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Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies.

ABSTRACT: STUDY DESIGN:Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG). PURPOSE:To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods. OVERVIEW OF LITERATURE:Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its etiopathology. Thus, a better understanding of anti-inflammatory agents and their role in IVD degeneration and pro-inflammatory cytokines expression is necessary for pain management and regeneration in IVD. METHODS:We performed primary cell culture of NP cells; immunocytochemistry; gene expression studies of cytokines, metalloproteases, extracellular proteins, and apoptotic markers using quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction (RT-PCR); cytotoxicity assay (MTT); and molecular docking studies using AutoDock 4.2 software (Molecular Graphics Laboratory, La Jolla, CA, USA) to confirm the binding mode of proteins and synthesized complexes. We calculated the mean±standard deviation values and performed analysis of variance and t-test using SPSS ver. 17.0 (SPSS, Inc., Chicago, IL, USA). RESULTS:Molecular docking showed that both Nar and NG bind to the selected genes of interest. Semi-quantitative RT-PCR analysis reveals differential gene expression of collagen (COL)9A1, COL9A2, COL9A3, COL11A2, COMT (catechol-O-methyltransferase), and THBS2 (thrombospondin 2); up regulation of ACAN (aggrecan), COL1A1, COL11A1, interleukin (IL)6, IL10, IL18R1, IL18RAP, metalloprotease (MMP)2, MMP3, MMP9, ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), IGF1R (insulin-like growth factor type 1 receptor), SPARC (secreted protein acidic and cysteine rich), PARK2 (parkin), VDR (vitamin D receptor), and BCL2 (B-cell lymphoma 2); down regulation of IL1A, CASP3 (caspase 3), and nine genes with predetermined concentrations of Nar and NG. CONCLUSIONS:The present study evaluated the anti-inflammatory and regenerative efficiencies of Nar and NG in degenerated human NP cells. Altered gene expressions of cytokines, metalloproteases, extracellular proteins, apoptotic genes were dose responsive. The molecular docking (in silico) studies showed effective binding of these native ligands (Nar and NG) with genes identified as potent inhibitors of inflammation. Thus, these natural flavonoids could serve as anti-inflammatory agents in the treatment of low back pain and sciatica.

SUBMITTER: Devraj VM

PROVIDER: S-EPMC6894971 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies.

Devraj Vijaya Madhuri VM Vemuri Satish Kumar SK Banala Rajkiran Reddy RR Gunda Shravan Kumar SK Av Gurava Reddy GR Gpv Subbaiah S

Asian spine journal 20191004 6

<h4>Study design</h4>Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG).<h4>Purpose</h4>To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods.<h4>Overview of literature</h4>Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its ...[more]

PMID: 31575107

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Project description:Study designIsolated human nucleus pulposus (hNP) cells from degenerated intervertebral disc were incubated under hydrostatic pressure and evaluated for regenerative potential.ObjectivesTo characterize metabolic turnover in hNP cells isolated from degenerated intervertebral discs classified by Pfirrmann grade under physiologically relevant hydrostatic pressure at high osmolality in vitro.Summary of background dataWe demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic hydrostatic pressure followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells.MethodshNP cells isolated from discarded tissue classified as Pfirrmann Grade 2-3 (n=13: age, 46.7±14.0) and Grade 4 (n=13: age, 53.0±11.5) were incubated under cyclic hydrostatic pressure at 0.2-0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules, catabolic and anti-catabolic proteins were evaluated.ResultsAggrecan and collagen type II expression was significantly more upregulated under HP in Grade 2-3 than in Grade 4 tissues (both, P<0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in Grade 2-3, while a negative correlation was found in Grade 4 (P<0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP.ConclusionsResident cells in mild-moderate degenerated disc classified as Pfirrmann grade 2-3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading.RelevanceThis study explored the potential of degenerated remnant NP cells under physiological environment, possibly leading to establishing strategies for IVD regeneration.

Project description:Intervertebral disc (IVD) degeneration is a pathological process, which may lead to lower back pain. The present study aimed to investigate the pathogenesis of IVD degeneration. GSE42611 was downloaded from Gene Expression Omnibus, including 4 nucleus pulposus samples isolated from degenerated IVDs and 4 nucleus pulposus samples separated from normal IVDs. The differentially expressed genes (DEGs) between the degenerated and normal samples were screened using the limma package in R. Functional and pathway enrichment analyses were conducted separately for the upregulated and downregulated genes, using Database for Annotation, Visualization and Integrated Discovery software. In addition, protein‑protein interaction (PPI) networks were constructed using the Search Tool for the Retrieval of Interacting Genes database and Cytoscape software. Finally, module analyses were conducted for the PPI networks using the MCODE plug‑in in Cytoscape. A total of 558 DEGs were identified in the degenerated nucleus pulposus cells: 253 upregulated and 305 downregulated. Pathway enrichment analysis revealed that downregulated thrombospondin 1 (THBS1) was enriched in extracellular matrix‑receptor interaction. Interleukin (IL)‑6 in the PPI network for the upregulated genes and vascular endothelial growth factor A (VEGFA) in the PPI network for the downregulated genes had higher degrees. Additionally, four modules (µM1, µM2, µM3 and µM4) were identified from the PPI network for the upregulated genes. Four modules (dM1, dM2, dM3 and dM4) were identified from the PPI network for the downregulated genes. In the dM2 module, collagen genes and integrin subunit α4 (ITGA4) may interact with each other. Additionally, functional enrichment indicated that collagen genes were enriched in extracellular matrix organization. In conclusion, IL‑6, VEGFA, THBS1, ITGA4 and collagen genes may contribute to the progression of IVD degeneration. These results suggested that the manipulation of these genes and their products may have potential as a novel therapeutic strategy for the treatment of patients with IVD.

Project description:Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Dataset Information

Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies.

Publications

Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies.

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