Gastrodin alleviates premature senescence of vascular endothelial cells by enhancing the Nrf2/HO-1 signalling pathway

Abstract

Endothelial dysfunction is an independent risk factor for stroke. The dysfunction of endothelial cells (EC) is closely concerned with EC senescence. Gastrodin (GAS) is an organic compound extracted from the dried root mass of the Orchidaceae plant Gastrodiae gastrodiae. It is used clinically to treat diseases such as vertebrobasilar insufficiency, vestibular neuronitis and vertigo. In the present study, we used hydrogen peroxide (H₂ O₂ )-induced human umbilical vein endothelial cells (HUVECs) to establish an in vitro EC senescence model and to investigate the role and mechanism of GAS in EC senescence. It's found that H₂ O₂ -treated HUVECs increased the proportion of senescence-associated β-galactosidase (SA β-gal) positive cells and the relative protein expression levels of senescence-associated cyclin p16 and p21. In addition, GAS reduced the proportion of SA β-gal positive cells and the relative protein expression levels of p16 and p21, and increased the proliferation and migration ability of HUVECs. Meanwhile, GAS increased the expression of the anti-oxidative stress protein HO-1 and its nuclear expression level of Nrf2. The anti-senescence effect of GAS was blocked when HO-1 expression was inhibited by SnPPIX. Furthermore, absence of HO-1 abolished the effect of GAS on HUVEC proliferation and migration. In conclusion, GAS ameliorated H₂ O₂ -induced cellular senescence and enhanced cell proliferation and migration by enhancing Nrf2/HO-1 signalling in HUVECs. These findings of our study expanded the understanding of GAS pharmacology and suggested that GAS may offer a potential therapeutic agent for stroke.

Keywords: Nrf2/HO-1; cell senescence; cerebrovascular disease; gastrodin; oxidative stress; proliferation.

FIGURE 1

Establishment and characterization of an in vitro HUVECs senescence model. (A) Different concentrations of H₂O₂ (0, 50, 100, 200, and 400 e) were applied to cultured HUVECs for 2 h. A CCK‐8 assay was used to evaluate cell viability. (B, C) SA β‐gal staining was performed to evaluate cell senescence. Senescent cells become larger and stained blue (Magnification ×100) (D) Determination of p16 and p21 protein expression by western blotting. (E, F) Densitometric quantification of p16 and p21 protein expression based on western blot assays. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. CCK‐8, Cell Counting Kit‐8; HUVECs, human umbilical vein endothelial cell; SA β‐gal, senescence‐associated β‐galactosidase.

FIGURE 2

Evaluation of GAS cytotoxicity on HUVECs. (A) Chemical formula of GAS. (B) CCK‐8 assay results on HUVECs exposed to different concentrations of GAS for 24 h. (C) Results of CCK‐8 assays determining the effect of GAS on the viability of H₂O₂‐treated HUVECs. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. CCK‐8, Cell Counting Kit‐8; GAS, Gastrodin; HUVECs, human umbilical vein endothelial cell.

FIGURE 3

Gastrodin inhibits senescence induced by H₂O₂ in HUVECs. (A) Determination of p16 and p21 protein expression by western blotting. (B, C) Densitometric analysis of p16 and p21 protein expression based on western blot assays. (D) Representative images of SA β‐gal staining in cultured HUVECs. Senescent cells become larger and stained blue (E) Quantification of SA β‐gal‐positive cells. (Magnification ×100) *p < 0.05, **p < 0.01 ***p < 0.001 ^**** p < 0.0001. HUVECs, human umbilical vein endothelial cells; SA β‐gal, senescence‐associated β‐galactosidase. Senescence: 100 μM H₂O₂.

FIGURE 4

Gastrodin sustains the proliferative and migratory ability of senescent HUVECs. (A) Representative images of EdU straining in cultured HUVECs. (B) Quantification of EdU‐positive cells, indicating active proliferation. (Magnification ×200) (C) Representative images from wound healing assays performed in cultured HUVECs. (Magnification ×50) (D) Quantification of wound closure rates. *p < 0.05, **p < 0.01, ***p < 0.0001. HUVECs, human umbilical vein endothelial cells.

FIGURE 5

Gastrodin activates Nrf2/HO‐1 signalling in H₂O₂‐treated HUVECs. (A) Western blot analysis of Nrf2 and HO‐1 protein expression. (B, C) Densitometric quantification of Nrf2 and HO‐1 protein expression based on western blot data. (D) Analysis of protein levels of HO‐1, p16, and p21 by western blotting. (E–G) Densitometric quantification of HO‐1 (E), p16 (F), and p21 (G) protein expression based on western blot data. (H) Representative images of SA β‐gal staining in cultured HUVECs. Senescent cells become larger and stained blue (Magnification × 100) (I) Quantification of SA β‐gal‐positive cells. *p < 0.05, **p < 0.01, ***p < 0.001. HO‐1, Heme oxygenase‐1; HUVECs, human umbilical vein endothelial cells; Nrf2, Nuclear factor erythroid 2‐related factor 2; SA β‐gal, senescence‐related β‐galactosidase.

FIGURE 6

Activation of Nrf2/HO‐1 signalling underlies gastrodin's proliferative and migratory effects on H₂O₂‐treated HUVECs. (A) Representative images of EdU staining (cell proliferation assay). (Magnification ×200) (B) Quantification of EdU‐positive cells. (C) Representative images from wound healing assays. (Magnification ×50) (D) Quantification of wound closure rates. *p < 0.01, **p < 0.001, ***p < 0.0001. HO‐1, Heme oxygenase‐1; HUVECs, human umbilical vein endothelial cells; Nrf2, Nuclear factor erythroid 2‐related factor 2.

FIGURE 7

Scheme summarizing the inhibition of H₂O₂‐induced HUVECs senescence by gastrodin through the Nrf2/HO‐1 signalling pathway. HUVECs, human umbilical vein endothelial cells; Nrf2, Nuclear factor erythroid 2‐related factor 2; HO‐1, Heme oxygenase‐1; Keap1, Kelch‐like ECH‐associated protein 1 (Keap1); ARE, antioxidant response elements.