Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88‐Dependent Inflammation

Figures

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  Figure 1. Sch B reduces HG‐induced cardiomyocyte hypertrophy and fibrotic responses. A) The chemical structure of Schisandrin B. B) Immunoblot analysis for β‐MyHc, Col‐1, and TGF‐β1 in H9C2 cells exposed to 33 × 10−3 m glucose (HG). Cells were pretreated with Sch B at indicated concentrations for 1 h and then stimulated with HG for 24 or 36 h. GAPDH was used as loading control. C) Immunoblot analysis for fibrosis markers in primary cardiac fibroblasts and hypertrophy marker in primary cardiomyocytes. Cells were pretreated with Sch B for 1 h before exposure to HG for 24 or 36 h. GAPHD was used as control. D,E) Rhodamine phalloidin staining of D) H9C2 and E) primary cardiomyocytes, showing the effect of Sch B on HG‐induced hypertrophy. Cells were treated as indicated for panel (B). Cells were counterstained with DAPI. Right panel shows the quantification of cell size, respectively [scale bar = 50 µm]. F,G) qPCR analysis of hypertrophic/fibrotic response genes in F) H9C2 cells and G) primary cardiomyocytes. Cells were pretreated with Sch B for 1 h and then stimulated with HG for 12 h. Levels of Col1a1, Tgfb1, Myh6, Anp, and Ska were normalized to Actb. Data in panels (D)–(G) shown as mean ±…

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  Figure 2. Sch B regulates the expression of inflammatory cytokines in H9C2 cells. A,B) H9C2 cells were pretreated with 10 × 10−6 m Sch B for 1 h before exposure to HG for 6 h. RNA sequencing was carried out and differentially expressed genes were identified by DESeq 2. Panel (A) shows the Venn diagram of differentially expressed genes between control versus HG and HG versus HG+Sch B. WikiPathway enrich analysis of overlap of differentially expressed genes in plane B. The inflammatory response pathway is highlighted by red. C) Schematic illustrating HG‐induced innate toll‐like receptor‐4 (TLR4) signaling leading to the induction of inflammatory factors. D) Heat‐map of inflammation cytokines detected by qPCR assay. H9C2 cells were pretreated with 10 × 10−6 m Sch B for 1 h and then exposed to HG for 8 h. Left panel shows inflammation cytokines that were suppressed by Sch B pretreatment. Right panel shows factors that were not suppressed by Sch B. E) qPCR verification of classical TLR4 proinflammatory cytokines (TNF‐α and IL6) and type 1 interferons (IFN‐α and ‐β) in H9C2 cells pretreated with Sch B before HG exposure. Cells were treated as indicated for panel (D). F) NF‐κB and IRES reporter…

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  Figure 3. Sch B exhibits differential regulation of TLR4/TRIF and TLR4/MyD88 pathways. A) Immunofluorescence staining of H9C2 cells for p65 subunit of NF‐κB (red). Cells were pretreated with 10 × 10−6 m Sch B for 1 h and then exposed to HG for 1 h. Cells were counterstained with DAPI (blue) (scale bar = 100 µm). B) Immunoblot analysis of IκBα in total cell lysates and p65 in nuclear fractions prepared from H9C2 cells. Cells were treated as indicated for panel (A). GAPDH was used as control for total cell lysates and lamin B for nuclear proteins. C) Western blot analysis of MAPK activation in H9C2 cells. Cells were treated as in panel (B). Total cell lysates were probed for phosphorylated (p‐) and total ERK, JNK, and p38. D) Levels of MyD88 pathway protein TAK1 in H9C2 cells exposed to HG. Cells were treated as indicated for panel (B). Upper blots showing H9C2 cells and lower blots showing primary cardiomyocytes. Densitometric quantification of blots is shown in right panel. E) Analysis of TRIF pathway showing levels of TBK1 and IRF3. H9C2 (upper blots) and primary cardiomyocytes (lower blots) were treated as indicated for panel (B). Densitometric quantification is shown on right. F)…

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  Figure 4. Sch B directly binds to MyD88. A) Western blot analysis of MyD88 knockdown in H9C2 cells. Cells were transfected with negative control siRNA (NC) or siRNA against MyD88 (siMyD88). GAPDH was used as loading control. (Values represent the mean ± SEM; n = 3; ***p < 0.001 compared to NC by unpaired two‐tailed Student's t‐test). B) qPCR analysis of inflammatory cytokines in H9C2 cells transfected with MyD88 siRNA. Transfected cells were treated with 10 × 10−6 m Sch B for 1 h before exposure to HG for 8 h. Untransfected (Ctrl) and cells transfected with negative control siRNA (NC) were used as control (values represent mean ± SEM; n = 3; ns = not significant; *p < 0.05; ***p < 0.001 compared to control, and #p < 0.05 compared to HG+siNC by unpaired two‐tailed Student's t‐test). C) H9C2 cells were transfected with Flag‐ and HA‐tagged MyD88. Cells were treated with 10 × 10−6 m Sch B for 1 h and then exposed to HG for 30 min. Flag was immunoprecipitated and HA was detected to examine MyD88 dimerization. D) Binding of biotinylated‐Sch B to MyD88 was determined by immunoblotting. Bio‐Sch B was added to streptavidin‐agarose beads. Untreated beads, unconjugated Sch B, and biotin alone were…

