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.

Keywords: Flavonoids; Inflammation; Intervertebral disc degeneration; Naringin and naringenin; Nucleus pulposus.

Fig. 1.

Culture of human nucleus pulposus cells from degenerated intervertebral disc. (A) Day 0: explant from the degenerated disk; (B) day 7: cells propagating out from the explants and; (C) day 21: culture flask became fully confluent. The cells were polygonal in shape like chondrocytes.

Fig. 2.

(A–C) Characterization of NP cells using CD24 marker on flow cytometer analysis. NP, nucleus pulposus.

Fig. 3.

Effect of naringin and naringenin as well as their combination on NP cells at various concentrations, including 5, 10, 20, 50, and 100 μg/mL and the combination of naringin and naringenin in the following three ratios: 1:1, 1:2, and 2:1 compared to control. We examined the growth enhancement effect of naringin and naringenin on NP cells. NP, nucleus pulposus.

Fig. 4.

MTT assay for assessing the cytotoxicity of individual and combinatorial flavonoid treatments in normal cell line (HEK 293).

Fig. 5.

Immunocytochemistry of NP cells stained with type II collagen and SOX2 (SRY-box transcription factor 6) antibody counterstained with DAPI (4’, 6-diamidino-2-phenylindole) nuclear stain visualized under confocal laser scanning microscope. (A) Untreated controls, (B) naringin (20 μg/mL), (C) naringenin (20 μg/mL), and (D) naringin+naringenin (1:1 ratio). Cross-section analyses along the flattened Np cells showing a width of cell fluorescent intensity histogram visualized using confocal microscopy. (E) Np cells after drug treatment, (F) depth coding, (G) histogram image, and (H) three-dimensional topographical image. NP, nucleus pulposus.

Fig. 6.

Mitochondria stained with Mitotracker Green, showing mitochondria distribution using fluorescent microscope. (A) Control. (B) Naringin. (C) Naringenin. (D) Naringin+naringenin.

Fig. 7.

Gene expression pattern of 25 genes with different concentration of drug using gel electrophoresis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ACAN, aggrecan; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5; IGFR, insulin-like growth factor receptor; IL, interleukin; COL, collagen; CASP, caspase; MMP, matrix metalloproteinase; COMT, catechol-O-methyltransferase; SPARC, secreted protein acidic and cysteine rich; THBS2, thrombospondin 2; BCL2, B-cell lymphoma 2; PARK2, parkin; VDR, vitamin D receptor.

Fig. 8.

(A) Trend line between the control and treated groups vs. that in different genes. Gene Expression of 25 discogenic genes involved in intervertebral disc. (B) Heat map effect between the control and treated group (different concentrations) vs. that in different genes. THBS2, thrombospondin 2; BCL2, B-cell lymphoma 2; VDR, vitamin D receptor; PARK2, parkin; COMT, catechol-O-methyltransferase; SPARC, secreted protein acidic and cysteine rich; IGFR, insulin-like growth factor receptor; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5; MMP, matrix metalloproteinase; CASP, caspase; IL, interleukin; COL, collagen; ACAN, aggrecan; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 9.

(A, B) Molecular docking conformations of naringin and naringenin with aggrecan and interleukin 1 alpha.

Fig. 10.

Molecular docking conformations among naringin, naringenin, and proteins. ACAN, aggrecan; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5; BCL, B-cell lymphoma; CASP, caspase; COL, collagen; COMT, catechol-O-methyltransferase; IGF1R, insulin-like growth factor type 1 receptor; IL, interleukin; MMP, matrix metalloproteinase; PARK2, parkin; VDR, vitamin D receptor. (Continued to the next page).