Targeting SIRT1 stability: arctiin ameliorates doxorubicin-induced cardiac injury by inhibiting SMURF2 binding and enhancing protective lipid metabolism

Figures

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  FIGURE 1. Arctiin prevented deterioration of cardiac function from DOX-induced injury. (A) Changes in body weight of four groups (n = 6). (B–D) Cardiac echocardiographic examination and analysis (n = 6). (E,F) H&E staining of heart and ratio of HW/TL on 7th day after exposure of DOX (n = 6). (G,H) mRNA level of Nppa and Nppb of four groups (n = 3). (I-K) Contents of cTnI, CK-MB, and LDH in serum of four groups (n = 6). Data were presented as mean ±S.D.

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  FIGURE 2. Arctiin alleviated oxidative injury lured by DOX in mice. (A–C) Representative western blot images and statistical results (n = 3). (D–F) D represents 4-HNE and DHE staining of hearts, E and F represent statistical results of of two kinds of staining respectively (n = 3). (G–K) The contents of GSH level, CAT activity, NADPH oxidase activity, SOD activity, and MDA in hearts (n = 6). (L) Representative of mRNA level of oxidative stress related genes (n = 3). Data were presented as mean ±S.D.

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  FIGURE 3. Arctiin suppressed endoplasmic reticulum stress after DOX hit in mice. (A) Representative western blot images and (B–G) statistical results of proteins related to ER stress, including Glucose-regulated protein-78 (GRP78), X-box binding protein-1 (XBP1), Phosphorylated-eukaryotic translation initiation factor-2 (p-eIF2), C/EBP homologous protein (CHOP), Activating transcription factor 6 (ATF6), and Caspase-12 (n = 3). Data were presented as mean ±S.D.

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  FIGURE 4. Arctiin attenuated DOX-induced cardiomyocyte apoptosis in mice. (A–E) Representative western blot images and Statistical results of proteins related to apoptosis, including P53, BAX, BCL2, and c-Caspase3 (n = 3). (F) Represent TUNEL staining and (G) quantitative statistical result (n = 3). White arrows represent TUNEL-positive cells. Data were presented as mean ±S.D.

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  FIGURE 5. Arctiin could attenuate DOX-induced cardiomyocytes injury in vitro. (A) Representative western blot images and (B–G) statistical results of proteins related to oxidative stress, endoplasmic reticulum stress, and apoptosis (n = 3). (H,I) The contents of SOD activity and MDA in hearts (n = 6). (J–L) Represent DCFH-DA staining, TUNEL staining and their quantitative statistical results (n = 3). White arrows represent TUNEL-positive cells. Data were presented as mean ±S.D.

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  FIGURE 6. Arctiin could directly bind to and stabilize SIRT1. (A,B) Representative Western blot and quantification of SIRT1 in cells treated with vehicle or Arctiin under PBS or DOX conditions (n-3). (C) Predicted binding mode of Arctiin in SIRT1. (D) Backbone RMSD of protein, ligand and complex. (E) Per-residue RMSF of the complex. (F) Radius of gyration (total and along X/Y/Z-axes). (G) Time evolution of solvent accessible surface area (SASA). (H) Number of protein–ligand hydrogen bonds over time. (I) 2D Gibbs free-energy landscape along PC1 and PC2. (J) 3D free-energy surface as a function of RMSD and Rg. (K) Per-residue free-energy contributions identifying key hot-spot residues. (L) Decomposition of binding free energy into MM, VDW, PB, SA, COU and total ΔG terms. (M) Time evolution of SIRT1 secondary-structure elements during the MD simulation. (N) Electrostatic potential surface (APBS; red, negative; blue, positive) highlighting the Arctiin-binding groove. (O) SIRT1 structure colored by B-factor/RMSF (blue to red from low to high flexibility). (P) Representative snapshots of the Arctiin–SIRT1 complex at 0, 50 and 100 ns. (Q) SIRT1 was incubated with biotin-Arctiin (10 μM) with or…

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  FIGURE 7. Arctiin inhibits SMURF2-mediated ubiquitination and degradation of SIRT1 (A) mRNA level of Sirt1 in DOX or Arctiin treated H9C2 cell (n = 3). (B) Western blots and (C) statistical result of SIRT1 in H9C2 cells pretreated with arctiin or vehicle, exposed to doxorubicin (DOX), and treated with cycloheximide or MG-132 (n = 3). (D) Western blots and (E) statistical result of SIRT1 in H9C2 cells pretreated with arctiin or vehicle, exposed to DOX, and treated with bafilomycin A1 (n = 3). (F) Co-IP assay and Western blot analysis of SIRT1 ubiquitination levels in H9C2 cells (n = 3). (G) Co-IP assay and Western blot analysis of the interaction between SIRT1 and SMURF2 in H9C2 cells (n = 3). Data were presented as mean ±S.D.

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  FIGURE 8. SIRT1 was responsible for Arctiin-mediated beneficial effect in DOX insult. (A) Western blot image of SIRT1 in H9C2 cells and (B) statistical result (n = 3). (C,D) Represent DCFH-DA staining and quantitative statistical result (n = 3). (E) Western blot images of endoplasmic reticulum stress-related proteins and (F,G) statistical result in H9C2 cells (n = 3). (H,I) Represent TUNEL staining and quantitative statistical result in H9C2 cells (n = 3). (J–L) Western blot images of downstream signaling proteins of SIRT-1 in H9C2 cells and their statistical results (n = 3). White arrows represent TUNEL-positive cells. Data were presented as mean ±S.D.

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  FIGURE 9. Knockout SIRT1 in cardiomyocytes blunted the beneficial effect of Arctiin in mice. (A) Western blot images and (B) statistical results of SIRT-1 in mice with different gene types (n = 3). (C,D) mRNA level of Nppa and Nppb of six groups (n = 3). (E–G) The ratio of HW/TL, EF, and FS of six groups (n = 6). (H–J) Contents of cTnI, CK-MB, and LDH in serum of six groups (n = 6). (K–M) Western blot images of endoplasmic reticulum stress-related proteins and partial statistical results in mice (n = 3). (N) 4-HNE staining and (P) TUNEL staining and (O,Q) statistical results (n = 3). (R–T) Western blot images of downstream signaling proteins of SIRT1 in mice and their statistical results (n = 3). White arrows represent TUNEL-positive cells. Data were presented as mean ±S.D.

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  FIGURE 10. SIRT1-dependent lipid metabolic remodeling by Arctiin after DOX injury. (A) PCA score plot shows distinct clustering of the indicated groups, reflecting divergent lipid profiles (n = 3). (B) Heatmap analysis indicates that the protective lipid remodeling by Arctiin requires SIRT1, as it is lost in Sirt1-cKO mice (n = 3).