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 (H2O2)‐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 H2O2‐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 H2O2‐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.


| INTRODUC TI ON
Cerebrovascular disease is one of the leading causes of death and disability worldwide. 1It is the first cause of death and the leading cause of adult disability in China. 2 Vascular endothelial cells (EC) play an important role in maintaining vascular homeostasis, such as maintaining blood flow, vascular tension, responding to hormones, neurotransmitters and physiological stimuli, and secreting factors related to vascular regulation. 3A growing body of research suggests that vascular EC dysfunction is critically associated with cerebrovascular disease. 4,5Senescent ECs exhibit functional deficits, adopt enlarged and flattened morphologies and lose their ability to replicate. 6is compromises the integrity of the endothelial barrier function and facilitates the onset of a range of cerebrovascular diseases, such as intracerebral haemorrhage, cerebral embolism and intracranial aneurysms. 7,8Cerebrovascular diseases impose a high social, familial and economic burden. 9Therefore, therapeutic prevention or attenuation of EC senescence has important implications for the treatment of cerebrovascular diseases.
Cellular senescence involves complex etiological and pathophysiological processes determined by a multitude of factors, including oxidative stress, inflammation and shorten length of telomeres. 10,11It is a state of irreversible growth arrest that cells enter into in response to various stressors or as a result of normal aging processes.It is characterized by a stable and permanent cessation of cell division, accompanied by distinct morphological and molecular changes in the cell 12 ; This process acts as a safeguard mechanism to prevent the proliferation of damaged or potentially harmful cells, such as those with extensive DNA damage or oncogenic mutations.By halting the division of these cells, cellular senescence helps to maintain genomic stability and suppress the development of cancer. 13In vascular ECs and other cell types, characteristic indicators of senescence include increased expression of senescence-associated β-galactosidase (SA β-gal), p16 and p21, and adoption of a senescence-associated secretory phenotype (SASP). 8,14Oxidation-reduction (redox) reactions contribute to fundamental physiological processes, regulate various aspects of cellular activity and maintain cellular and organismal homeostasis.When the intracellular redox reaction is broken, mitochondrial function is impaired and a large amount of ROS is produced.Excessive ROS cannot be removed in time, damaging the DNA integrity and the length of telomerase in cells and triggering the senescence-related process. 12Ultimately, these senescenceinducing signals will trigger the p53/p21 and p16/ retinoblastoma protein tumour suppressor pathways as final effectors of the aging program. 15Therefore, disruption of the redox reactions homeostasis can lead to cell dysfunction and death. 11,16me oxygenase-1(HO-1) is a pivotal stress-inducible enzyme that protects cells from oxidative injury by catalysing the conversion of heme into biliverdin, carbon monoxide and iron ions. 17-1 expression is induced in most cell types upon oxidative and inflammatory stimuli. 18,19Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that acts as a master regulator of the expression of HO-1 and other antioxidant genes. 20,21en cells are exposed to oxidative stress, Nrf2 dissociates from the cytosolic repressor protein Kelch-like ECH-associated protein 1 (Keap1). 22Subsequently, Nrf2 is translocated into the nucleus, where it binds DNA sequences known as antioxidant response elements (ARE) to regulate the expression of various downstream genes, promoting the restoration of redox homeostasis. 20,23erefore, we speculated that Nrf2/HO-1 signalling pathway activation exerts an essential role in protecting ECs from cell senescence induced by oxidative stress.
Based on an increasing number of studies characterizing the bioactive ingredients and pharmacological properties of herbal extracts and polyherbal formulations used in traditional Chinese medicine (TCM), the therapeutic potential of TCM for prevention and treatment of cardiovascular and cerebrovascular diseases has attracted considerable attention in recent years. 24Gastrodin (GAS), a phenolic glycoside, is the main active compound of a Chinese herbal medicine obtained from the roots of rhizoma Gastrodiae, and is also produced by chemical synthesis.GAS has been employed clinically as a TCM therapy for vascular and neurological conditions such as hypertension, cerebral ischemia and epilepsy. 25Zhang et al. reported that GAS exposure alleviates H 2 O 2 -induced injury in mouse liver sinusoidal ECs through p38 MAPK phosphorylation and activation of Nrf2 signalling. 26More recently, GAS treatment was shown to alleviate acetaminopheninduced liver injury in mice through inhibition of MAPK and stimulation of Nrf2-dependent pathways. 27However, whether GAS exerts anti-senescence effects on vascular ECs remains unknown.Thus, in this work we characterized the effects of GAS on the expression of senescence markers, the proliferation potential and the migratory capacity of cultured ECs challenged with H 2 O 2 , and investigated also the underlying molecular mechanisms.

