão, hipercolesterolemia, diabetes, obesidade (índice de massa corporal − IMC >30) ou uso de analgésicos durante a semana anterior ao procedimento.

Níveis da dosagem sérica do ácido hialurônico

O plasma dos pacientes e controles foram testados quanto ao AH por ensaio fluoro não competitivo e não isotópico. Este método é baseado na afinidade de ligação de uma proteína globular específica, proteína de ligação, responsável pela interação entre a molécula de AH com o esqueleto proteico do agrecam, que detecta quantidades de AH entre 0,2 a 500 μ g/L. Esse método é muito específico e sensível (<0,2 µ g/L), dando coeficientes de variação intra e interensaio de 2 a 5% e 3 a 14%, respectivamente, em plasma de indivíduos normais.⁽¹⁶⁾

Concentração séricas de interleucina 6

Os níveis de IL-6 foram determinados por ELISA utilizando kit humano IL-6 ELISA Ready-Set-Go!^®(eBioscience™ Inc, CA, EUA), conforme instruções do fabricante.

Atividade catepsina B

A CatB foi quantificada por fluorescência utilizando o substrato Z-FR-MCA fluorogênico (Sigma, MO, EUA) em 37°C. A intensidade fluorescente foi monitorada por leitor ( microplate reader ) com excitação e emissão de ondas com frequência estabelecida entre 365 e 420nm, respectivamente. O ensaio foi pré-incubado e as amostras séricas com enzima ativadora de ditiotreitol (DTT) 2mM por 20 minutos. Após, foi adicionado inibidor irreversível E-64, azocaseína (Sigma, MO, EUA), como previamente descrito por Almeida et al.⁽⁹⁾

Coloração imuno-histoquímica

Região lombar de discos intervertebrais degenerados foram escolhidas com base nos resultados da coloração por hematoxilina-eosina das secções dos tecidos. Secções de 3 µ m de espessura de tecidos embebidos em parafina fixados em formalina foram desparafinizadas e reidratadas. Os anticorpos primários para IL-6 (Santa Cruz Biotechnology, CA, EUA) e CatB (Calbiochem, EUA) foram diluídos a 1:350 e incubados overnight . Um anticorpo biotinilado secundário (LSAB^®, DakoCytomation^®, Glostrup, Dinamarca) foi aplicado durante 30 minutos, e as lâminas foram subsequentemente incubadas com complexo de estreptavidina marcado com peroxidase (LSAB^®, DakoCytomation^®, Glostrup, Dinamarca) por 30 minutos adicionais. As secções foram desenvolvidas usando 3,3'-diaminobenzidina (DAB) como o cromógeno durante 1 minuto e posteriormente contracoradas com hematoxilina.

Quantificação digital

As placas foram analisadas utilizando um microscópio Nikon Eclipse^®TS100 para identificar as áreas que melhor representassem coloração imuno de IL-6 e CatB (locais de calor). Em cada caso, a quantificação do grau de imunocoloração foi realizada usando método digital de análise de imagem assistida por computador. Fotomicrografias (640×480 pixels ) foram obtidas de campos consecutivos não coincidentes para cada caso, com ampliação de 400 vezes por câmera digital Nikon Coolpix^®4300 ajustada aos mesmos parâmetros. As imagens resultantes foram analisadas no sistema de processamento e análise de imagens ImageLab^®(Softium Informática^®, São Paulo, Brasil) ajustado à escala do micrômetro. As imagens foram confeccionadas na mesma escala de ampliação (400 vezes). O índice de percentagem (IP) positiva de células marcadas, o índice de expressão da intensidade de imunocoloração (ItC) e o índice de expressão (IE) foram obtidos, conforme descrito por Matos et al.⁽¹⁷⁾

Análise estatística

As medidas de tendência central e dispersão de valor das amostras e os testes estatísticos para comparação entre elas foram baseadas no tipo de distribuição de dados. As distribuições foram determinadas como paramétricas pelo teste de Kolmogorov-Smirnov. Cada valor de variável contínua obtido no estudo foi expresso como média e desvio padrão. Utilizaram-se frequências absolutas e relativas para categorização. Para comparar as médias das populações amostrais, utilizaram-se o teste de análise de variância ANOVA e o teste t de Student . As comparações entre percentagens foram conduzidas pelo teste do χ². Adotou-se nível de significância baseado na chance de ocorrência de erro tipo I menor que 5% (p<0,05). Para todas as análises, adotou-se o Statistical Package for Social Science (SPSS), versão 19.0 (SPSS Inc.; Illinois, EUA).

RESULTADOS

A tabela 1 mostra a comparação das características gerais dos pacientes com degeneração de DDI, fratura de coluna e indivíduos não afetados (Grupo Controle), demonstrando que não houve diferença significativa entre os grupos incluídos no estudo em relação a idade, gênero, tabagismo ou trabalho manual.

Tabela 1. Características gerais da população do estudo.

