Leonurine Ameliorates Doxorubicin‐Induced Cardiotoxicity via STING/NF‐κB/NLRP3 Inflammasome Signaling Pathway

Figures

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  FIGURE 1. LEO ameliorates cardiac function in DIC mice. (A) Evaluation of cardiac function (n = 10). (B) Gross view of the heart, WGA, HE, MASSON staining, as well as immunohistochemistry for CD68, F4/80, COL‐I, and α‐SMA (n = 3). (C) Establishment of the model and treatment regimen. (D‐H) Body weight (BW)‐time curve, heart mass (HW), general heart area, heart mass‐to‐body mass ratio (HW/BW), and heart mass‐to‐tibia length ratio (HW/TL) in mice (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 2. LEO downregulates the cGAS/STING/NF‐κB/NLRP3 pathway in cardiac endothelial cells of DIC mice. (A) Volcano plot of differentially expressed genes between the DOX group and the LEO group (n = 3). (B) KEGG enrichment analysis of differentially expressed genes between the DOX group and the LEO group (n = 3). (C & D) Protein expression levels of cGAS, p‐STING/STING, p‐TBK1/TBK1, p‐NF‐κB/NF‐κB, NLRP3, and GSDMD‐FL/GSDMD‐NT in the hearts of mice (n = 3). (E) Immunofluorescence and statistical analysis of P‐STING and CD31 in the hearts of DIC mice (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 3. LEO ameliorates DOX‐mediated cardiac vascular endothelial cell (CMVEC) injury and downregulates the cGAS/STING/NF‐κB/NLRP3 pathway. (A) IC50 of LEO on CMVEC. (B) Effects of LEO on CMVEC viability (n = 3). (C) Wound healing assay of CMVEC treated with LEO (n = 3). (D) Tube formation assay of CMVEC treated with LEO. (E) TUNEL staining and statistical analysis. (F) Annexin V‐FITC/PI flow cytometry analysis of CMVEC treated with LEO. (G, H) WB experiments and statistical analysis of cGAS, p‐STING/STING, p‐TBK1/TBK1, p‐NF‐κB/NF‐κB, NLRP3, and GSDMD‐FL/GSDMD‐NT. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 4. LEO binds to the STING protein via the TYR261 amino acid residue. (A) 3D conformational diagram of molecular docking between LEO and human STING protein, along with the binding affinity of relevant residues. (B) CETSA results for LEO binding to STING. (C) SPR results for LEO binding to human STING protein. (D) Molecular dynamics simulation of Ligand RMSD for LEO and the STING protein mutant. (E) Molecular dynamics simulation of the RoG for LEO and the STING protein mutant. (F) Molecular dynamics simulation of MM‐GBSA for LEO and the STING protein mutant. (G) 3D diagram of molecular docking between STINGTYR261A and LEO. (H) Molecular dynamics simulation of RMSD for STINGTYR261A and LEO. (I) Molecular dynamics simulation of RMSF (Root Mean Square Fluctuation) for STINGTYR261A and LEO. (J) Molecular dynamics simulation of MM‐GBSA for STINGTYR261A and LEO. (K) Amino acid sequence alignment of the STING TYR261 site across multiple species. (L) CO‐IP results for Bio‐LEO with STINGWT/STINGTYR261A. (M) Pull‐down assay results for Bio‐LEO with human STING/STINGTYR261A protein. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 5. LEO inhibits the oligomerization of the STING protein and the formation of the STING/TBK1 protein heterodimer. (A) Position of TYR‐261 in the STING dimer. (B) In vitro and in vivo WB experiments examining the effect of LEO on the oligomerization of STING/STINGTYR261A. (C) Molecular dynamics simulation of RMSD for LEO and the human STING protein. (D) Molecular dynamics simulation of RMSF for LEO and the human STING protein. (E) Molecular dynamics simulation of ROG for LEO and the human STING protein. (F, G) Molecular dynamics simulation of MM‐GBSA for LEO and the human STING protein. (H) Molecular dynamics simulation of RMSD for LEO and the STING/TBK1 dimer. (I) 3D diagrams of molecular docking between LEO and the STING/TBK1 dimer. A: Native docking structure of wild‐type STING and TBK1; B: Docking structure of wild‐type STING and TBK1 in the presence of LEO; C: Docking structure of STINGTYR261A and wild‐type TBK1 in the presence of LEO. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 6. LEO reduces the 2',3'‐cGAMP‐induced specific activation of the STING/NLRP3 signaling pathway in CMVEC. (A) Levels of IL18, IL1β, TNFα, ICAM‐1, ICAM‐2, and CASP1 in the culture supernatant (n = 10). (B) TUNEL staining (n = 3). (C–E) Immunofluorescence staining of STING, TBK1, co‐localization of STING/TBK1, NLRP3, CASP1, co‐localization of NLRP3/CASP1, and NF‐κB (n = 3). (F) Statistical analysis of IF and Tunel staining results (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 7. LEO inhibits mitochondrial dysfunction in cardiomyocytes induced by pyroptosis‐derived EVs from endothelial cells via targeting the STING/GSDMD pathway. (A) Transmission electron microscopy (TEM) results of CMVEC. Arrows indicate cellular holes or membrane defects (n = 3). (B) TEM examination of EVs in the supernatant of CMVEC. (C) NTA of vesicles in the supernatant of CMVEC. (D) Detection of the effects of vesicle transplantation on cardiomyocytes by ROS assay (n = 3). (E) PCR analysis of PINK, PARKIN, DRP1, MT‐CO1, MT‐CO2, and MT‐ATP6 levels in cardiomyocytes (n = 3). (F–H) JC‐1 staining, Mitotracker staining, and statistical analysis of cardiomyocytes (n = 3). (I) Detection of ECAR, OCR, ATP/ADP ratio, NAD+/NADH ratio, and mitochondrial respiratory chain complex (I‐V) activity in cardiomyocytes (n = 10). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 8. STING‐KO improves cardiac function in DIC mice and shows no additive effect with LEO treatment. (A) Assessment of cardiac function in mice. (B) Levels of CKMB, LDH, Myo and cTNT in mouse serum. (C) Gross observation of mouse hearts, along with WGA staining, HE staining, MASSON staining, and immunohistochemical examination. (D) Measurement of HW/BW, cardiomyocyte area, cardiac inflammation, and fibrosis levels. (E) Levels of serum inflammatory factors and vascular endothelial damage factors. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.01 and ns: Not significant.

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  FIGURE 9. Schematic Diagram of the Mechanism of LEO in Treating DIC.