Syringin exerts anti‐inflammatory and antioxidant effects by regulating SIRT1 signaling in rat and cell models of acute myocardial infarction

Figures

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  Figure 1. Effect of syringin administration of different concentrations in H2O2‐treated H9c2 cells. H9c2 cells were challenged with 200 μM H2O2 for 6 h, followed by coconditioning with syringin at the indicated concentrations. Cell viability was assessed by CCK‐8 assay. *p < .05, **p < .01, ***p < .001. CCK‐8, cell counting kit‐8.

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  Figure 2. Effect of syringin administration on SIRT1 and PGC‐1α expression and confirmation of SIRT1 silencing in H2O2‐treated H9c2 cells. H9c2 cells were transfected with shRNA‐control (siCtrl) or shRNA‐SIRT1 (siSIRT1) for 12 h and then challenged with 200 μM H2O2 for 6 h, followed by coconditioning with 3 μM syringin. (A, B) qPCR data show the mRNA expression levels of SIRT1 and PGC‐1α in different groups of H9c2 cells. (C) Western blot indicates the protein expression levels of SIRT1 and PGC‐1α in different groups of H9c2 cells. **p < .01, ***p < .001. PGC‐1α, peroxisome proliferator‐activated receptor gamma coactivator 1 alpha; qPCR, quantitative PCR; shRNA, short hairpin RNA; SIRT1, sirtuin 1.

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  Figure 3. Effect of syringin and SIRT1 silencing on cell viability and apoptotic signaling in H2O2‐treated H9c2 cells. H9c2 cells were transfected with shRNA‐control (siCtrl) or shRNA‐SIRT1 (siSIRT1) for 12 h and then challenged with 200 μM H2O2 for 6 h, followed by coconditioning with 3 μM syringin. (A) The viability of H9c2 cells administered different treatments was assessed by CCK‐8 assay. (B) Flow cytometry with annexin V‐FITC and PI staining was conducted to examine the percentage of early apoptotic cells. (C) Protein levels of Bax, as well as cleaved caspase 3 and caspase 8, were examined by WB. *p < .05, **p < .01. CCK‐8, cell counting kit‐8; shRNA, short hairpin RNA; SIRT1, sirtuin 1; WB, western blot.

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  Figure 4. Effect of syringin and SIRT1 silencing on inflammation in H2O2‐treated H9c2 cells. H9c2 cells were transfected with shRNA‐control (siCtrl) or shRNA‐SIRT1 (siSIRT1) for 12 h and then challenged with 200 μM H2O2 for 6 h, followed by coconditioning with 3 μM syringin. ELISA was performed to detect the levels of released (A) IL‐1β, (B) IL‐6, and (C) TNF‐α. (D) Western blot indicates the protein expression levels of TLR4 and NF‐κB and the phosphorylation levels of Jak1 and Stat3. *p < .05, **p < .01, ***p < .001. shRNA, short hairpin RNA; SIRT1, sirtuin 1; TNF, tumor necrosis factor.

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  Figure 5. Effect of syringin and SIRT1 silencing on oxidative stress and the antioxidant response in H2O2‐treated H9c2 cells. H9c2 cells were transfected with shRNA‐control (siCtrl) or shRNA‐SIRT1 (siSIRT1) for 12 h and then challenged with 200 μM H2O2 for 6 h, followed by coconditioning with 3 μM syringin. (A) DCFH‐DA staining was performed to measure the ROS production. (B, C) Activity of the antioxidative enzymes SOD and catalase was determined using the corresponding assay kits. (D) Protein levels of NRF2 and HO‐1 were detected by western blot. *p < .05, **p < .01. NRF2, nuclear erythroid‐related factor 2; ROS, reactive oxygen species; shRNA, short hairpin RNA; SIRT1, sirtuin 1; SOD, superoxide dismutase.

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  Figure 6. Syringin treatment protects against ischemia/reperfusion (I/R) injury in vivo. Syringin was administered intraperitoneally for 7 days in rats after I/R injury was induced. (A) Representative images of myocardial infarct in rats detected by triphenyltetrazolium chloride staining. The percentage of myocardial area affected by infarct is also shown. (B) Myocardial tissues of mice stained with Masson (×200). (C) Representative images of TUNEL staining and quantitative results. (D) Protein levels of Bax, as well as cleaved caspase 3 and caspase 8, were examined by WB. *p < .05, **p < .01. TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling; WB, western blot.

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  Figure 7. Syringin treatment upregulates myocardial Sirt1 and Pgc‐1α expression in ischemia/reperfusion (I/R) rats. Syringin was administered intraperitoneally for 7 days in rats after I/R injury was induced. (A, B) qPCR data show the mRNA expression levels of SIRT1 and Pgc‐1α in the homogenate of rat myocardial tissue. (C) Western blot shows the protein expression levels of SIRT1 and Pgc‐1α in the homogenate of rat myocardial tissue. *p < .05. PGC‐1α, peroxisome proliferator‐activated receptor gamma coactivator 1 alpha; qPCR, quantitative PCR; SIRT1, sirtuin 1.

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  Figure 8. Syringin treatment attenuates myocardial inflammation and oxidative stress in ischemia/reperfusion (I/R) rats. Syringin was administered intraperitoneally for 7 days in rats after I/R was induced. ELISA was performed to detect (A) IL‐1β, (B) IL‐6, and (C) TNF‐α levels in rat myocardial tissue homogenate. (D) Western blot (WB) indicates the protein expression levels of TLR4 and NF‐κB and the phosphorylation levels of Jak1 and Stat3. (E) DCFH‐DA staining was performed to measure ROS production in rat myocardium. (F, G) Activity of the antioxidative enzymes SOD and catalase was determined using the corresponding assay kits. (H) Protein levels of myocardial NRF2 and HO‐1 in rats were detected by WB. *p < .05, ***p < .001. NRF2, nuclear erythroid‐related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF, tumor necrosis factor.

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  Figure 9. Schematic diagram for the mechanism of action of syringin on MI. Syringin protects heart function via the SIRT1/PGC‐1α signaling pathway in ischemia/reperfusion rats and H2O2‐treated H9c2 cells. Syringin reduces MI injury and ameliorates apoptosis, inflammation, and OS. These effects in vitro were abrogated through the silencing of SIRT1. MI, myocardial infarction; OS, oxidative stress; PGC‐1α, peroxisome proliferator‐activated receptor gamma coactivator 1 alpha; SIRT1/PGC‐1α, sirtuin 1/peroxisome proliferator‐activated receptor gamma coactivator 1 alpha.