CM1, a Chrysin Derivative, Protects from Endotoxin-Induced Lethal Shock by Regulating the Excessive Activation of Inflammatory Responses

Abstract

Sepsis, a leading cause of death worldwide, is a harmful inflammatory condition that is primarily caused by an endotoxin released by Gram-negative bacteria. Effective targeted therapeutic strategies for sepsis are lacking. In this study, using an in vitro and in vivo mouse model, we demonstrated that CM1, a derivative of the natural polyphenol chrysin, exerts an anti-inflammatory effect by inducing the expression of the ubiquitin-editing protein TNFAIP3 and the NAD-dependent deacetylase sirtuin 1 (SIRT1). Interestingly, CM1 attenuated the Toll-like receptor 4 (TLR4)-induced production of inflammatory cytokines by inhibiting the extracellular-signal-regulated kinase (ERK)/MAPK and nuclear factor kappa B (NF-κB) signalling pathways. In addition, CM1 induced the expression of TNFAIP3 and SIRT1 on TLR4-stimulated primary macrophages; however, the anti-inflammatory effect of CM1 was abolished by the siRNA-mediated silencing of TNFAPI3 or by the genetic or pharmacologic inhibition of SIRT1. Importantly, intravenous administration of CM1 resulted in decreased susceptibility to endotoxin-induced sepsis, thereby attenuating the production of pro-inflammatory cytokines and neutrophil infiltration into the lung compared to control mice. Collectively, these findings demonstrate that CM1 has therapeutic potential for diverse inflammatory diseases, including sepsis.

Keywords: CM1; TNFAIP3; Toll-like receptor 4; inflammation; sepsis; sirtuin 1.

Figure 1

Chemical structures and cytotoxicity in chrysin and its derivatives. (A–C) BMDMs were incubated with different concentration of chrysin, CM1, or CM2 for 18 h. Cell viability measured by a Cell Counting Kit 8 (CCK-8) assay. Data are representative of three independent experiments and are presented as means ± standard deviation (SD). *** p < 0.001, compared with control cells (two-tailed Student’s t-test). SC, solvent control (0.01% dimethylsulfoxide).

Figure 2

CM1 inhibits lipopolysaccharide (LPS)-induced inflammatory responses in bone marrow-derived macrophages (BMDMs). (A–C) BMDMs were incubated with LPS (100 ng/mL) for the indicated times. (A,B) mRNA levels of Tnfα and Il6 analysed by reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR (qPCR). (C) Protein levels of TNF-α and IL-6 in culture medium measured by enzyme-linked immunosorbent assay (ELISA). (D,E) BMDMs were stimulated with LPS and co-treated with chrysin (0.5 or 2.5 µg/mL), CM1 (0.5 or 2.5 µg/mL), or CM2 (5 or 10 µg/mL) for 18 h. (D) mRNA levels of Tnfα and Il6 evaluated by RT-PCR (top) and qPCR (bottom). (E) Protein levels of TNF-α and IL-6 in culture medium investigated by ELISA. Data are representative of three independent experiments and are presented as means ± SD. *** p < 0.001, compared with control cells (two-tailed Student’s t-test). U, untreated cells; SC, solvent control (0.01% DMSO).

Figure 3

CM1 inhibits LPS-induced extracellular-signal-regulated kinase (ERK) phosphorylation and nuclear factor kappa B (NF-κB) activation. (A,D) BMDMs were incubated with LPS (100 ng/mL) for the indicated times. Mitogen-activated protein kinase (MAPK) and NF-κB activation were determined by immunoblotting. (B,C,E) BMDMs were stimulated with LPS only, LPS and chrysin (0.1, 0.5, or 2.5 µg/mL), or LPS and CM1 (0.1, 0.5, or 2.5 µg/mL) for 30 min. (B,E) Protein levels determined by immunoblotting. (C) Densitometric analysis of p-ERK, p-JNK, and p-p38 expression with normalisation to β–tubulin. (F) Cells were fixed and stained for NF-κB p65 (green); nuclei were stained with 4′,6-diamidino-2-phenylindol (DAPI) (blue). Nuclear translocation of NF-κB p65 analysed by confocal microscopy. Scale bar: 20 µm. Data are representative of three independent experiments and are presented as means ± SD. *** p < 0.001, compared with control cells (two-tailed Student’s t-test). U, untreated cells; SC, solvent control (0.01% DMSO).