| HUVECs culture and treatment protocols
Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection.All cells were cultured in endothelial cell medium (ECM) (ScienCell 1001; USA) supplemented with 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement, 1% penicillin/streptomycin (P/S) at 37°C in 5% CO 2 .To select a suitable concentration of H 2 O 2 for the HUVEC senescence model, cell viability assays were conducted in cells exposed for 2 h to different concentrations of H 2 O 2 (0, 50, 100, 200 and 400 μM) in serum-free medium.GAS (purity >98%) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd.To assess the effect of GAS on HUVECs viability, different concentrations of GAS (0, 6.25, 12.5, 25, 50,100 and 200 μM) were in complete medium were added to HUVECs for 24 h at 37°C.After determining optimal, non-cytotoxic concentrations of H 2 O 2 and GAS, the effects of GAS on senescence markers and on cell proliferation and migration abilities were assessed by incubating HUVECs with GAS (50 or 100 μM) 6 h before and over the 24 h that followed H 2 O 2 addition.This experiment is grouped as follows: Control group (HUVECs), senescence group (HUVECs exposed to 100 μM H 2 O 2 ), 50 μM gastrodin + senescence group, 100 μM gastrodin + senescence group To evaluate the role of HO-1 in the GAS-mediated cytoprotection, prior to GAS treatment, HUVECs were pre-treated with 20 μM tin-protoporphyrin IX (SnPPIX; MedChemExpress), a specific HO-1 inhibitor, for 2 h.All experiments were repeated three times.

| Cell viability assay
Cell Counting Kit-8 (CCK-8; Dojindo Co., Kumamoto, Japan) was used to analyse cell viability according to the manufacturer's instructions.In brief, 8 × 10 3 HUVECs per well were cultured in 96well plates, and incubated with ECM at 37°C with 5% CO 2 for 24 h.Depending on the experiment, the cells are treated and grouped accordingly.After the cells in each group were treated as required, cells were washed with PBS and 10 μL of CCK-8 solution plus 90 μL of FBS-free ECM were added to each well.After 2 h, optical density was measured at 450 nm in a microplate reader (BioTek Instruments, Inc.).

| Senescence-associated β -galactosidase staining
H 2 O 2 -induced cellular senescence was detected by using a SA β-gal staining kit (Beyotime Biotechnology, Shanghai, China), following to the manufacturer's protocol.Briefly, HUVECs were cultured in six-well plates, treated with or without H 2 O 2 , washed three times with PBS, and fixed with 1 mL of fixative solution 15 min.Each well was then incubated with 1 mL of SA β-gal staining mixture overnight at 37°C staining-mixture.The percentage of SA β-gal -positive cells was calculated from images taken on a bright field microscope (TS100; Nikon Corporation) at 100× magnification using ImageJ software (National Institutes of Health, USA).Senescent cells become larger and stained blue.Three fields were randomly selected for each group.Count at least 400 cells in each field and then count the ratio of positive cells to total cells All experiments were repeated for three times.

| Cell proliferation assay
Cell proliferation was detected by EdU-488 staining using a BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai).
In briefly, following experimental treatments, HUVECs were incubated with 10 μM EdU for 2 h.The medium was then removed and the cells washed with PBS.Cells were then fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.3% Triton X-100 for 15 min and incubated with the Click Additive Solution for 30 min in the dark.After, nuclear staining with Hoechst 33342, images were captured by a fluorescence microscope.The percentage of EdU positive cells was calculated by ImageJ software.Three fields were randomly selected for each group.The ratio of the number of proliferating cells (cells stained green) to the total number of cells (cells stained blue) was calculated for each field of view.All experiments were repeated for three times.

| Cell migration assay
Cell migration potential was assessed by scratch wound-healing assays.Briefly, HUVECs were plated in six-well plates (2 × 10 5 cells/ well; three replicates per treatment) cultured at 37°C for 24 h and treated for 2 h with or without H 2 O 2 .Cells were then left untreated or incubated with 50 or 100 μM GAS until cell confluence was >90%.
A sterile 200-μL pipette tip was used to scratch the cell monolayer cells across the center of the well.The wells were then washed with PBS and fresh culture medium was added.HUVECs were photographed at both baseline (0 h) and after 12 h under a light microscope at 40× magnification, and migration rate was quantitatively measured with ImageJ software using the formula: Wound closure (%) = (A0-A1)/A0 × 100, where A0 and A1 represent the wound area at baseline and after 12 h, respectively.anti-GAPDH (Cat.no.380626; 1:1000 dilution; ZENBIO Chengdu, China), Histone H3 (Cat.no.9715s; 1:1000 dilution; CST, USA).All the antibodies were diluted using primary antibody dilution buffer for western blot (Beyotime, Shanghai).Following three washes in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Cat.no.A21020; 1:10,000 dilution; Abbkine, Wuhan, China) for 2 h at room temperature.Bands were visualized using ECL detection system reagents (Pierce; Thermo Fisher Scientific, Inc.) and grey values for each band quantified and normalized against GAPDH expression by densitometry using ImageJ software.

