Trichodimerol inhibits inflammation through suppression of the nuclear transcription factor-kappaB/NOD-like receptor thermal protein domain associated protein 3 signaling pathway

Abstract

Excessive inflammation causes chronic diseases and tissue damage. Although there has been drug treatment, its side effects are relatively large. Searching for effective anti-inflammatory drugs from natural products has become the focus of attention. First isolated from Trichoderma longibraciatum, trichodimerol is a natural product with TNF inhibition. In this study, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were used as a model to investigate the anti-inflammatory activity of trichodimerol. The results of nitric oxide (NO) detection, enzyme-linked immunosorbent assay (ELISA), and reactive oxygen species (ROS) showed that trichodimerol could reduce the production of NO, ROS, and the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α. Western blotting results showed that trichodimerol could inhibit the production of inflammatory mediators such as cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) and the protein expression of nuclear transcription factor-kappaB (NF-κB), p-IKK, p-IκB, Toll-like receptor 4 (TLR4), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteinyl aspartate specific proteinase (Caspase)-1, and ASC, which indicated that trichodimerol may inhibit inflammation through the NF-κB and NLRP3 pathways. At the same time, molecular docking showed that trichodimerol can directly combine with the TLR4-MD2 complex. Hence, trichodimerol inhibits inflammation by obstructing the interaction between LPS and the TLR4-MD2 heterodimer and suppressing the downstream NF-κB and NLRP3 pathways.

Keywords: NF-κB; NLRP3; inflammation; molecular docking; trichodimerol.

Figure 1

The structure and cytotoxicity of trichodimerol. (A) The structure of trichodimerol. (B) Cell viability. Cell viability was tested with CCK8 reagent 48 h after RAW264.7 macrophages were administered different amounts of trichodimerol, and dimethyl sulfoxide (DMSO) was used as a blank group.

Figure 2

The effect of trichodimerol on inflammation. (A) Nitric oxide (NO) production in the supernatant of RAW264.7 macrophages after trichodimerol administration. (B) RAW264.7 macrophages were treated with different concentrations of trichodimerol and induced by lipopolysaccharide (LPS). The expression of tumor necrosis factor (TNF)-α and IL-6 in the supernatant was detected by ELISA. (C) mRNA expression levels of IL-6 and TNF-α in RAW264.7 macrophages in different treatment groups. All data are expressed as the mean ± SD. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001, compared with the DMSO group. ^&&p < 0.01, ^&&&p < 0.001, and ^&&&&p < 0.0001, compared with the trichodimerol 15 μM group. ^*p < 0.05, ^**p < 0.01, and ^****p < 0.0001, compared with the LPS group.

Figure 3

The effect of trichodimerol on the inflammatory mediators. (A) The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in RAW264.7 macrophages treated with different concentrations; GAPDH was used as an internal reference. (B) Quantitative statistical results of the protein expression of iNOS and COX-2. (C) mRNA expression levels of iNOS and COX-2 in RAW264.7 macrophages in different treatment groups. (D) Reactive oxygen species (ROS) production in RAW264.7 macrophages induced by LPS 24 h after trichodimerol treatment. Green fluorescence represents intracellular ROS stained by DCFH-DA, blue fluorescence represents nucleus, white stripe = 50 μm. All data are expressed as the mean ± SD. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001, compared with the DMSO group. ^&p < 0.05, ^&&&p < 0.001, and ^&&&&p < 0.0001, compared with the trichodimerol 15 μM group. ^*p < 0.05, ^**p < 0.01, and ^****p < 0.0001, compared with the LPS group.

Figure 4

The accumulation of ROS in zebrafish. (A) Images of reactive oxygen species (ROS) levels in zebrafish exposed to different amounts of trichodimerol. Green fluorescence represents intracellular ROS stained by DCFH-DA. (B) ImageJ was used to quantify the fluorescence intensity, which was statistically analyzed. All data are expressed as the mean ± SD. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, compared with the DMSO group. ^***p < 0.001, compared with the LPS group.

Figure 5

The effect of trichodimerol on the NLRP3 pathway. (A) RAW264.7 macrophages were treated with different concentrations of trichodimerol (5, 10, and 15 μM) for 2 h, followed by LPS (1 μg/ml) for 24 h. The protein expression of ASC, Caspase-1, and NLRP3 was detected by Western blotting; GAPDH was used as an internal reference. (B) Quantitative statistical results of the protein expression of ASC, Caspase-1, and NLRP3. (C) mRNA expression levels of IL-1β. All data are expressed as the mean ± SD. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001, compared with the DMSO group. ^&p < 0.05, ^&&p < 0.01, and ^&&&&p < 0.0001, compared with the trichodimerol 15 μM group. ^*p < 0.05, ^**p < 0.01, and ^****p < 0.0001, compared with the LPS group.

Figure 6

The effect of trichodimerol on the NF-κB pathway. (A) Nuclear translocation of RAW264.7 macrophages induced by LPS after 12 h of trichodimerol treatment. Red fluorescence represents the NF-κB signal, blue fluorescence represents the nucleus, white stripe = 50 μm. (B) The protein expression of TLR4, NF-κB, p-NF-κB, p-IKKα/β, and p-IκB in RAW264.7 macrophages treated with different concentrations; Tubulin was used as an internal reference. (C) Quantitative statistical results of TLR4 protein expression. (D) Quantitative statistical results of the protein expression of NF-κB. (E) Quantitative statistical results of the protein expression of p-NF-κB. (F) Quantitative statistical results of the protein expression of p-IKKα/β. (G) Quantitative statistical results of the protein expression of p-IκB. All data are expressed as the mean ± SD. ^#p < 0.05, ^##p < 0.01, compared with the DMSO group. ^&p < 0.05, compared with the trichodimerol 15 μM group. ^*p < 0.05, ^**p < 0.01, compared with the LPS group.

Figure 7

Molecular docking results of trichodimerol and TLR4-MD2. Molecular docking is simulated under the lowest energy conformation. Hydrogen bond interactions are represented by dotted lines.