Vaccarin alleviates endothelial inflammatory injury in diabetes by mediating miR-570-3p/HDAC1 pathway

Abstract

Vaccarin is a flavonoid glycoside, which has a variety of pharmacological properties and plays a protective role in diabetes and its complications, but its mechanism is unclear. In this study, we aim to investigate whether histone deacetylase 1(HDAC1), a gene that plays a pivotal role in regulating eukaryotic gene expression, is the target of miR-570-3p in diabetic vascular endothelium, and the potential molecular mechanism of vaccarin regulating endothelial inflammatory injury through miR-570-3p/HDAC1 pathway. The HFD and streptozotocin (STZ) induced diabetes mice model, a classical type 2 diabetic model, was established. The aorta of diabetic mice displayed a decrease of miR-570-3p, the elevation of HDAC1, and inflammatory injury, which were alleviated by vaccarin. Next, we employed the role of vaccarin in regulating endothelial cells miR-570-3p and HDAC1 under hyperglycemia conditions in vitro. We discovered that overexpression of HDAC1 counteracted the inhibitory effect of vaccarin on inflammatory injury in human umbilical vein endothelial cells (HUVECs). Manipulation of miRNA levels in HUVECs was achieved by transfecting cells with miR-570-3p mimic and inhibitor. Overexpression of miR-570-3p could decrease the expression of downstream components of HDAC1 including TNF-α, IL-1β, and malondialdehyde, while increasing GSH-Px activity in HUVECs under hyperglycemic conditions. Nevertheless, such phenomenon was completely reversed by miR-570-3p inhibitor, and administration of miR-570-3p inhibitor could block the inhibition of vaccarin on HDAC1 and inflammatory injury. Luciferase reporter assay confirmed the 3'- UTR of the HDAC1 gene was a direct target of miR-570-3p. In summary, our findings suggest that vaccarin alleviates endothelial inflammatory injury in diabetes by mediating miR-570-3p/HDAC1 pathway. Our study provides a new pathogenic link between deregulation of miRNA expression in the vascular endothelium of diabetes and inflammatory injury and provides new ideas, insights, and choices for the scope of application and medicinal value of vaccarin and some potential biomarkers or targets in diabetic endothelial dysfunction and vascular complications.

Keywords: HDAC1; diabetes; endothelial dysfunction; inflammation; microRNA-570-3p; vaccarin.

FIGURE 1

Chemical structure of vaccarin.

FIGURE 2

Vaccarin can significantly improve the physiological state and blood glucose level of T2DM mice. (A). The weight of the mice at the terminal of the experiment. (The first 5 weeks were the modeling period and the last 6 weeks were the administration period.) (B). Weekly weight changes after the start of the experiment. (C). Fasting blood glucose. After 6 weeks of treatment, fasting blood glucose was tested in the mice. (D). Weekly changes of fasting blood glucose after successful modeling. (E). Oral glucose tolerance test (OGTT). (F). Area under the curve (AUC) level of OGTT. (G). Insulin tolerance test (ITT). (H). Area under the curve (AUC) level of ITT. (I–M). Content of serum TG, LDL, NEFA, ALT and AST. Values are mean ± SEM. n = 3-6 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. MOD.

FIGURE 3

Vaccarin improved aortic inflammatory injury in T2DM mice. (A). Representative photomicrographs of aortic tissue with H&E (200X, 1000X). (B–D). The effects of vaccarin on the TNF-α mRNA, IL-1β mRNA, and HDAC1 mRNA expression in aortic tissue in comparison with T2DM mice. (E–H). Western blotting analysis of HDAC1, TNF-α, and IL-1β protein expression levels in aortic tissue. Gray value of western blotting. (I). Representative photomicrographs of aortic tissue with immunohistochemical staining for TNF-α and IL-1β (200X). (J). Content of serum MDA. (K). Activity of serum GSH-Px. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. MOD.

FIGURE 4

Vaccarin attenuated HG-induced inflammatory injury in HUVEC cells. (A,B). The effects of vaccarin or metformin on the TNF-α mRNA and IL-1β mRNA expression in HG-induced HUVEC cells. (C–E). Western blotting analysis of TNF-α and IL-1β protein expression levels in HG-induced HUVEC cells. Gray value of western blotting. (F). Content of MDA. (G). Activity of GSH-Px. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. NG; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG.