| Gastrodin increases the viability in H 2 O 2 -treated HUVECs
Prior to assessing the potential protective effects of GAS (Figure 2A) against H 2 O 2 -induced senescence in HUVECs, possible cytotoxicity was ruled out by incubating HUVECs with various concentrations of GAS (0, 6.25, 12.5, 25, 50, 100 and 200 μM) for 24 h.CCK-8 assay results showed that none of the concentrations tested was cytotoxic to HUVECs (Figure 2B).Therefore, HUVECs were pre-incubated with various GAS concentrations for 6 h, exposed to H 2 O 2 for 2 h, and further supplemented with GAS (0, 6.25, 12.5, 25, 50, 100 and 200 μM) for an additional 24 h.Results of CCK-8 assays indicated that GAS significantly improved the viability of senescent HUVECs with the increase of GAS, at concentrations of 50 μM and higher significantly improved the viability of senescent HUVECs (Figure 2C).Therefore, we selected GAS concentrations of 50 and 100 μM to treat cells in the following experiments.

| Gastrodin inhibits H 2 O 2 -induced senescence in HUVECs
Next, we evaluated whether the increase in cell viability mediated by GAS in H 2 O 2 -treated HUVECs was associated with antisenescence effects.Western blot analysis showed that in the presence of 50 or 100 μM GAS, after H 2 O 2 exposure the protein expression of p16 and p21 was significantly decreased.In line with these findings, upon incubation with GAS, the percentage of SA β-gal-positive HUVECs was significantly reduced (Figure 3D,E).
These results indicate that GAS inhibits H 2 O 2 -induced senescence in HUVECs.

| Gastrodin sustains proliferation and migration in H 2 O 2 -treated HUVECs
Next, the effects of GAS on the proliferation and migration of senescent HUVECs were evaluated through EdU and wound healing assays, respectively.The results of EdU assays demonstrated that compared to the control group, the rate of proliferation was decreased by H 2 O 2 , and this effect was significantly attenuated after treatment with 50 or 100 μM GAS (Figure 4A,B).In turn, as shown in Figure 4C,D, wound healing assay results demonstrated that cell migration capacity was obviously decreased by H 2 O 2 , and this effect was significantly attenuated in cells co-incubated with 100 μM GAS.The effect of gastrodin on the migration capacity of ECs was further verified in the Transwell assay.In Figure S1 A,B, it was found that the migration capacity of ECs was significantly reduced in the senescent group compared with the control group.It was further found that gastrodin increased the migration ability of ECs in a concentration-dependent manner.These results indicated that GAS sustains the proliferative and migratory capacity of vascular ECs during oxidative stress.

| Gastrodin counteracts H 2 O 2 -mediated senescence in HUVECs by activating the Nrf2/ HO-1 antioxidant pathway
Since oxidative stress is the main determinant of H 2 O 2 -induced cellular senescence, we speculated that the pro-survival effects of GAS on H 2 O 2 -exposed HUVECs are mediated by activation of antioxidant responses.To test this hypothesis, we evaluated the effect of GAS on the protein expression of Nrf2, a key regulator of antioxidant gene expression and HO-1, a main Nrf2 effector.
Western blot analyses showed that compared to control cells, the protein expression of both Nrf2 and HO-1 was increased in HUVECs incubated with H 2 O 2 alone, and both proteins were further upregulated in cells incubated with H 2 O 2 in the presence of GAS (Figure 5A-C).To investigate whether GAS-mediated HO-1 upregulation influences the expression of senescence markers in H 2 O 2 -treated HUVECs, SnPPIX, an inhibitor of HO-1, was applied to HUVEC cultures before H 2 O 2 exposure.As shown in Figure 5D,E, SnPPIX pre-treatment abolished the enhancing effect of GAS on HO-1 expression.Supporting a key role for HO-1 in GAS-mediated anti-senescence effects, additional western blot assays indicated that SnPPIX pre-treatment negated the suppressive effect of GAS on the expression of the senescence-related proteins p16 and p21 in H 2 O 2 -challenged HUVECs (Figure 5F,G).
To further explore whether Nrf2/HO-1 signalling activation plays an essential role in GAS-mediated protective effects

