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

Abstract

Study design: Developing an in vitro model for assessing the anti-inflammatory properties of curvularin.

Purpose: To evaluate the efficacy of natural fungal macrolactone as a therapeutic drug against lipopolysaccharide (LPS)-induced inflammation in primary human nucleus pulposus cells (NPCs) in vitro.

Overview of literature: Lumbar disk disease is a common cause of lower back pain (LBP) and sciatica. It is an established fact that inflammation, rather than mechanical compression on the nerve root, plays a role in the cause of LBP and sciatica. Current treatment options for reducing inflammation are either nonsteroidal anti-inflammatory drugs or steroids, prolonged use of which can potentially lead to adverse effects such as gastrointestinal disturbances and renal and cardiac issues. Hence, there is a need for better antiinflammatory drugs with no or minimal complications for treating inflammation-induced LBP and sciatica. Curvularin (Cur), a fungal macrolactone, is known for its anti-inflammatory activity, but nothing is known about its impact on inflammation due to disk pathologies.

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.

Keywords: Curvularin; Cytokines; Inflammation; Intervertebral disc degeneration; Lipopolysaccharide; Macrolactone; Nonsteroidal anti-inflammatory drugs.

Fig. 1.

(A, B) Culture of degenerated human nucleus pulposus cells.

Fig. 2.

(A–C) Flow cytometry and immunocytochemistry studies for the characterization of primary human nucleus pulposus cells using the CD24 antibody.

Fig. 3.

MTT assay for assessing the cytotoxicity of individual and combined macrolactone and other steroid treatments on a normal cell line (human embryonic kidney-293). LPS, lipopolysaccharide; Cur, curvularin; Beta, betamethasone; Dexa, dexamethasone.

Fig. 4.

Gene expression of pro-inflammatories (TNF-α, IL-1β, and IL-6), metalloproteases (MMP-2, MMP-3; ADAMTS-5), anti-inflammatories (IL-2 and IL-10), cell death marker (CYT-C), stress-cum-repair marker (IGF1R), collagen 9A1, and endogenous control (GAPDH) in all experimental groups. TNF, tumor necrosis factor; IL, interleukin; MMPs, matrix metalloproteinases; CYT-C, cytochrome c; IGF1R, insulin-like growth factor 1 receptor; COL9A1, collagen 9 alpha 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenas.

Fig. 5.

(A–F) The effect of Cur, Beta, and Dexa, and their combination, on LPS-induced inflammation and loss of NPCs, compared to the untreated control group. We examined the growth enhancement and protective effects of Cur, Beta, and Dexa on NPCs. Cur, curvularin; NPCs, nucleus pulposus cells; LPS, lipopolysaccharide; Beta, betamethasone; Dexa, dexamethasone.

Fig. 6.

The of individual and combinatorial anti-inflammators against LPS treated degenerated human NPC using cell proliferation assay. LPS, lipopolysaccharide.

Fig. 7.

(A–K) Graphical representation of varied gene expressions using real-time polymerase chain reaction. LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; MMPs, matrix metalloproteinases; CYT-C, cytochrome c; IGF1R, insulin-like growth factor 1 receptor; COL9A1, collagen 9 alpha 1. *p<0.05. **p<0.01. ***p<0.001.