Anti-inflammatory Activity of β-Carotene, Lycopene and Tri- n-butylborane, a Scavenger of Reactive Oxygen Species

Abstract

Background/aim: The polyene carotenoids β-carotene and lycopene are antioxidants that not only quench singlet oxygen but also inhibit lipid peroxidation. Tri-n-butyl borane (TBB) is used as an initiator for dental resin materials and is extremely reactive with oxygen and reactive oxygen species (ROS). This reactionability of TBB may be analogous to that of carotenoids with ROS. To clarify the biological activity of such ROS scavengers, we investigated the anti-inflammatory activity of β-carotene, lycopene and TBB in terms of the expression of RNA for lipopolysaccharide (LPS)-induced cyclooxygenase-2 (Cox2), nitric oxide synthase 2 (Nos2) and tumor necrosis factor-alpha (Tnfa), and mRNA expression and up-regulation of heme oxygenase 1 (Hmox1) mRNA in RAW264.7 cells.

Materials and methods: mRNA expression was investigated using real-time reverse transcriptase-polymerase chain reaction (PCR). The antioxidant activity of carotenoids was evaluated using the induction period method in the azobisisobutyronitrile or benzoyl peroxide-methyl methacrylate system.

Results: Hmox1 mRNA, but not Cox2 and Nos2 mRNA, was up-regulated by 100 μM β-carotene and lycopene, and by 0.125% TBB. LPS-stimulated Cox2, Nos2 and Tnfa gene expression was inhibited by 50 μM β-carotene and lycopene, and by 0.5-1% TBB. Both β-carotene and lycopene had weak antioxidant activity, but β-carotene showed pro-oxidant activity at higher concentrations.

Conclusion: The anti-inflammatory activity of β-carotene, lycopene and TBB may be related to their ROS-scavenging activity. Additionally, the activity of carotenoids and TBB may be attributed to the electrophilicity of ROS-induced carotenoid intermediates and boranes, respectively. Their anti-inflammatory activity may be attributable to enhancement of the potency of the electrophile/antioxidant response element transcription system in view of their up-regulation of Hmox1 mRNA expression.

Keywords: COX2; HMOX1; LPS; NOS2; RAW264.7 cells; lycopene; tri-n-butyl borane; β-Carotene.

Figure 1. The chemical structures of β-carotene and lycopene

Figure 2. Stimulation of heme oxygenase 1 (Hmox1) gene expression by β-carotene and lycopene in RAW264.7 cells. The cells were incubated for3.5 h with the indicated doses of β-carotene or lycopene, and then their total RNAs were prepared. Each cDNA was synthesized, and the expressionlevel of Hmox1 mRNA was determined by real-time polymerase chain reaction and standardized against the expression of 18s rRNA. The resultsare presented as means±standard error (SE) of three independent experiments, SE<15%. Significant differences between samples for β-caroteneand lycopene were observed for stimulation of Hmox1 gene expression. *Significantly different at p<0.01 vs. control group.

Figure 3. Stimulation of heme oxygenase 1 (Hmox1) gene expression by tri-n-butyl borane (TBB) in RAW264.7 cells. The cells were incubated for 3.5 h with the indicated doses of TBB, and then their total RNAs were prepared. Each cDNA was synthesized, and the expression level of Hmox1 mRNA was determined by real-time polymerase chain reaction and standardized against the expression of 18s rRNA. The results are presented as means±standard error (SE) of three independent experiments, SE<15%. Significant differences between samples for eachTBB were observed for stimulation of Hmox1 gene expression.*Significantly different at p<0.01 vs. control group

Figure 4. Inhibitory effects of β-carotene and lycopene on lipopolysaccharide (LPS)-stimulated cyclooxygenase-2 (Cox2) (A), nitric oxide synthase 2 (Nos2) (B) and tumor necrosis factor-alpha(Tnfa) (C) gene expression in RAW264.7 cells. The cells were pretreated for 30 min with the indicated doses of β-carotene or lycopene. They were then incubated for 3 h with or without LPS at 100 ng/ml, and their total RNAs were prepared. Each cDNA was synthesized, and the expression level of Cox2, Nos2 and Tnfa mRNAs were determined by real-time polymerase chain reaction and standardized against the expression of 18s rRNA. The results are presented as means±standard error (SE) of three independent experiments, SE<15%. Significant differences between samples for β-carotene and lycopene were observed for inhibition of Cox2, Nos2 and Tnfa gene expression. *Significantly different at p<0.01 vs. control group.

Figure 5. Inhibitory effects of tri-n-butyl borane (TBB) on lipopolysaccharide (LPS)-stimulated cyclooxygenase-2 (Cox2) (A), nitric oxide synthase 2 (Nos2) (B) gene expression in RAW264.7 cells. The cells were pretreated for 30 min with the indicated doses of TBB. Theywere then incubated for 3 h with or without LPS at 100 ng/ml, and their total RNAs were prepared. Each cDNA was synthesized, and the expression level of Cox2 and Nos2 mRNAs were determined by real-time polymerase chain reaction and standardized against the expression of 18s rRNA. The results are presented as means±standard error (SE) of three independent experiments, SE<15%. Significant differences between samples for each TBB were observed for inhibition of Cox2 and Nos2 gene expression. *Significantly different at p<0.01 vs. control group.