| Inhibitor blunts gastrodin's proliferative and migratory effects on H 2 O 2 -treated HUVECs
Finally, we asked whether GAS-mediated HO-1 expression contributes to enhanced proliferative and migratory capacity in HUVECs challenged with H 2 O 2 .As shown in Figure 6A,B, EdU assay results indicated that the proliferative effect of GAS on H 2 O 2 -treated HUVECs was inhibited when the cells were pre-incubated with SnPPIX.Similarly, results of wound healing assays showed that SnPPIX pre-treatment abolished the pro-migratory effect of GAS on H 2 O 2 -exposed HUVECs (Figure 6C,D).The effect of gastrodin on EC senescence was also abolished by the HO-1 inhibitor SnPPIX in the Transwell assay (Figure S1C,D).Collectively, these data imply that the Nrf2/HO-1 axis plays a major protective role in GAS-treated, H 2 O 2 -exposed HUVECs.In conclusion, our study demonstrated that gastrodin alleviates hydrogen peroxide-induced senescence of human umbilical vein ECs through enhancing the Nrf2/HO-1 signalling pathway (Figure 7).

| DISCUSS ION
In this study, we demonstrated that GAS exposure alleviates oxidative stress-induced senescence in vascular ECs and enhances their proliferative and migratory potential by stimulating Nrf2/ HO-1 signalling.Two lines of evidence support this conclusion.Cellular senescence is a multifactorial process, influenced by multiple environmental and genetic factors and characterized by progressive decline in physiological functions, such as growth, proliferation and migration. 28Senescent ECs usually present with flattened, enlarged and increasingly polyploid nuclei. 6,29Although many endogenous and exogenous factors can promote cellular senescence, they are typically and strongly associated with an imbalance in redox reactions. 30terestingly, an increasing number of studies have found that Nrf2, is a member of the Cap 'n' collar/basic leucine zipper (CNC-bZIP) family of transcription factors and acts as a master regulator of cellular redox status in eukaryotic cells and of phase II detoxification responses in mammals. 13,31Dose-and time-dependent Nrf2 activation has been detected in a variety of cells upon H 2 O 2 stimulation. 32,33ring oxidative stress, the nuclear expression of Nrf2 was increased and activates the transcription of a series of antioxidant genes, including HO-1, which catalyses the degradation of heme to biliverdin, iron ions and carbon monoxide. 31Since this effect is associated with antioxidant, anti-inflammatory and anti-apoptotic actions, induction of HO-1 expression through pharmacological interventions is considered an important therapeutic strategy in vascular diseases.
Consistent with activation of the Nrf2/HO-1 signalling axis by oxidative stress, we observed that the protein expression of Nrf2 and HO-1 was upregulated in HUVECs following H 2 O 2 exposure.However, at the H 2 O 2 concentration tested (100 mM), the increase in Nrf2 and HO-1 expression was clearly insufficient to prevent the induction of senescence markers and features in these cells.A previous study demonstrated that GAS can alleviate mouse liver sinusoidal EC damage induced by H 2 O 2 by stimulating the Nrf2/ HO-1 signalling pathway. 26Likewise, Lin et al. showed that following oxidative stress, GAS-mediated activation of Nrf2/HO-1 signalling exerts an essential protective role against oxidative damage in HUVECs in vitro and accelerates also wound healing in vivo. 20Our results confirmed that GAS activates the Nrf2/HO-1 pathway in H 2 O 2 -treated HUVECs and demonstrated also that GAS-mediated HO-1 induction is a major determinant of its cytoprotective actions.However, in this work, the effects of GAS on EC senescence were evaluated only in vitro.Thus, further in vivo studies are required to confirm and expand the present findings.

| CON CLUS IONS
Our research showed that GAS counteracts H 2 O 2 -induced senescence and dysfunction in HUVECs by inhibiting SA β-gal, p16 and p21 protein expression and by sustaining cell proliferation and migration capacities via enhanced activation of the Nrf2/HO-1 axis.
These findings suggest that GAS may be a valuable therapeutic agent to prevent and treat stroke and other age-related cerebrovascular diseases.

1 |
Figure 1B,C, the percentage of SA β-gal-positive cells increased in a H 2 O 2 dose-dependent manner.Additionally, western blot analysis confirmed that the expression of two cell senescence marker proteins p16 and p21 was also increased after H 2 O 2 treatment (Figure 1D).As shown in Figure 1E,F, relative p16 and p21 protein expression levels were comparable among cells treated with 100 and 200 μM H 2 O 2 .However, since viability was lower in cells exposed to 200 μM H 2 O 2 , an H 2 O 2 concentration of 100 μM was selected to establish the HUVEC senescence model for subsequent experiments.

First| 9 of 12 TONG
, GAS treatment decreased the expression of characteristic senescence markers, namely SA β-gal, p16 and p21, and enhanced the proliferation and migration ability of H 2 O 2 -challenged HUVECs.Second, Second, GAS exposure upregulated Nrf2 and HO-1 protein F I G U R E 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.et al. expression levels in H 2 O 2 -treated HUVECs, and inhibitor blunted its anti-senescence, pro-proliferative and pro-migratory effects.