	Controles	Degeneração do disco	Fratura	Valor de p
Pacientes	33	83	6
Idade	36,48±7,83	36,41±9,80	33,83±5,12	0,794
Gênero (feminino/masculino)	18/15	32/51	2/4	0,260
Tabagistas	5 (15,2)	20 (24,1)	1 (16,7)	0,511
Trabalhadores que realizavam atividade manual	6 (18,2)	31 (37,3)	2 (33,3)	0,136

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Resultados expressos por n, média±desvio padrão ou n (%).

Análise inmuno-histoquímica de tecido do disco invertebral

A expressão proteica da IL-6 em tecidos discais degenerativos e em discos de pacientes com fratura acidental da coluna foi obtida por quantificação digital de reações imuno-histoquímicas. A imunorreatividade para a IL-6 foi mais intensa no ânulo fibroso (AF) e no NP dos discos degenerativos do que na fratura da coluna ( Figura 1A ). Pode-se demonstrar que o AF dos discos degenerativos apresentou imunorreatividade mais intensa para a IL-6 na MEC em comparação com a marcação nos fibroblastos. Imunorreatividade forte para IL-6 também estava presente nos condrócitos de NP de discos degenerativos, enquanto nenhuma marcação pôde ser observada nas MEC ( Figura 1A ). O IE da IL-6 foi significativamente maior nos discos degenerativos em comparação com a DDI da fratura ( Figura 1B ). A expressão de CatB também foi investigada nos discos intervertebrais degenerativos e na fratura da coluna usando imuno-histoquímica. A expressão demonstrou marcação CatB de baixa intensidade no AF e NP dos discos de fratura ( Figura 2A ), enquanto que discos intervertebrais com degeneração apresentaram imunoreatividade intensa ( Figura 2A ). A marcação intensa de CatB tanto nos condrócitos quanto na MEC foi observada na NP de discos degenerativos ( Figura 2A ). Assim, a intensidade proteica da CatB foi significativamente maior na DDI degenerativa em comparação com os discos de fratura ( Figura 2B ).

Figura 1 — * p<0,05. AF: ânulo fibroso; NP: núcleo pulposo; IE: índice de expressão.

Figura 2 — * p<0,05. AF: ânulo fibroso; NP: núcleo pulposo; IE: índice de expressão.

Níveis de circulação de interleucina-6, catepsina B e ácido hialurônico

Os níveis circulantes de IL-6 nos pacientes com degeneração discal e fratura foram estatisticamente maiores em comparação ao Grupo Controle (p<0,001), conforme demonstrado na figura 3A . Não houve diferença significativa comparando os níveis de IL-6 entre DDI e pacientes com fratura ( Figura 3A ). A mensuração da atividade de CatB em plasma de pacientes com DDI, fratura do disco intervertebral e indivíduos não afetados (Grupo Controle) demonstrou inexistência de diferença significativa entre os grupos ( Figura 3B ). No entanto, os níveis séricos de AH foram estatisticamente maiores nos pacientes com DDI (p<0,001) comparados ao controle ( Figura 4 ), e não houve diferença significativa nos níveis circulantes de AH entre os pacientes com fratura e Grupo Controle ( Figura 4 ).

Figura 4 — * p<0,05. AH: ácido hialurônico.

DISCUSSÃO

A CatB é uma protease que acredita-se ter papel importante na degradação de proteínas de MEC de cartilagem, como agrecano e colágeno.⁽¹⁰^,¹²^,¹⁸⁾A expressão aumentada de CatB tem sido relatada em condrócitos de pacientes com osteoartrite.⁽¹⁹⁾Além disso, células degeneradas intervertebrais secretam citocinas pró-inflamatórias, incluindo IL-6.⁽²⁰⁾A atividade pró-inflamatória persistente da IL-6 favorece o acúmulo de células mononucleares no local da lesão, a angioproliferação e funções antiapoptóticas de células T.⁽²¹^-²³⁾

Os dados sugerem que tanto a IL-6 quanto a CatB estão intimamente relacionadas à fisiopatologia da DDI. Em nosso estudo, a expressão da proteína CatB foi significativamente aumentada nos tecidos discais degenerativos em comparação com os discos de fratura. A imunorreatividade intensa para CatB foi encontrada nas MEC de espécimes de disco degenerativo, sugerindo possível microambiente acídico causado pelo processo inflamatório que favoreceu a secreção de CatB. Além disso, os níveis de proteína IL-6 foram maiores quando comparados aos discos intervertebrais do Grupo Controle.

Não foi encontrada diferença significativa da atividade de CatB e da IL-6 nos grupos quando os plasmas foram analisados. As concentrações circulantes de IL-6 e CatB em pacientes com fratura da coluna vertebral também foram significativamente maiores quando comparadas ao Grupo Controle, mostrando que o aumento de IL-6 e CatB no soro não é específico da DDI. O aumento da concentração sérica de IL-6 e CatB em pacientes com fratura pode ser devido à inflamação aguda causada pela fratura, especialmente se considerarmos que o sangue periférico foi coletado logo após o trauma.

