Asian Spine J.  2019 Dec;13(6):875-889. 10.31616/asj.2019.0073.

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

• Devraj VM
• Vemuri SK
• Banala RR
• Gunda SK
• AV GR
• GPV S

• KMID: 2467417
• DOI: http://doi.org/10.31616/asj.2019.0073

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.

Keyword

Intervertebral disc degeneration; Flavonoids; Inflammation; Nucleus pulposus; Naringin and naringenin

MeSH Terms

Anti-Inflammatory Agents
Caspase 3
Collagen
Cysteine
Cytokines
Down-Regulation
Flavonoids
Gene Expression
Humans*
Immunohistochemistry
In Vitro Techniques
Inflammation
Interleukin-10
Interleukins
Intervertebral Disc
Intervertebral Disc Degeneration
Ligands
Low Back Pain
Lymphoma
Metalloproteases
Models, Molecular*
Pain Management
Polymerase Chain Reaction
Primary Cell Culture
Regeneration
Sciatica
Thrombospondins
Up-Regulation
Anti-Inflammatory Agents
Caspase 3
Collagen
Cysteine
Cytokines
Flavonoids
Interleukin-10
Interleukins
Ligands
Metalloproteases
Thrombospondins