FIGURE 5

Vaccarin alleviated HG-induced inflammatory injury in HUVEC cells by inhibiting HDAC1 expression. (A). The effects of vaccarin on HDAC1 mRNA expression in HG-induced HUVEC cells. (B,C). Western blotting analysis of HDAC1 protein expression levels in HG-induced HUVEC cells. Gray value of western blotting. (D–F). The expression of HDAC1 mRNA, TNF-α mRNA, and IL-1β mRNA in HUVEC cells transfected with siRNA NC or HDAC1 siRNA. (G–J). Western blotting analysis of HDAC1, TNF-α, and IL-1β protein expression levels in HUVEC cells transfected with siRNA NC or HDAC1 siRNA. Gray value of western blotting. (K). The expression of HDAC1 mRNA in HUVEC cells transfected with pcDNA3.1-HDAC1. (L). Western blotting analysis of Flag protein expression levels in HUVEC cells transfected with pcDNA3.1-HDAC1. (M,N). The effects of vaccarin on TNF-α mRNA and IL-1β mRNA expression in HUVEC cells transfected with pcDNA3.1 or pcDNA3.1-HDAC1. (O–Q). Western blotting analysis of TNF-α and IL-1β protein expression levels in HUVEC cells transfected with pcDNA3.1 or pcDNA3.1-HDAC1. Gray value of western blotting. (R). Content of MDA. (S). Activity of GSH-Px. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. NG; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG.

FIGURE 6

Vaccarin inhibited the HG-induced decrease of miR-570-3p in vitro. (A). Upset graph is used to represent overlapping sets of elements for miRNAs. (B–C). The effects of vaccarin on the expression of miR-570-3p miRNA in HG-induced HUVEC cells and aortic tissue. (D). Complementary bases between miR-570-3p and the 3′ UTR of HDAC1. (E). Luciferase activity was detected in the HUVEC cells. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. NG or Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. HG or MOD.

FIGURE 7

Overexpression of miR-570-3p inhibited HDAC1 expression and inflammatory injury in HUVEC cells. (A). The expression of miR-570-3p miRNA in HUVEC cells transfected with miR-570-3p NC or miR-570-3p mimics. (B–D). The expression of HDAC1 mRNA, TNF-α mRNA, and IL-1β mRNA in HUVEC cells transfected with miR-570-3p NC or miR-570-3p mimics. (E–H). Western blotting analysis of HDAC1, TNF-α, and IL-1β protein expression levels in HUVEC cells transfected with miR-570-3p NC or miR-570-3p mimics. Gray value of western blotting. (I). Content of MDA. (J). Activity of GSH-Px. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

Vaccarin inhibited the expression of HDAC1 and inflammatory injury by up-regulating miR-570-3p. (A). The effects of vaccarin on the expression of miR-570-3p miRNA in HUVEC cells transfected with miR-570-3p NC or miR-570-3p inhibitor. (B–D). The effects of vaccarin on the expression of HDAC1 mRNA, TNF-α mRNA, and IL-1β mRNA in HUVEC cells transfected with miR-570-3p NC or miR-570-3p inhibitor. (E–H). Western blotting analysis of HDAC1, TNF-α, and IL-1β protein expression levels in HUVEC cells transfected with miR-570-3p NC or miR-570-3p inhibitor. Gray value of western blotting. (I). Content of MDA. (J). Activity of GSH-Px. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 9

Effect of vaccarin on the inflammatory injury when co-transfected with miR-570-3p mimic and pcDNA3.1-HDAC1. (A,B). The effects of vaccarin on the expression of TNF-α mRNA and IL-1β mRNA in HUVEC cells transfected with miR-570-3p NC or miR-570-3p mimics or pcDNA3.1 or pcDNA3.1-HDAC1. (C,E). Western blotting analysis of TNF-α and IL-1β protein expression levels in HUVEC cells transfected with miR-570-3p NC or miR-570-3p mimics or pcDNA3.1 or pcDNA3.1-HDAC1. Gray value of western blotting. (F). Content of MDA. (G). Activity of GSH-Px. Values are mean ± SEM. n = 3 in each group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 10

Protective mechanisms of vaccarin against HG-induced endothelial inflammatory injury.