In Vitro Assessment of Anti-Adipogenic and Anti-Inflammatory Properties of Black Cumin (Nigella sativa L.) Seeds Extract on 3T3-L1 Adipocytes and Raw264.7 Macrophages

Abstract

Background and Objectives: This study evaluated the in vitro anti-adipogenic and anti-inflammatory properties of black cumin (Nigella sativa L.) seed extract (BCS extract) as a potential candidate for developing herbal formulations targeting metabolic disorders. Materials and Methods: We evaluated the BCS extract by assessing its 2,2-diphenyl-1-picrohydrazyl (DPPH) radical scavenging activity, levels of prostaglandin E₂ (PGE₂) and nitric oxide (NO), and mRNA expression levels of key pro-inflammatory mediators. We also quantified the phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) signaling molecules. To assess anti-adipogenic effects, we used differentiated 3T3-L1 cells and BCS extract in doses from 10 to 100 μg/mL. We also determined mRNA levels of key adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/BEPα), adipocyte protein 2 (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), and sterol-regulated element-binding protein 1c (SREBP-1c) using real-time quantitative polymerase chain reaction (qPCR). Results: This study showed a concentration-dependent DPPH radical scavenging activity and no toxicity at concentrations up to 30 μg/mL in Raw264.7 cells. BCS extract showed an IC₅₀ of 328.77 ± 20.52 μg/mL. Notably, pre-treatment with BCS extract (30 μg/mL) significantly enhanced cell viability in lipopolysaccharide (LPS)-treated Raw264.7 cells. BCS extract treatment effectively inhibited LPS-induced production of PGE₂ and NO, as well as the expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), interleukin (IL)-1β and IL-6, possibly by limiting the phosphorylation of p38, p65, inhibitory κBα (I-κBα), and c-Jun N-terminal kinase (JNK). It also significantly attenuated lipid accumulation and key adipogenic genes in 3T3-L1 cells. Conclusions: This study highlights the in vitro anti-adipogenic and anti-inflammatory potential of BCS extract, underscoring its potential as a promising candidate for managing metabolic disorders.

Keywords: 3T3-L1 cells; Raw264.7 cells; adipogenic differentiation; oil red O; pro-inflammatory mediators.

Figure 1

HPLC analysis of thymoquinone in the BSC extract (A) and standard thymoquinone (B).

Figure 2

DPPH radical scavenging activity of BCS extract. DPPH-radical: 2,2-diphenyl-1-picrohydrazyl radical; BCS extract: Black cumin seeds extract. ^a p < 0.01: Significant as compared to control.

Figure 3

BCS extract-mediated inhibition in NO production in LPS-stimulated Raw264.7 cells. (A) Effect of BCS extract (3–100 μg/mL) on LPS-mediated cytotoxicity of Raw264.7 cells, (B) Effect of DEXA, a reference drug, on cell cytotoxicity, (C) NO production, and (D) mRNA expression of iNOS quantified by qPCR analysis. BCS extract: Black cumin seeds extract; NO: Nitric oxide; DEXA: Dexamethasone; LPS: Lipopolysaccharide; iNOS: Inducible nitric oxide synthase. ^a p < 0.01 and ^b p < 0.05: Significant as compared to control; ^c p < 0.01: Significant as compared to LPS.

Figure 4

BCS extract-mediated decrease in PGE₂ production in LPS-stimulated Raw264.7 cells. (A) PGE₂ production in BCS extract (3–30 μg/mL) pre-treated Raw264.7 cells, (B) mRNA expression of COX-2 mRNA quantified by qPCR. BCS extract: Black cumin seeds extract; PGE2: Prostaglandin E2; COX-2: Cyclooxygenase 2; DEXA: Dexamethasone; LPS: Lipopolysaccharide. ^a p < 0.01 and ^b p < 0.05: Significant as compared to control; ^c p < 0.01 and ^d p < 0.05: Significant as compared to LPS.

Figure 5

BCS extract-mediated reduction in pro-inflammatory cytokines production in LPS-stimulated Raw264.7 cells. mRNA expression levels of TNF-α (A), IL-1β (B), IL-6 (C), and MCP-1 (D) quantified by ELISA or qPCR. Raw264.7 cells were treated with BCS extract (3–30 μg/mL) for 0.5 h and then LPS (0.3 μg/mL) for 6 h (A-right, B–D) or 18 h (A-left). BCS extract: Black cumin seeds extract; TNF-α: Tumor necrosis factor-α; DEXA: Dexamethasone; LPS: Lipopolysaccharide; IL: Interleukin; MCP-1: Monocyte chemoattractant protein 1. ^a p < 0.01 and ^b p < 0.05: Significant as compared to control; ^c p < 0.01 and ^d p < 0.05: Significant as compared to LPS.

Figure 6

BCS extract-mediated inhibition in MAPKs and NF-κB phosphorylation in LPS-stimulated Raw264.7 cells. Raw264.7 cells were exposed to BCS extract (3–30 μg/mL) for 0.5 h and then LPS (0.3 μg/mL) for 0.5 h. Phosphorylation levels of MAPKs (A) and NF-κB (B) were quantified by immunoblotting. BCS extract: Black cumin seeds extract; JNK: c-Jun N-terminal kinase; p38 MAPK: DEXA: Dexamethasone; LPS: Lipopolysaccharide; p38 mitogen-activated protein kinase; ERK: Extracellular signal-regulated kinase; I-κBα: Inhibitory κBα; MAPK: Mitogen-activated protein kinases; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells. ^a p < 0.01: Significant as compared to control; ^b p < 0.01 and ^c p < 0.05: Significant as compared to LPS.

Figure 7

BCS extract-medicated inhibition in lipid accumulation during 3T3-L1 cell differentiation. (A) Cell viability of 3T3-L1 cells differentiated into adipocyte-like cells in the presence of either BCS extract (10–100 μg/mL) or RA (10 μM). (B) Quantification of lipid accumulation after oil red O staining. BCS extract: Black cumin seeds extract; 3T3-L1: Murine pre-adipocyte cell line; RA: All trans retinoic acid; MDI: Methylisobutylxanthine, dexamethasone, and insulin differentiation medium. ^a p < 0.01: Significant as compared to control; ^b p < 0.01: Significant as compared to MDI.

Figure 8

BCS extract-mediated inhibition in mRNA expression of adipogenic genes during 3T3-L1 cell differentiation. qPCR quantified mRNA expressions of C/EBPα (A), PPARγ (B), SREBP-1c (C), FAS (D), aP2 (E), and LPL (F) in 3T3-L1 differentiated into adipocyte-like cells in the presence of either BCS extract (10–100 μg/mL) or RA (10 μM). BCS extract: Black cumin seeds extract; 3T3-L1: Murine pre-adipocyte cell line; C/BEPα: CCAAT/Enhancer binding protein α; RA: All trans retinoic acid; PPARγ: Peroxisome proliferator-activated receptor γ; SREBP-1c: Sterol regulatory element binding protein 1c; aP2: Adipocyte protein 2; LPL: Lipoprotein lipase. ^a p < 0.01 and ^b p < 0.05: Significant as compared to control; ^c p < 0.01 and ^d p < 0.01: Significant as compared to MDI.