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. 2024 Mar 12;14(3):334.
doi: 10.3390/biom14030334.

Limonene Exerts Anti-Inflammatory Effect on LPS-Induced Jejunal Injury in Mice by Inhibiting NF-κB/AP-1 Pathway

Sarmed H Kathem  1 , Yasameen Sh Nasrawi  2 , Shihab H Mutlag  1 , Surya M Nauli  3
Affiliations

Limonene Exerts Anti-Inflammatory Effect on LPS-Induced Jejunal Injury in Mice by Inhibiting NF-κB/AP-1 Pathway

Sarmed H Kathem et al. Biomolecules. .

Abstract

The human gastrointestinal system is a complex ecosystem crucial for well-being. During sepsis-induced gut injury, the integrity of the intestinal barrier can be compromised. Lipopolysaccharide (LPS), an endotoxin from Gram-negative bacteria, disrupts the intestinal barrier, contributing to inflammation and various dysfunctions. The current study explores the protective effects of limonene, a natural compound with diverse biological properties, against LPS-induced jejunal injury in mice. Oral administration of limonene at dosages of 100 and 200 mg/kg was used in the LPS mouse model. The Murine Sepsis Score (MSS) was utilized to evaluate the severity of sepsis, while serum levels of urea and creatinine served as indicators of renal function. Our results indicated that LPS injection induced renal function deterioration, evidenced by elevated serum urea and creatinine levels compared to control mice. However, pretreatment with limonene at doses of 100 and 200 mg/kg mitigated this decline in renal function, evidenced from the reduced levels of serum urea and creatinine. Limonene demonstrated anti-inflammatory effects by reducing pro-inflammatory cytokines (TNF-α, IL-1β, COX-2), suppressing the TLR4/NF-κB/AP-1 but not IRF3 signaling pathways, and modulating oxidative stress through Nrf2 activation. The results suggest that limonene holds promise as a potential therapeutic agent for mitigating intestinal inflammation and preserving gastrointestinal health.

Keywords: IL-1β; IRF3; TLR4; TNF-α; iNOS; intestinal injury; monoterpenes.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic presentation of experimental animal design.
Figure 2
Figure 2
Effects of limonene on pro-inflammatory cytokines in LPS-induced intestinal injury in mice. Data represent mean ± SEM for mRNA expression of pro-inflammatory cytokines measured 24 h after induction with LPS (10 mg/kg) in mice jejunal tissue: (A) IL-1β; (B) TNF-α; (C) iNOS; (D) COX-2. Limonene treatment used in 2 doses (100 mg/Kg and 200 mg/kg) for 5 consecutive days before LPS injection. Calculations of gene expression performed relative to GAPDH as a control gene. n = 6 in each group. Analysis of data was performed with Prism GraphPad 5. * p < 0.05; ** p < 0.01; **** p < 0.0001 indicate statistical significance. Control: formula image; LPS: formula image; limonene 100 mg/kg: formula image; limonene 200 mg/kg: formula image.
Figure 3
Figure 3
Effects of limonene on inflammatory pathways in LPS-induced intestinal injury in mice. Data represent mean ± SEM for mRNA expression of inflammatory markers measured 24 h after induction with LPS (10 mg/kg) in mice jejunal tissue: (A) TLR4; (B); NF-κB; (C) IRF3; (D) AP-1. Limonene treatment used in 2 doses of 100 mg/kg and 200 mg/kg for 5 consecutive days before LPS injection. Calculations of gene expression performed relative to GAPDH as a control gene. n = 6 in each group. Analysis of data was performed with Prism GraphPad 5. * p < 0.05; ** p < 0.01; **** p < 0.0001 indicate statistical significance. Control: formula image; LPS: formula image; limonene 100 mg/kg: formula image; limonene 200 mg/kg: formula image.
Figure 4
Figure 4
Effect of limonene on the jejunal expression of Nrf2. Data represent mean ± SEM for mRNA expression of Nrf2 measured 24 h after induction with LPS (10 mg/kg) in mice jejunal tissue. Limonene treatment used in 2 doses (100 mg/kg and 200 mg/kg) for 5 consecutive days before LPS injection. Calculations of gene expression performed relative to GAPDH as a control gene. n = 6 in each group. Analysis of data was performed with Prism GraphPad 5. * p < 0.05; ** p < 0.01; *** p < 0.001 indicate statistical significance. Control: formula image; LPS: formula image; limonene 100 mg/kg: formula image; limonene 200 mg/kg: formula image.
Figure 5
Figure 5
Effect of limonene on mice mortality and Murine Sepsis Score (MMS) in LPS-induced intestinal injury. Data represented as mean ± SEM for Murine Sepsis Score (MMS) and percentage for mortality. (A) MSS; (B) mortality. Limonene treatment used in 2 doses (100 mg/Kg and 200 mg/kg) for 5 consecutive days before LPS injection. n = 6 in each group. Analysis of data was performed with Prism GraphPad 5. * p < 0.05; **** p < 0.0001 indicate statistical significance. Control: formula image; LPS: formula image; limonene 100 mg/kg: formula image; limonene 200 mg/kg: formula image.
Figure 6
Figure 6
Effect of limonene on urea and creatinine in LPS-induced intestinal injury. Data represent mean ± SEM for urea and creatinine measured 24 h after induction with LPS (10 mg/kg) in mice jejunal tissue: (A) urea; (B) creatinine. Limonene treatment used in 2 doses (100 mg/Kg and 200 mg/kg) for 5 consecutive days before LPS injection. n = 6 in each group. Analysis of data was performed with Prism GraphPad 5. **** p < 0.0001 indicate statistical significance. Control: formula image; LPS: formula image; limonene 100 mg/kg: formula image; limonene 200 mg/kg: formula image.
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