The Anti-Inflammatory and Cytoprotective Efficiency of Curvularin, a Fungal Macrolactone against Lipopolysaccharide-Induced Inflammatory Response in Nucleus Pulposus Cells: An In Vitro Study

Methods@#Primary NPCs were cultured and characterized by flow cytometry and immunocytochemistry using the CD24 antibody and treated with 10 μg/mL LPS for 36 hours and then treated with Cur, betamethasone, and dexamethasone (10 μg/mL) for 48 hours, after which cell cycle analysis, cell viability assay, and gene expression studies (quantitative polymerase chain reaction [PCR] and quantitative real-time-PCR) were conducted. The NPCs treated with Cur downregulated the expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6); matrix metalloproteinases (MMPs; MMP-2 and MMP-3), ADAMTS; and apoptotic marker (cytochrome c). @*Results@#In our study, Cur-treated cells showed enhanced expression of collagen 9A1 and insulin-like growth factor receptor 1, indicating the recovery of NPCs from inflammatory assault. @*Conclusions@#Based on observations, the anti-inflammatory properties of Cur render it an excellent drug molecule for treating disk degeneration nonsurgically, by direct injection into spinal disks when treating LBP and sciatica.

The Anti-Inflammatory and Cytoprotective Efficiency of Curvularin, a Fungal Macrolactone against Lipopolysaccharide-Induced Inflammatory Response in Nucleus Pulposus Cells: An In Vitro Study

Methods@#Primary NPCs were cultured and characterized by flow cytometry and immunocytochemistry using the CD24 antibody and treated with 10 μg/mL LPS for 36 hours and then treated with Cur, betamethasone, and dexamethasone (10 μg/mL) for 48 hours, after which cell cycle analysis, cell viability assay, and gene expression studies (quantitative polymerase chain reaction [PCR] and quantitative real-time-PCR) were conducted. The NPCs treated with Cur downregulated the expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6); matrix metalloproteinases (MMPs; MMP-2 and MMP-3), ADAMTS; and apoptotic marker (cytochrome c). @*Results@#In our study, Cur-treated cells showed enhanced expression of collagen 9A1 and insulin-like growth factor receptor 1, indicating the recovery of NPCs from inflammatory assault. @*Conclusions@#Based on observations, the anti-inflammatory properties of Cur render it an excellent drug molecule for treating disk degeneration nonsurgically, by direct injection into spinal disks when treating LBP and sciatica.

Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD).

BACKGROUND CONTEXT: One of the common causes of low back pain is intervertebral disc degeneration. The pathophysiology of disc degeneration involves apoptosis of nucleus pulposes cells and degradation of extra cellular matrix (ECM). Caspase 3 plays a central role in apoptosis and the ADAMTS5 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 5) gene plays a critical role in ECM degradation. Hence, we hypothesized that if one can silence these two genes, both apoptosis and ECM degradation can be prevented, thereby preventing the progression and even reverse disc degeneration. PURPOSE: The purpose of this study is to demonstrate the regenerative potential of small interfering RNA (siRNA) designed against Caspase 3 and ADAMTS5 genes in an in vitro and animal model of disc degeneration. STUDY DESIGN: In vitro study followed by in vivo study in a rabbit model. METHODS: In vitro studies were done using the human hepatocellular carcinoma (Hep G2) cell line for validating the efficacy of liposomal siRNA in controlling the expression of genes (Caspase 3 and ADAMTS5). Later, siRNA's validation was done in a rabbit annular punctured model by administering siRNA's individually (Caspase 3 and ADAMTS5) and in combination Caspase3-ADAMTS5) for assessing their synergistic effect in down regulating the gene expression in the degenerative discs. Annular punctured intervertebral discs of the rabbit were injected with siRNA formulations (single and dual) and phosphate buffer saline, one week after initial puncture. Magnetic resonance imaging (MRI) scans were done before and after siRNA treatment (1, 4 and 8 weeks) for assessing the progression of disc degeneration. The histopathology and real time polymerase chain reaction (RT-PCR) studies were done for evaluating their efficacy. We did not receive any funding for conducting the study, and we do not have a conflict of interest with any researchers or scientific groups. RESULTS: The observations made from both in vitro and in vivo studies indicate the beneficial effects of siRNA formulation in down regulating the expression of Caspase 3 and ADAMTS5 genes. The MRI and histopathological evaluation showed that the disc degeneration was progressive in phosphate buffer saline and AT5-siRNA injected discs but the discs that received Caspase 3-siRNA and dual siRNA (Cas3-AT5-siRNA) formulation showed signs of recovery and regeneration 4 and 8 weeks after injection. The efficacy of siRNA designed against Cas3 and AT5 was also assessed in both in vitro and in vivo experiments by using RT-PCR analysis and the results showed downregulation of Caspase 3 gene in Caspase 3-siRNA group, but there was no significant downregulation of ADAMTS5 gene in ADAMTS5-siRNA group (ie, indicated by fold change). Synergistic effect was observed in the group that received dual siRNA (Cas3-AT5 siRNA) formulation. CONCLUSIONS: This experiment suggests that intervention by siRNA treatment significantly reduced the extent of apoptosis in the discs. CLINICAL SIGNIFICANCE: Delivery of siRNA directly into spinal discs has a potential in treating disc degeneration nonsurgically.

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

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.

Development of Novel Animal Model for Studying Scoliosis Using a Noninvasive Method and Its Validation through Gene-Expression Analysis

STUDY DESIGN: To induce scoliosis in young female Wistar rats using a noninvasive method and to validate this model. PURPOSE: To induce scoliosis in a rat model noninvasively by bracing and to study the corresponding gene-expression profile in the spine and different organs. OVERVIEW OF LITERATURE: Scoliosis involves abnormal lateral curvature of the spine, the causes of which remain unclear. In the literature, it is suggested that scoliosis is genetically heterogeneous, as there are multiple factors involved directly or indirectly in its pathogenesis. Clinical and experimental studies were conducted to understand the etiology of anatomical alterations in the spine and internal organs, as the findings could help clinicians to establish new treatment approaches. METHODS: Twelve female Wistar rats aged 21 days were chosen for this study. Customized braces and real-time polymerase chain reaction (RT-PCR) primers for rats were designed using Primer 3 software. Radiological analysis (X-rays), histopathological studies, SYBR green, and RT-PCR analysis were performed. RESULTS: The spines of six rats were braced in a deformed position, which resulted in a permanent structural deformity as confirmed by X-ray studies. The remaining rats were used as controls. Quantitative studies of the expression of various genes (osteocalcin, pleiotrophins, matrix metalloproteinase-2 [MMP2] and MMP9, TIMP, interleukins 1 and 6, tumor necrosis factor-α) showed their differential expression and significant upregulation (p < 0.05) in different organs of scoliotic rats in comparison to those in control rats. Histopathological findings showed tissue necrosis and fibrosis in the brain, retina, pancreas, kidney, liver, and disc of scoliotic rats. CONCLUSIONS: Bracing is a noninvasive method for inducing scoliosis in an animal model with 100% reliability and with corresponding changes in gene expression. Scoliosis does not just involve a spine deformity, but can be referred to as a systemic disease on the basis of the pathological changes observed in various internal organs.