As análises quantitativas da AH sérica são úteis no diagnóstico de várias doenças inflamatórias.⁽¹⁶^,²⁴^,²⁵⁾A degradação e o turnover da MEC resultam na liberação de fragmentos de AH e AH na circulação sistêmica.⁽²⁶⁾Diferentemente, em pacientes após tratamento inflamatório, níveis baixos de AH são encontrados na circulação.⁽²³^,²⁶⁾

Podemos sugerir que níveis aumentados de AH sugerem processo de degeneração. No entanto, outros estudos são necessários para confirmar essa indicação, uma vez que o presente estudo representa resultado preliminar.

Sabe-se que a degradação dos componentes da MEC no processo de DDI é modulada por várias enzimas proteolíticas, das quais as metaloproteinases da matriz (MMPs) e as agrecanases desempenham papel crítico na ativação de IL-6, IL-8, ciclo-oxigenase-2 (COX2), MMP1/13 e receptores do tipo Toll (TLR2).⁽²⁷^,²⁸⁾

É importante mencionar que, ultimamente, várias estratégias que visam à regeneração do disco intervertebral têm sido utilizadas. Tais estratégias, com resultados promissores, e envolvem terapias celulares e biomateriais, como o arcabouço do AH.⁽²⁸^,²⁹⁾

Os resultados podem contribuir para estudos adicionais, que permitam o uso dessas moléculas-alvo para o processo de DDI.

CONCLUSÃO

Os níveis séricos de ácido hialurônico em pacientes com degeneração do disco intervertebral são significativamente maiores do que os de pacientes não afetados e com fratura da coluna vertebral. A interlecuina 6 e a catepsina B estão possivelmente relacionadas à fisiopatologia da degeneração do disco intervertebral. No entanto, não representam um biomarcador no plasma.

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PERMALINK

Inflammatory biomarkers in sera of patients with intervertebral disc degeneration

Luciano Miller Reis Rodrigues

Lilian Zerbinatti de Oliveira

Mariane de Barros Ribeiro da Silva

Camila de Melo Accardo

Adriana Braz Del Giglio

Maria Aparecida da Silva Pinhal

ABSTRACT

Objective:

Methods:

Results:

Conclusion:

INTRODUCTION

OBJECTIVE

METHODS

Study population

Study design

Study limitations

Radiographic and clinical evaluation

Serum hyaluronic acid levels

Serum interleukin 6 concentration

Cathepsin B activity

Immunohistochemical staining

Digital quantification

Statistical analysis

RESULTS

Table 1. General characteristics of the study population.

Immunohistochemistry analysis of intervertebral disc tissue

Circulating levels of interleukin-6, cathepsin B and hyaluronic acid

Figure 3. Serum levels of interleukin-6 (IL-6) and cathepsin B activity. Control refers to healthy patient. Disc degeneration refers to patient with intervertebral disc degeneration. Fracture refers to patient with spine fracture.

Figure 4. Serum levels of hyaluronic acid. Control refers to healthy patient. Disc degeneration refers to patient with intervertebral disc degeneration. Fracture refers to patient with spine fracture.

DISCUSSION

CONCLUSION

REFERENCES

Biomarcadores inflamatórios no plasma de pacientes com degeneração do disco intervertebral

Luciano Miller Reis Rodrigues

Lilian Zerbinatti de Oliveira

Mariane de Barros Ribeiro da Silva

Camila de Melo Accardo

Adriana Braz Del Giglio

Maria Aparecida da Silva Pinhal

RESUMO

Objetivo:

Métodos:

Resultados:

Conclusão:

INTRODUÇÃO

OBJETIVO

METÓDOS

População do estudo

Desenho do estudo

Limitação do estudo

Avaliação radiográfica e clínica

Níveis da dosagem sérica do ácido hialurônico

Concentração séricas de interleucina 6

Atividade catepsina B

Coloração imuno-histoquímica

Quantificação digital

Análise estatística

RESULTADOS

Tabela 1. Características gerais da população do estudo.

Análise inmuno-histoquímica de tecido do disco invertebral

Níveis de circulação de interleucina-6, catepsina B e ácido hialurônico

Figura 3. Níveis séricos de interleucina 6 (IL-6) e atividade da catepsina B. Controle se refere a paciente saudável. Degeneração do disco se refere a paciente com degeneração do disco intervertebral. Fratura abrange paciente com fratura da coluna vertebral.

Figura 4. Níveis séricos de ácido hialurônico. Controle se refere a paciente saudável. Degeneração do disco se refere a paciente com degeneração do disco intervertebral. Fratura abrange paciente com fratura da coluna vertebral.

DISCUSSÃO

CONCLUSÃO

ACTIONS

PERMALINK

RESOURCES

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