Emodin Alleviates Sepsis-Induced Multiorgan Damage by Inhibiting NETosis through Targeting Neutrophils BCL-10

Abstract

Sepsis is a life-threatening condition caused by dysregulated host responses to infection, characterized by excessive inflammation and abnormal coagulation. Neutrophil extracellular traps (NETs) formation bridges these two pathological processes. Through both in vivo and in vitro experiments, it is observed that Emodin, a natural anthraquinone derivative derived from Dahuang, significantly ameliorates the cytokine storm and coagulation abnormalities induced by sepsis, demonstrating remarkable efficacy in inhibiting NETs formation. Furthermore, through protein microarrays, surface plasmon resonance (SPR), pull-down assays, and molecular docking analyses, BCL-10 is established as a direct target of Emodin, providing protective effects in both in vivo and in vitro settings. Through conditional knockout of BCL-10 in neutrophils, alongside single-cell sequencing analyses, it is confirmed that BCL-10 is key in promoting excessive NET formation in sepsis. Additionally, Emodin exerts powerful protective effects by modulating the function of the BCL-10/MALT1 complex, thereby alleviating the NF-κB signaling activation and inhibiting NETs formation. Collectively, these findings provide pharmacological evidence that Emodin targeted BCL-10 regulates the BCL-10/MALT1 complex and suppresses NF-κB activation, ultimately conferring significant multiorgan protective effects in sepsis. The conduct of this study provides new clues for the translational research of Emodin and its target BCL-10 in sepsis.

Keywords: BCL‐10/MALT1; Emodin; NETosis; NF‐κB; sepsis.

Figure 1

Emodin alleviated mortality and multiorgan injury in CLP‐induced septic mice. A) Schematic overview of the experimental design, including the modeling, group allocation, and intervention timeline. B) Kaplan–Meier survival curves over a 7‐day period comparing the Sham, CLP septic group, Emodin treatment group, and DXMS treatment group. C–E) Histopathological images showing tissue morphology and injury in the lung (C), liver (D), spleen (E), and kidney (F) across the four experimental groups.

Figure 2

Emodin‐reduced inflammatory response and coagulation abnormalities in CLP‐induced septic mice. A) Expression levels of various inflammatory cytokines and chemokines in the peripheral plasma of CLP‐induced septic mice at 24 and 48 h postsurgery. B) Comparative expression levels of inflammatory cytokines and chemokines in the peripheral plasma between the CLP‐induced septic group and the Emodin treatment group at 24 h postsurgery. C) Time course analysis of multiple coagulation‐related factors in peripheral plasma of CLP‐induced septic mice at 6, 12, 24, 48, and 72 h postsurgery. E,F) WB analysis of protein expression levels for TF (E), GPVI (F), and Shh (G) in liver tissues of the four experimental groups at 48 h postsurgery. G1, G2, G3, and G4 represent the Sham group, CLP model group, Emodin intervention group, and DXMS intervention group, respectively.

Figure 3

Emodin inhibited the formation of NETs in CLP‐induced septic mice. A) Bar graph showing the expression levels of cfDNA in different groups of mice at various time points (24, 48, and 72 h). B) Line graph illustrating the expression levels of cfDNA in different groups of mice at 24, 48, and 72 h. C) WB analysis of NE, MPO, and CitH3 protein expression in liver tissues of different groups of mice at various time points (24, 48, and 72 h). D,E) Bar graph (D) and line graph (E) showing the expression levels of NE protein in liver tissues of mice at different time points (24, 48, and 72 h). F,G) Bar graph (F) and line graph (G) depicting the expression levels of CitH3 protein in liver tissues of mice at different time points (24, 48, and 72 h). H,I) Bar graph (H) and line graph (I) represent the expression levels of MPO protein in liver tissues of mice at different time points (24, 48, and 72 h). J) Multiplex IF images of liver tissues from different groups stained for MPO and CitH3. G1, G2, and G3 represent the sham group, CLP model group, and Emodin intervention group, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., P > 0.05 (no significant difference).

Figure 4

Emodin significantly inhibited NETs formation in vitro. A) CCK‐8 assay to evaluate the effect of Emodin on the viability of dHL‐60 cells. B) CCK‐8 assay assessing the impact of Emodin on dHL‐60 cells after PMA induction. C) Dichlorodihydrofluorescein diacetate (DCFH‐DA) probe detection of ROS activity in dHL‐60 cells after PMA induction, with or without Emodin treatment. D) PicoGreen fluorescence assay measuring cfDNA levels in cell supernatants across different groups. E) MPO content was measured using a colorimetric MPO assay kit in various groups of cells. F,G) Enzyme‐linked immunosorbent assay (ELISA) quantification of NE (F) and CitH3 (G) levels in the cell supernatants of different groups. H) Sytox Green fluorescence staining of cells across different experimental groups. I) Immunofluorescence staining detecting NE protein expression in the various groups of cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., P > 0.05 (no significant difference).

