Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway

Abstract

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.

Keywords: Cav-1/eNOS; Gastrodin; Nrf2; homocysteine; oxidative stress.

FIGURE 1

Gas inhibited Hcy‐induced HUVECs oxidative stress and cytotoxicity. HUVECs were incubated with Hcy (1 mmol/L) for 24 h in the absence or presence of Gas (200‐800 μg/mL). (A) Cell viability was measured by MTT assay; (B) ATP content in cells was examined using CellTiter‐LumiTM luminescent Cell Viability Assay Kit. (C‐D) Cells oxidative damage were measured through MDA and LDH detection. (E) T‐AOC were measured by kit assays; (F) Cellular ROS was measured by DHE (2 μmol/L) staining. Imaged at 10 × magnification. Scale bars = 200 μm. Mean fluorescence was quantified using Image J software. (G) Western Blot showed that Gas regulated Hcy‐induced bax, bcl‐2 and cleaved caspase 3 expression. Data are Mean ± SEM (three independent experiments). *P < 0.05 vs. control group. #P < 0.05 vs. model group

FIGURE 2

Gas regulated Nrf‐2/ARE pathway against Hcy‐induced endothelial injury. HUVECs were incubated with Hcy (1 mmol/L) for 24 hours in the absence or presence of Gas (200‐800 μg/mL) (A) Gas inhibited Hcy‐induced Nrf2 transferred to cytoplasm; (B) Immunfluorescent staining of HUVECs with anti‐Nrf‐2 antibody (Red) and DAPI (blue). Scale bars = 5 μm. Histograms (mean ± SEM) show Nrf2 fluorescence intensity signal quantification in the nucleus; Gas regulated Nrf‐2/ARE pathway downstream enzymes SOD‐1, HO‐1 and Catalase in mRNA (C) and protein (D) level. Data are Mean ± SEM (three independent experiments). *P < 0.05 vs. control group. #P < 0.05 vs. model group

FIGURE 3

Gas decrease Hcy‐induced in NO production and increase vasodilatory effect in thoracic aorta rings dependent on Cav‐1/eNOS pathway. (A) HUVECs cultured with Hcy (1 mmol/L) were treated with Gas (50‐800 μg/mL) for 24 h, then the NO production was measured by kit assay. (B) HUVECs cultured with Hcy (1 mmol/L) were treated with Gas (200‐800 μg/mL) for 24 h, then incubated with DAF‐FM (5 μmol/L)for 30 min at 37℃. Imaged at 10 × magnification. Scale bars = 200 μm. NO levels were determined from the mean fluorescence and quantified using Image J software. (C) Quantification of Cav‐1, p‐eNOS and eNOS protein are measured by using Western blotting in Gas‐treated HUVECs with Hcy. (D) HUVECs exposed to L‐NAME (100 μmol/L) 30 min before Hcy (1 mmol/L) and Gas (800 μg/mL) treated, then the NO production was measured. (E) HUVECs were transfected with Cav‐1 siRNA or control siRNA. After 24 h, HUVECs were incubated with Gas or Hcy for another 24 h, then the NO production was measured. (F) Rings were pre‐treated with L‐NAME (100 μmol/L) or vehicle for 20 min, and then treated with Hcy, finally by Gas or Ach. Data are Mean ± SEM (three independent experiments). *P < 0.05 vs. control group. #P < 0.05 vs. Hcy group

FIGURE 4

Gas partially protected the HUVECs through the PI3K/Akt pathway. (A) HUVECs cultured with Hcy (1 mmol/L) were treated with Gas (200‐800 μg/mL) for 24 h, quantification of PI3K and p‐Akt/Akt protein expression in Gas‐treated HUVECs with Hcy. (B) HUVECs exposed to MK‐2206 (5 μmol/L) with Hcy (1 mmol/L) and Gas (800 μg/mL) treated, then Nrf2 subcellular localization was determined by Western Blot. HUVECs exposed to MK‐2206 (5 μmol/L) with Hcy (1 mmol/L) and Gas (800 μg/mL) treated, then (C) ROS were detected by DHE (2 μmol/L) staining, and (D‐E) NO production was detected by DAF‐FM(5 μmol/L) staining and kit assay, respectively. Imaged at 10 × magnification. Scale bars = 200 μm. Mean fluorescence was quantified using Image J software. Data are Mean ± SEM (three independent experiments). *P < 0.05, **P < 0.01 vs. control group. #P < 0.05, ##P < 0.01 vs. Hcy group

FIGURE 5

Model for Gas ameliorated endothelial cell damage partly through PI3K/Akt/eNOS and Nrf‐2/ARE pathway