Hesperetin, a Promising Dietary Supplement for Preventing the Development of Calcific Aortic Valve Disease

Figures

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  Figure 1. Hesperetin supplementation protects the mice from wire-injury-induced aortic valve stenosis. (A) mice treated with direct wire injury or sham operation were administered vehicle (CMC-Na) or hesperetin daily with oral gavage for 7 days before wire injury surgery and sacrificed on the 60th day; (B) representative echocardiogram of aortic valve peak velocity; (C) hematoxylin and eosin staining (HE) of aortic valve leaflets; scale bar = 200 μm; n = 5; (D) the Runx2 protein levels (red) in mouse aortic valves were detected by immunofluorescence staining, nuclear staining was performed with Hoechst (blue); scale bar = 100 μm; n = 5; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

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  Figure 2. Hesperetin supplementation alleviates inflammation caused by DWI in vivo or lipopolysaccharide (LPS)-induced inflammation in vitro. (A) KEGG analysis showing that the effect of hesperetin is related to the NF-κB signaling pathway; (B) the ICAM-1 protein levels (red) in mouse aortic valves were detected by immunofluorescence staining, nuclear staining was performed with Hoechst (blue), and WGA (green) was used to show the morphology of the valve leaflet; scale bar = 50 μm; n = 5; (C) molecular structure of hesperetin; (D) cell viability after treatment with different concentrations of hesperetin for 24 h (0–50 μM), n = 5; (E) immunofluorescence staining was performed to confirm that hesperetin inhibits the LPS-induced cell nuclear translocation of P65 (green), nuclear staining was performed with DAPI (blue), and Vimentin (green) was used to show the morphology of the VICs; scale bar = 50 μm; n = 5; (F) VICs were pretreated with or without 3 μM hesperetin for 24 h and then stimulated with or without LPS for 24 h, and the protein expression levels of ICAM-1, MCP-1, and p-p65 were determined by Western blotting; n = 5; (G) the levels of IL-6 and RANTES were detected by using ELISA, n…

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  Figure 3. Hesperetin supplementation alleviates LPS-induced apoptosis in VICs and DWI-induced apoptosis in vivo. (A) GEO analysis determined that hesperetin exerts its protective effects by inhibiting cell apoptosis; (B) the cleaved-caspase3 protein levels (red) in mouse aortic valves were determined by immunofluorescence; scale bar = 50 μm; n = 5; (C) immunoblotting images images of cleave-caspase3 and BAX in human aortic VICs subjected to different treatments: control, LPS, and LPS + Hst and their corresponding quantification; n = 5; (D) immunofluorescence staining showed that pretreatment with hesperetin depressed the LPS-induced protein levels of cleaved-caspase3 (green) in VICs; nuclear staining was performed with DAPI (blue), and Vimentin (red) was used to show the morphology of the VICs; scale bar = 100 μm; n = 5; (E) TUNEL (red) staining of VICs under LPS or LPS + Hst stimulation; scale bar = 100 μm; n = 5; (F) the cell apoptosis of VICs was tested via flow cytometry. n = 5; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

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  Figure 4. Hesperetin supplementation alleviates LPS-induced OS in VICs or DWI-induced apoptosis in vivo, and LPS-induced inflammation and apoptosis in VICs are dependent on ROS. (A) the HO-1 protein levels (red) in mouse aortic valves were detected by immunofluorescence staining, nuclear staining was performed with Hoechst (blue), and WGA (green) was used to show the morphology of the valve leaflet; scale bar = 50 μm; n = 5; (B) the reactive oxygen species (ROS) levels (green) in mouse aortic valves were detected with immunofluorescence, and nuclear staining was performed with Hoechst (blue); scale bar = 50 μm; n = 5; (C,D) fluorescence images of VICs stained with JC-1 and TMRM; scale bar = 100 μm; n= 5; (E) the levels of NQO1 and HO-1 were determined by immunoblotting; n = 5; (F) representative images show that NAC treatment reduced the ICAM-1, MCP-1, BAX, and cleaved-caspase3 levels in VICs, n = 5; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

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  Figure 5. Hesperetin supplementation upregulates the Nrf2–ARE signaling. (A,B) human aortic VICs were divided into three treatment groups: control, LPS, and LPS + Hst. The Nrf2 protein levels (green) in human aortic VICs were determined via immunofluorescence, nuclear staining was performed with DAPI (blue), and Vimentin (red) was used to show the morphology of the VICs; scale bar = 75 μm; n = 5; and immunoblotting, n = 5; (C) representative images show the extranuclear localization of Nrf2 (red) in VICs stimulated with ML385 and the intranuclear localization of Nrf2 after human VICs were treated with hesperetin; scale bar = 20 μm; (D–F) representative immunoblotting images and quantification of the levels of p-Nrf2, NQO1, HO-1, MCP1, and BAX in VICs treated or not treated with ML385 and Hst (n = 5); * indicates p < 0.05, ** indicates p < 0.01, *** p< 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

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  Figure 6. Sirt7 is involved in the protective effects of hesperetin in VICs. (A) the protein expression of Sirt7 (red) in CAVD patients and non-CAVD patients was detected via immunofluorescence, nuclear staining was performed with Hoechst (blue), and WGA (green) was used to show the morphology of the valve leaflet; scale bar = 100 μm; n = 3; (B) representative immunoblots and corresponding quantification of Sirt7 in human aortic VICs, n = 3; (C) docking pose of hesperetin (carbon atoms in green) into the human Sirt7 binding pocket. The key residues are displayed as sticks and colored gray. Hydrogen bonds are displayed with red dashed lines; (D) VICs were cultured under three different conditions (control, LPS, and LPS + Hst), and the expression of Sirt7 was then detected by Western blotting. * indicates p < 0.05, ** indicates p < 0.01, *** p < 0.001, **** indicates p < 0.0001 and ns indicates p > 0.05.

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  Figure 7. Sirt7 inhibits inflammatory responses and apoptosis by activating Nrf2 in VICs. (A) protein levels of Sirt7 and Nrf2 in VICs transfected with siRNA targeting Sirt7; n = 5; (B,C) the protein levels of cleaved-caspase3, BAX, MCP-1, and ICAM-1 in LPS and hesperetin-supplemented human VICs in which Sirt7 was knocked down were measured by Western blotting. A scrambled siRNA was transfected in VICs as a control; n = 3; (D) immunofluorescence staining confirmed that hesperetin inhibited the LPS-induced cell nuclear translocation of P65 (red) and that the knockdown of Sirt7 abolished this effect, nuclear staining was performed with DAPI (blue), and Vimentin (green) was used to show the morphology of the VICs; scale bar = 20 μm; (E) the apoptosis of VICs was tested via flow cytometry; n = 3; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

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  Figure 8. Sirt7 inhibits oxidative response by activating Nrf2 in VICs. (A) the protein levels of HO-1 and NQO-1 in LPS and hesperetin supplemented human VICs in which Sirt7 was knocked down were measured by Western blotting. A scrambled siRNA was transfected in VICs as a control; n = 3; (B) DCFH-DA (green) staning showed ROS production, n = 4; scale bar = 50 μm; (C) TMRM (red) staining showed MCMP function, n = 4; scale bar = 50 μm; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates < 0.0001, and ns indicates p > 0.05.