Figure 5

Identification of BCL‐10 as a binding protein of Emodin. A) Chemical structures of Emodin and biotin‐labeled Emodin (Bio‐Emodin). B) Schematic representation of the steps involved in identifying Emodin‐binding proteins using a recombinant human protein microarray. C,D) Representative images of the protein array showing positive control (green arrows) and negative control (blue arrows) spots, as well as the spot corresponding to BCL‐10 (yellow arrow). E) An enlarged image displaying the binding of Bio‐Emodin to the BCL‐10 spot on the protein array, indicating SNR. F) SPRi fitting curve for the interaction between Emodin and BCL‐10. G. Pull‐down experiments showing the interaction of BCL‐10 protein with Emodin in liver tissues from CLP sepsis mice. H,I) Molecular docking images of Emodin with BCL‐10, including the structural diagram of the lowest binding energy conformation (H) and a representation of the key interacting residues (I).

Figure 6

Single‐cell sequencing showed that Emodin targets BCL‐10 to inhibit NETs formation. A) Schematic diagram of the single‐cell sequencing process. B) Visualization of high‐dimensional data of whole blood samples from different experimental groups (sham group, CLP model group, and Emodin intervention group) using UMAP technology. C) KEGG analysis displaying the enrichment signaling pathways of upregulated genes in the CLP model group. D) UMAP plots of the NETs signaling pathway in the sham group, CLP model group, and Emodin intervention group. E–G) UMAP plots showing the distribution of CAMP (E), HMGB1 (F), and PAD4 (G) in the Sham group, CLP model group, and Emodin intervention group. H,I) UMAP visualization of the distribution and enrichment of BCL‐10 in all cells (H) and neutrophils (I).

Figure 7

The absence of BCL‐10 alleviated organ damage and NETs formation induced by CLP sepsis. A) Schematic representation of the construction, modeling, and grouping of BCL‐10^f/f and BCL‐10^−/− mice. B) Kaplan–Meier survival curves over a 7‐day period comparing the survival rates among BCL‐10^f/f CLP, BCL‐10^f/f sham, BCL‐10^−/− CLP, and BCL‐10^−/− sham groups. The survival curves of the BCL‐10^f/f sham group and BCL‐10^−/− sham group exhibit complete overlap due to 100% survival rates in both cohorts. C‐G. Histopathological images of the liver, lungs, heart, spleen, and kidneys from BCL‐10^f/f CLP, BCL‐10^f/f sham, BCL‐10^−/− CLP, and BCL‐10^−/− sham groups. H) Expression levels of multiple inflammatory cytokines and chemokines in BCL‐10^f/f CLP and BCL‐10^−/− CLP groups. I,J) IF images showing CitH3 in the liver (I) and lung (J) tissues of BCL‐10^f/f CLP, BCL‐10^f/f sham, BCL‐10^−/− CLP, and BCL‐10^−/− sham groups. G1, and G2 represent the BCL‐10^−/− CLP group and BCL‐10^f/f CLP group.

Figure 8

Single‐cell sequencing showed that the absence of BCL‐10 reduces neutrophil enrichment and NETs formation in CLP sepsis mice. A) UMAP visualization of whole blood samples from BCL‐10^f/f CLP and BCL‐10^−/− CLP mice. B) Bar graph showing the proportions of different cell types in the whole blood samples of BCL‐10^f/f CLP and BCL‐10^−/− CLP mice. C) KEGG analysis displaying the enrichment signaling pathways of upregulated genes in the BCL‐10^f/f CLP model group compared to the BCL‐10^−/− CLP group. D–F) UMAP plots showing the expression of CAMP (D), HMGB1 (E), and PAD4 (F) in the BCL‐10^f/f CLP and BCL‐10^−/− CLP groups.

Figure 9

Emodin regulated NF‐κB activation and inhibits NETs formation by affecting the function of the BCL‐10/MALT1 complex. A) Heatmap of regulon activity in neutrophils from the Sham group, CLP model group, and Emodin intervention group, analyzed using SCENIC software. B) WB analysis of the phosphorylation levels of NF‐κB p65, IκBα, and IKKα/β in liver tissues from the Sham group, CLP model group, and Emodin intervention group at different time points (24 h/48 h/72 h). C,D) WB analysis of BCL‐10 protein and MALT1 expression levels in liver tissues from the Sham group, CLP model group, and Emodin intervention group at different time points (24 h/48 h/72 h). E) IF imaging showing the expression of the BCL‐10/MALT1 complex in liver tissues from different mouse groups. F) Heatmap of regulon activity in neutrophils from BCL‐10^f/f CLP and BCL‐10^−/− CLP groups, analyzed using SCENIC software. G) WB analysis of p‐p65, MPO, and NE expression levels in liver tissues from BCL‐10^f/f and BCL‐10^−/− mice across different groups. G1, G2, and G3 represent the sham group, CLP model group, and Emodin intervention group, respectively.