Bisdemethoxycurcumin attenuates myocardial fibrosis in heart failure with preserved ejection fraction by targeting TGFBR1 and oxidative stress

Figures

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  Fig. 1. Flow chart of pharmacological targets of BDMC in the treatment of HFpEF based on network pharmacological method and proteomics (BDMC: Bisdemethoxycurcumin, HFpEF: Heart Failure with Preserved Ejection Fraction).

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  Fig. 2. BDMC exerts protective effects on HFpEF in vivo. A. Chemical structure of BDMC. B. HFpEF mice was established by administrating high fat diet (HFD) and L-NAME for 16 weeks on male C57BL/6 J mice. BDMC was administered after HFpEF for 4 weeks. C. Running distance of mice after admistrating BDMC. n = 6. D-G. Impact of BDMC on cardiac left ventricular ejection fraction (LVEF) (D, E), E/E’ (F) and Interventricular Septal Diameter (IVS, D) (G). n = 6. H. Heart weight to Tibia length ratio (HW/Tibia) of mice after admistrating BDMC. n = 6. I. Lung Weight (LW) wet/dry of mice after admistrating BDMC. n = 6. (CTL:Control group).

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  Fig. 3. BDMC exerts anti-fibrotic effect in vivo. A. Representative gross images of whole hearts, staining with H&E, Masson, wheat germ agglutinin (WGA) of mice after admistrating BDMC. B. Quantification of fibrosis area percent of different group. n = 6. C. Mean cross-sectional area of different group. n = 6. D. qPCR analysis of fibrosis-related genes Acta2, Col3a1, Postn, Fn1 in vivo in the indicated groups. E-F. Systolic Blood Pressure (SBP) (E) and Diastolic Blood Pressure (DBP) (F) of mice after admistrating BDMC. n = 6. (CTL:Control group).

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  Fig. 4. BDMC exerts anti-fibrotic effect in iCFs in vitro. A. Impact of BDMC on TGF-β induced stimulation of immortalized cardiac fibroblasts (iCF) was tested in vitro. B. qPCR analysis of fibrosis-related genes Acta2, Col3a1, Postn, Fn1 in vitro in the indicated groups. C. Representative images of α-smooth muscle actin (α-SMA) immunofluorescence of iCF in the indicated groups. D. Expression of α-smooth muscle actin (α-SMA) of iCF in the indicated groups. n = 6. E. Representative images of EDU assay of iCF in the indicated groups. F. Quantification of EDU positive percent in the indicated groups. (CTL:Control group).

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  Fig. 5. Identification of therapeutic targets for BDMC against HFpEF. A.Venn diagram of potential therapeutic targets. B. Classification of the identified targets. 49 therapeutic targets for BDMC against HFpEF can be obtained by the intersection of 3066 drug targets and 1507 HFPEF-related targets and classified into 6 functional categories. C. Diseases-targets-functional annotations-signaling pathways network. GO analysis results are on the left. The dark green quadrangles represent cellular components. The blue quadrilateral represents biological processes. The pink quadrilateral represents the function of the molecule. The yellow circle in the middle indicates targets for BDMC against HFpEF. The central red quadrilateral represents the enriched KEGG pathway. Disease enrichment analysis is represented by the blue diamond on the right. (GO: Gene ontology, KEGG: Kyoto Encyclopedia of Genes Genomics). D. mRNA interaction network of therapeutic targets for BDMC against HFpEF. E. Protein–protein interaction network of therapeutic targets for BDMC against HFpEF. F. Target molecules located on the cell membrane. G. Molecular docking binding energy of BDMC with target proteins. H. Relative mRNA…

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  Fig. 6. Integrating proteomics analysis and network pharmacology analysis of BDMC against HFpEF. A. Heatmap of all the protein expression in the three groups. B. Volcano plots of the enriched proteins. C. Venn diagram of the integrated proteins of proteomics analysis and network pharmacology analysis. D. Heatmap of the enriched different expression proteins of up-regulated in HFpEF and down-regulated in HFpEF-BDMC compared with HFpEF. E. Enriched differential bubble chart of proteins in Figure D.

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  Fig. 7. Expression of TGF-β/SMAD pathway-related fibrotic proteins in BDMC treated HFpEF. A. Protein bands of TGFBR1, p-TGFBR1, COL3A1, PCNA, MMP9, SMAD3, p-SMAD2/3 in the indicated groups. B. Relative protein expression of targets in different treatment groups of mice. n = 3. C. Protein bands of TGFBR1, p-TGFBR1, COL3A1, PCNA, MMP9, SMAD3, p-SMAD2/3 in the indicated groups. D. Relative protein expression of targets in different treatment groups of mice. n = 3. E. Immunofluorescence Staining of TGF-β, TGFBR1, DAPI in the indicated groups. F. The 3-dimensional map of the binding sites between BDMC with TGFBR1. G. Molecular dynamics (MD) simulation of BDMC binding to TGFBR1. H. Root Mean Square Deviation (RMSD) of BDMC and TGFBR1. I. Root Mean Square Fluctuation (RMSF) of BDMC and TGFBR1.

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  Fig. 8. BDMC inhibited mitochondrial-related oxidative stress in vivo and in vitro. A. Gene Set Enrichment Analysis (GSEA) of HFpEF-BDMC (indicated PC4-HFpEF). B. ROS production was quantified by DCFH-DA in vitro. n = 6. C. ATP content in the indicated groups. n = 6. D. Representative transmission electron microscopy (TEM) images. n = 6. E. Mitochondrial length (major axis) in the indicated groups. n = 6. F. Representative mitochondrial immunofluorescence using Mitotracker (red). n = 6. G. Mitochondrial volume (fold over CTL) in the indicated groups. n = 6. H. Number of mitochondrial/cell in the indicated groups. n = 6. I. Representative images of mitoSOX assay of iCF in the indicated groups. n = 6. J. Quantification of mitoSOX positive percent in the indicated groups. n = 6. K. Representative protein bands of MFN1, MFN2, DRP1, TOMM20 and ACTIN in the indicated groups. L. Relative protein expression of targets in different treatment groups. n = 3.