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  Figure 5. Sch B suppresses cardiac hypertrophy and fibrosis in db/db mice. A) Body weight of mice at time points indicated. B) Blood glucose levels in mice. C,D) Levels of serum C) LDH and D) CK‐MB in mice. E,F) Representative H&E‐stained images of mouse heart tissues showing E) transverse and F) longitudinal sections. Right panel showing quantification of cardiomyocyte size determined from transverse sections (scale bar = 50 µm). G) Representative images of Sirius Red staining of heart tissues. Quantification of collagen levels is shown on right (scale bar = 50 µm). H) Representative images of Masson's trichrome staining of heart tissues. Quantification of fibrosis is shown on right (scale bar = 50 µm). I) Immunoblotting of Col‐1, TGF‐β1, β‐MyHc in heart tissue. GAPDH was used as loading control. J) qPCR analysis of cardiac fibrosis and hypertrophy genes in heart tissues. mRNA levels were normalized to Actb. Data in all panels are shown as mean ± SEM (n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 compared to db/m and #p < 0.05, ##p < 0.01 compared to db/db by one‐way ANOVA followed by Bonferroni's multiple comparisons test).

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  Figure 6. Sch B reduces inflammatory responses in db/db mice by inhibiting MyD88. A) Co‐immunoprecipitation showing formation of MD2/TLR4, TLR4/MyD88, and TLR4/TRIF complexes in heart tissues of db/db mice. TLR4 was immunoprecipitated (IP) and levels of MD2, TRIF, and MyD88 were detected by immunoblotting (IB). Densitometric quantification is shown on right. B) Western blot analysis of pathway activation downstream of MyD88 in heart tissues. Phosphorylated TAK1, TBK1, and IRF3 were detected. Total proteins were used as control. Densitometric quantification shown on right. C) Analysis of MAPK and NF‐κB activation in heart tissues of db/db mice. Phosphorylated ERK, JNK, and p38 were detected. Total proteins were used as control. IκB‐α was used to measure NF‐κB activity, with GAPDH as the loading control. Right panel shows densitometric quantification. D) mRNA levels of Tnf, Il6, and Ifng in heart tissues of mice. Transcripts were normalized to Actb. E) Protein levels of IL‐6 and TNF‐α in serum of db/db mice were determined by enzyme‐linked immunoassay (ELISA). Data in all panels are shown as mean ± SEM (n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 compared to db/m; #p < 0.05, ##p <…

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  Figure 7. Sch B treatment suppresses cardiac injury in STZ‐induced model of type 1 diabetes. A,B) Representative images of A) Masson's trichrome and B) Sirius red staining of heart tissues (scale bar = 50 µm). C) mRNA levels of Cola1, Anp, and Ska in heart tissues. Transcripts were normalized to Actb. D) TLR4 was immunoprecipitated (IP) from mouse heart lysates and MD2, TRIF, and MyD88 were detected by immunoblotting (IB). Right panel shows the densitometric quantification. E) Western blot analysis of pathway activation downstream of MyD88 in heart tissues. Phosphorylated TAK1, TBK1, and IRF3 were detected. Total proteins were used as control. F) Analysis of MAPK and NF‐κB activation in heart tissues of STZ‐induced T1DM mice. Phosphorylated ERK, JNK, and p38 were detected. Total proteins were used as control. IκB‐α was used to measure NF‐κB activity, with GAPDH as the loading control. G) mRNA levels of Tnfa, Il6, and Ifng in heart tissues of mice. Transcripts were normalized to Actb. H) Protein levels of IL‐6 and TNF‐α in serum of mice were examined by ELISA. Data in panels (C), (D), (G), and (H) are shown as mean ± SEM (n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 compared to…

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  Figure 8. Cardiomyocyte‐specific MyD88 knockout prevents STZ‐induced cardiac inflammation and injury. Male MyD88f/f and MyD88f/fMyh6Cre mice were injected with STZ to induce T1DM. A) Fasting blood glucose and B) body weight were recorded weekly for 16 weeks. Heart tissue and blood samples were collected at termination for the determination of C) heart weight/tibia length (HW/TL) ratios, D) serum LDH, and E) serum CK‐MB activity. Representative images (400X magnification) of F) H&E staining (longitudinal and transverse), G) Sirius red and Masson's trichrome staining of the heart tissues were shown (scale bar = 50 µm). Representative H) western blot and I) qPCR analysis of MyHc, Col‐1, and TGF‐β in the heart tissue were shown. J) Levels of p‐ERK/ERK, p‐JNK/JNK, and IκB‐α were determined by western blot. GAPDH was loading control. K) qPCR analyses of Tnf, Il6, and Il1b in the heart tissues were shown. All data are shown as mean ± SEM (n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 compared to MyD88f/f; ns = not significant, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to MyD88f/f+T1DM by one‐way ANOVA followed by Bonferroni's multiple comparisons test).