Figure 4

CM1 attenuates LPS-induced inflammatory responses through TNFAIP3 upregulation. (A,B) BMDMs were stimulated with LPS only (100 ng/mL) or LPS with CM1 (1 µg/mL) for the indicated times. (A) Protein levels of tumour necrosis factor alpha-induced protein 3 (TNFAIP3) determined by immunoblotting. (B) Densitometric analysis of TNFAIP3 expression with normalisation to β–tubulin. (C,D) HeLa cells were transfected with control siRNA or siTNFAIP3. (C) Transfected cells were stimulated with LPS only or LPS and CM1 for 18 h. mRNA levels of Tnfα and IL6 were determined by qPCR. RT-PCR was performed to assess transfection efficiency (inset). (D) Transfected cells were co-stimulated with LPS and CM1 for 30 min. Protein levels of p-ERK, p-IKKαβ, total IκBα, and TNFAIP3 were evaluated by immunoblotting. Data are representative of three independent experiments and are presented as means ± SD. *** p < 0.001, compared with control cells (two-tailed Student’s t-test). U, untreated cells; SC, solvent control (0.01% DMSO); siNS, non-specific siRNA; siTNFAIP3, specific siRNA for TNFAIP3.

Figure 5

CM1 reduces LPS-induced inflammatory responses by enhancing SIRT1 activity. (A) BMDMs were stimulated with LPS only (100 ng/mL) or LPS and CM1 (1 µg/mL) for the indicated times. NF-κB p65 acetylation and SIRT1 expression were determined by immunoblotting. (B–E) BMDMs were pre-treated with increasing concentrations of sirtinol (5, 15, or 30 µM; B and D) or EX-527 (5, 15, or 30 µM; (C,E)) for 2 h, followed by exposure to LPS only or LPS and CM1 for 30 min (B,C) or 18 h (D,E). (B,C) Immunoblotting was performed to evaluate the acetylation of NF-κB p65. (D,E) mRNA levels of Tnfα and Il6 determined by qPCR. (F,G) BMDMs from mSirt1^+/+ and mSirt1^−/− mice stimulated with LPS only or LPS and CM1 (0.1, 0.5, or 2.5 µg/mL) for 30 min (for (F)) or 18 h (for (G)). (F) NF-κB p65 acetylation was determined by immunoblotting. (G) mRNA levels of Tnfα and Il6 measured by qPCR. Data are representative of three independent experiments and are presented as means ± SD. ** p < 0.01, *** p < 0.001, compared with control cells (for (D,E)) or cells isolated from Sirt1^+/+ mouse (two-tailed Student’s t-test). U, untreated cells; SC, solvent control (0.01% DMSO).

Figure 6

CM1 contributes to the protection of mice against lethal shock. (A–E) Mice (n = 10 per group) were intravenously injected with either vehicle control or CM1 (10 mg/kg) once daily for 3 days before endotoxin stimulation (intraperitoneal injection, 30 mg/kg). (A) Survival rates of each group were monitored for 168 h. (B–E) Mice were sacrificed at 24 h post-LPS injection (n = 5 per group). (B) Serum samples were collected from vehicle control-treated or CM1-treated mice. Levels of TNFα and IL-6 were determined using ELISA. (C) The expression of Tnfα and Il6 in lung (left) and spleen (right) was analysed using real-time qPCR. (D) Immunohistochemical analysis of the lung tissue was performed to determine neutrophil infiltration. Scale bar: 50 µm. (E) The number of infiltrating neutrophils was counted from 8 random fields. The experiments were conducted in triplicate to ensure reproducibility, with the results expressed as the means ± SD. Statistical significance of mean differences was determined using either a log-rank test (A) or a two-tailed Student’s t-test (B,C,E). * p < 0.05, *** p < 0.001, compared with control mice stimulated with LPS. U, untreated; SC, solvent control.

Figure 7

Inhibitory effect of CM1 on TLR4-induced inflammation and its molecular mechanisms. Upon the binding of LPS to the TLR4/MD2/CD14 complex, the downstream signalling cascade is activated, leading to the ubiquitination of TRAF6 and subsequently activation of TAK1 and the IKK complex, which phosphorylates IκB, leading to the release of NF-κB p65 into the nucleus. This process results in the transcription of pro-inflammatory cytokines such as TNF-α and IL-6. CM1 directly upregulates the expression of TNFAIP3/A20, which inhibits the NF-κB p65 pathway and enhances the activity of SIRT1. Increased SIRT1 activity, in turn, deacetylates p65, suppressing its ability to transcribe pro-inflammatory genes. The dual action of CM1 suggests its therapeutic potential for LPS-induced inflammation.