Aims. This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results. Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24, collagen type II, and aggrecan. After treatment with hydrogen peroxide, the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3), cleaved CASP-1, N-terminal fragment of gasdermin D (GSDMD-N), interleukin (IL)-18, and IL-1β in NPCs were upregulated, and the number of propidium iodide (PI)-positive cells was also increased, which was able to be alleviated by pretreatment with melatonin. The protective effect of melatonin on pyroptosis was blunted by both the melatonin receptor antagonist luzindole and the nuclear factor erythroid 2–related factor 2 (Nrf2) inhibitor ML385. In addition, the expression of the
Aims. This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD). Methods. The gene expression profile of GSE23130 was downloaded from the Gene Expression Omnibus (GEO) database. Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases, and we used Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the functions and pathways of EP-DEGs. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and identify hub EP-DEGs. NetworkAnalyst was used to analyze
Study purpose and background. Novel regenerative therapies have the potential to restore function and relieve pain in patients with low back pain (LBP) caused by intervertebral disc (IVD) degeneration. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation into nucleus pulposus (NP) cells of the IVD, which have potential for IVD regeneration. We have also shown that GDF6 stimulation activates the Smad1/5/8 and ERK1/2 signalling cascades. The aim of this study was to progress our understanding of the immediate/early response mechanisms in ASCs (N=3) which may direct GDF6-induced differentiation. Methods and results. RNAseq was used to perform transcriptome-wide analysis across a 12-hour time course, post-stimulation. Gene ontology analysis revealed greater
Objective. Modic changes (MC), a form of intervertebral disc degeneration visible as subchondral and vertebral bone marrow changes on spine magnetic resonance (MR), are known to be associated with low back pain. This study aimed to identify genes contributing to the development of MC using genome-wide association study. Methods. Presence of MC was evaluated in lumbar MR images in the Northern Finland Birth Cohort 1966 (NFBC1966, N=1182) and TwinsUK (N=647). Genome-wide association analyses were carried out in the cohorts separately using a linear regression model fitted to test for additive effects of SNPs and adjusting for age, sex, BMI, and either family relatedness via a kinship matrix (TwinsUK) or population stratification using principal components (NFBC1966). Meta-analysis of the two studies was carried out using the inverse-variance weighting approach. Results. A locus associated with MC reaching genome-wide significance (p<5e-8) was found on chromosome 9 with the lead SNP rs1934268 in intron 6 of the PTPRD gene. The SNP is located in the region of binding for a number of
Background. Degeneration of the intervertebral disc (IVD) is a leading cause of lower back pain, and a significant clinical problem. Inflammation mediated by IL-1β and TNF-α drives IVD degeneration through promoting a phenotypic switch in the resident nucleus pulposus (NP) cells towards a more catabolic state, resulting in extracellular matrix degradation. Bone marrow mesenchymal stem cells (MSCs) produce bioactive factors that modulate local tissue microenvironments and their anti-inflammatory potential has been shown in numerous disease models. Thus MSCs offer a potential therapy for IVD degeneration. In a clinical setting, adipose-derived stem cells (ASCs) might represent an alternative and perhaps more appealing cell source. However, their anti-inflammatory properties remain poorly understood. Methods. Here we assess the anti-inflammatory properties of donor-matched human ASCs and MSCs using qPCR and western blotting. Results. We demonstrate that stimulating ASCs or MSCs with IL-1β and/or TNF-α elicits a strong anti-inflammatory response with increased expression of IL-1 receptor antagonist (IL-1Ra), cyclooxygenase-2 (COX-2) and the tissue protective protein tumour-necrosis factor stimulated gene-6 (TSG-6). ASCs produced significantly higher levels of IL-1Ra and TSG-6 than their matched MSCs at both gene and protein levels, indicating that ASCs are potentially a more potent anti-inflammatory cell type. This anti-inflammatory response was also observed upon co-culture with degenerate NP cells without exogenous cytokine. Signalling analyses suggested this difference between cell types might be mediated through differences in the activation of inflammation-associated
Introduction. From the many human studies that attempt to identify genes for adolescent idiopathic scoliosis (AIS), the view emerging is that AIS is a complex genetic disorder with many predisposing genes exhibiting complex phenotypes through environmental interactions. Although advancements in genomic technology are transforming how we undertake genetic and genomic studies, only some success has been reached in deciphering complex diseases such as AIS. Moreover, the present challenge in AIS research is to understand the causative and correlative effects of discovered genetic perturbations. An important limitation to such investigations has been the absence of a method that can easily stratify patients with AIS. To overcome these challenges, we have developed a functional test that allows us to stratify patients with AIS into three functional subgroups, representing specific endophenotypes. Interestingly, in families with multiple cases of AIS, a specific endophenotype is shared among the affected family members, indicating that such a transmission is inherited. Moreover, increased vulnerability to AIS could be attributable to sustained exposure to osteopontin (OPN), a multifunctional cytokine that appears to be at the origin of the Gi-coupled receptor signalling dysfunction discovered in AIS. We examined the molecular expression profiles of patients with AIS and their response to OPN. Methods. Osteoblasts isolated from patients with AIS were selected for each functional subgroup and compared with osteoblasts obtained from healthy matched controls. We used the latest gene chip human genome array Affymetrix (HuU133 Plus 2.0 array) that allows for the analysis of the expression level of 38 000 well characterised human genes. Raw data were normalised with robust multiarray analysis method. Statistical analysis was done by the EB method with FlexArray software. Selection criteria for in-depth analysis include the magnitude of change in expression (at least □} 3-fold) and 5% false discovery rate as stringency selection. Validation of selected candidate genes was done by qPCR and at the protein level by Western blot and ELISA methods. Plasma OPN concentrations were measured by ELISA on a group of 683 consecutive patients with AIS and were compared with 262 healthy controls and 178 asymptomatic offspring, born from at least one scoliotic parent, and thus considered at risk of developing the disorder. The regulation of OPN signalling pathway in normal and AIS cells were validated in vitro by cellular dielectric spectroscopy (CDS). Results. Of 38 000 human genes tested, we have found eight genes specifically associated with the functional subgroup 1, 16 genes with the functional subgroup 2, and 11 genes with the functional subgroup 3. Interestingly, only 19 genes were shared and affected to the same extent in all AIS functional subgroups exhibiting a similar curve pattern (double major), suggesting their role in the formation of this curve pattern. Indeed, most of these genes encode for regulatory proteins such as
Mesenchymal stem-cell based therapies have been
proposed as novel treatments for intervertebral disc degeneration,
a prevalent and disabling condition associated with back pain. The
development of these treatment strategies, however, has been hindered
by the incomplete understanding of the human nucleus pulposus phenotype
and by an inaccurate interpretation and translation of animal to
human research. This review summarises recent work characterising
the nucleus pulposus phenotype in different animal models and in
humans and integrates their findings with the anatomical and physiological
differences between these species. Understanding this phenotype
is paramount to guarantee that implanted cells restore the native
functions of the intervertebral disc. Cite this article:
