Tetrahydroxy stilbene glycoside attenuates endothelial cell premature senescence induced by H2O2 through the microRNA-34a/SIRT1 pathway

Numerous studies have demonstrated that endothelial cell senescence plays a decisive role in the development and progression of cardiovascular diseases (CVD). Our previous results confirmed that Tetrahydroxy stilbene glycoside (TSG) can alleviate the human umbilical vein endothelial cells (HUVECs) senescence induced by H2O2 through SIRT1. It has been reported that miR-34a is a translational suppressor of SIRT1. In this study, we aimed to explore whether TSG regulates SIRT1 through miR-34a to ameliorate HUVECs senescence. H2O2 was used to induce premature senescence in HUVECs, and miR-34a mimic or inhibitor were transfected to over-express or suppress the expression level of miR-34a. Results revealed that TSG apparently decreased the miR-34a expression level in H2O2-induced premature senescence of HUVECs. When SIRT1 expression was inhibited by EX527, the attenuation of TSG on the expression level of miR-34a were abolished. When miR-34a expression was knockdown, the effect of TSG on HUVECs senescence could be enhanced. While miR-34a mimic could reverse the effect of TSG on HUVECs senescence. In conclusion, we demonstrated that TSG could attenuated endothelial cell senescence by targeting miR-34a/SIRT1 pathway.

www.nature.com/scientificreports/ binding site with the 3'UTR of SIRT1 11,12 . Whether miR-34a is involved in the effects of TSG on ECs senescence induced by H 2 O 2 needs further study. This study aimed to investigate that the effect of miR-34a and its target protein SIRT1 expression on the process of TSG against HUVECs senescence.

TSG inhibited miR-34a expression in premature senescence of HUVECs induced by H 2 O 2 .
To clarify the effect of TSG on miR-34a, TSG (40 μg/ml, 20 μg/ml) was administered to the H 2 O 2 -induced HUVECs. qRT-PCR analyses ( Fig. 1) indicated that miR-34a was elevated upon stimulation with H 2 O 2 . Compared with the H 2 O 2 group, it led to a significantly decreased expression of miR-34a with TSG pretreatment. And the 40 μg/ml of TSG had been used for subsequent experiments.
TSG inhibited miR-34a causing the activation of SIRT1. Studies have shown that miR-34a family was an upstream regulator of SIRT1. To determine whether the regulating effects of TSG on SIRT1 in H 2 O 2 -induced HUVECs were mediated by miR-34a, the HUVECs were treated with H 2 O 2 , in the presence of TSG, TSG and SIRT1 inhibitor EX527 or EX527. As shown in Fig. 3a, TSG group significantly inhibited miR-34a expression enhanced by the SIRT1 inhibitor EX527. Next, the following study explored the effect of miR-34a mimic or inhibitor on SIRT1 within TSG treatment in H 2 O 2 -induced HUVECs. As shown in Fig. 3b, c, compared with the H 2 O 2 group, SIRT1 protein was less expressed in the miR-34a mimic group, and TSG elevated the expression of SIRT1 reduced by miR-34a mimic. While compared with the H 2 O 2 + TSG group, SIRT1 expression was up-regulated after the adding miR-34a inhibitor. This demonstrated that miR-34a inhibitor increased SIRT1 protein level, and enhanced the promotion effect of TSG on SIRT1. These results indicated that in H 2 O 2 -induced premature HUVECs, TSG increased SIRT1 by inhibiting miR-34a expression.
TSG suppressed the apoptosis in H 2 O 2 -induced HUVECs mediated by miR-34a. In order to determine the effects of miR-34a on TSG inhibiting the HUVECs apoptosis, we analyzed the proportion of apoptosis cells when miR-34a was over-expressed or down-expressed. As shown in Fig. 4a, b, compared with the H 2 O 2 group, the TSG and miR-34a inhibitor alone could significantly inhibit cell apoptosis, and the combination To assess the effect of TSG on miR-34a expression, after pretreatment with TSG (40 μg/ml) or TSG (20 μg/ml) for 24 h, HUVECs were exposed by H 2 O 2 for 2 h, total RNA was extracted and the level of miR-34a was detected by qRT-PCR. Results are expressed as the means ± SD. www.nature.com/scientificreports/ of them had an enhanced effect. Whereas, compared with H 2 O 2 + TSG group, over-expression of miR-34a by miR-34a mimic increased the number of apoptosis cells, and could significantly reverse the down-regulation of TSG on cell apoptosis. These data indicated that TSG alleviated cell apoptosis through miR-34a.  Compared with the H 2 O 2 group, miR-34a inhibitor had an inhibitory effect on stopping the cell cycle in the G0-G1 phase, similar as TSG (Fig. 5a, b). Meanwhile, compared with H 2 O 2 + TSG group, miR-34a mimic could significantly reverse the percentage of G0-G1 cells decreased by TSG. Therefore, the results indicated that TSG could reduce the percentage of cells in the G0-G1 phase through inhibiting the expression of miR-34a.
Protective effects of TSG against H 2 O 2 -induced HUVECs aging regulating miR-34a/SIRT1 signaling pathway. Our previous result suggested that TSG attenuated H 2 O 2 -induced HUVECs cell senescence by regulating SIRT1. To further study the effect of miR-34a on endothelial dysfunction and senescence, we analyzed the expression of senescence-associated proteins when miR-34a was over-expressed or suppressed, such as PAI-1 and p21 proteins. Compared with the H 2 O 2 group (Fig. 6a-c), we found that the PAI-1 and p21 protein levels were significantly decreased by TSG, and miR-34a inhibitor had the similar effect. In contrast, miR-34a mimic significantly increased the PAI-1 expression level. Compared with miR-34a mimic group, TSG could   www.nature.com/scientificreports/ cause a significant decrease in PAI-1 and p21 expression increased by miR-34a mimic. Compared with miR-34a inhibitor group, TSG significantly promoted the inhibition effect of miR-34a inhibitor on PAI-1. TSG could further promote the miR-34a inhibitor down-regulation of p21, but the difference was not significant. Thus, these results showed that TSG attenuated cellular senescence through miR-34a.

Discussion
Cell senescence is a process of cellular physiological function decline caused by oxidative stress. Aging of endothelial cells will disrupt the cell balance of the body, leading to endothelial dysfunction, which is a major risk factor for accelerating the development of cardiovascular diseases [13][14][15] . Therefore, anti-endothelial cell senescence is of great significance to the prevention and treatment of cardiovascular diseases. TSG has been recognized as a potential therapeutic agent in the treatment of cardiovascular-related diseases 16 . We recently published that TSG exerted inhibition effect on H 2 O 2 -induced senescence in HUVECs 5 . In this present study, we demonstrated the protective mechanism that TSG inhibited cell senescence via the miR-34a/SIRT1 signal axis. miRNA is a class of small non-coding RNA that could regulate gene expression levels during multiple cellular processes 17 . miRNA, primarily acting as a negative regulator of mRNA translation, is involved in the regulation of cell cycle regulation, stress response, differentiation, aging, apoptosis by binding to the complementary sequences in the 3'untranslated region (3'UTR) 18 . Thus, aberrant expression of miRNA affects multiple biological processes, including cell differentiation, proliferation, and apoptosis 19 . More importantly, certain miRNA have been shown to be important regulators of aging-related gene expression and play an important role in cell senescence 20 . Based on previous studies, miR-34a is up-regulated in many aging-related diseases 21 . Li et al. 22 and Cui et al. 23 have proved that miR-34a plays a key role in the process of cell senescence as a pro-senescence factor. Consequently, we detected the activation status of miR-34a genes during the induction of cell senescence in this experiment. Our results showed that miR-34a was highly expressed in H 2 O 2 -induced cell senescence model, indicating miR-34a elevated is associated with pathological events of senescence in vascular endothelial cells, which was consistent with the above research conclusions. Notably, this effect was regulated by TSG treatment. This prompted us to further explore the role of miR-34a in TSG in the treatment of cell senescence.
To test our speculation of miR-34a crucially contributing to the anti-aging effects of TSG. We modulated miR-34a expression in H 2 O 2 -induced premature senescence HUVECs. As expected, we found that miR-34a over-expression in HUVECs markedly induced senescence related alternations, such as increased apoptosis, retarded cell cycle progression with the majority of cells arrested in G1, whereas TSG treatment improved it, similar as miR-34a inhibitor. PAI-1 is a serine protease inhibitor, which is a mediator and marker of cell aging 24 . Studies have shown that PAI-1 is highly expressed in senescent cells, and there is a direct correlation between www.nature.com/scientificreports/ increased PAI-1 level and senescence related galactosidase-positive cells. p21 is a main cell cycle regulator which can bind cyclin/CDK complexes and regulate cell cycle progression negatively 25 . In our study, compared with that in H 2 O 2 -induced cell senescence group, the TSG group and the miR-34a inhibitor group had similar effects, which could down-regulate the contents of senescence related factors PAI-1 and p21. With all the above results, we suggested that the protective effect of TSG on cellular senescence in H 2 O 2 -induced premature HUVECs could be compensated through the down-regulation of miR-34a. Studies have confirmed that the activation of miR-34a/SIRT1/p53 signaling was involved in endothelial cell dysfunction and apoptosis 26 . Our previous studies had confirmed that TSG could prevent HUVECs from senescence by activating SIRT1 and decreasing p53 expression 5 . It has been demonstrated that miR-34a negatively regulates SIRT1 expression, which is also reflected by our study, where miR-34a expression was significantly enhanced under the intervention of the SIRT1 inhibitor EX527. The function of miR-34a/SIRT1 pathway in H 2 O 2 -induced cell damage has been demonstrated by Guo et al. 27 . Here, our results indicated that TSG up-regulated SIRT1 while suppressed miR-34a expression in HUVECs, and these disturbances were inhibited by EX527 and miR-34a mimic. Conversely, miR-34a inhibitor could enhance the up-regulation of SIRT1 by TSG. With the combination of TSG and miR-34a inhibitor, it showed higher SIRT1 expression in HUVECs, compared to cells treated with TSG alone. Collectively, these results strongly supported that activated miR-34a/SIRT1 signaling was required for TSG to intervene in HUVECs premature senescence induced by H 2 O 2 .
In conclusion, our current study has revealed that TSG inhibited HUVECs premature senescence induced by H 2 O 2 through targeting miR-34a/SIRT1. However, our data were mainly obtained from the in vitro model of HUVECs, deeper studies on delineating the anti-aging function of TSG and the control of miR-34a/SIRT1 network on endothelial cell senescence through in vivo methods needs to be elucidated later, because there are other aging-related regulatory mechanisms that may also be involved. Additionally, the interaction between TSG and miR-34a is also worth dedicated studies.

Conclusions
Collectively, our data suggested that miR-34a/SIRT1 pathway was involved in the TSG inhibiting aging process of HUVECs. TSG may be used as a promising therapy drug to reduce vascular senescence and aging.

Methods
Materials. The HUVECs were purchased from the Institute of Cell Biology, Chinese Academy of Sciences.
The cells were randomly divided into groups. MTS assay. HUVECs were cultured in 96-well plates at a density of 3,000 per well for 24 h. And then they were treated with different treatment factors according to the grouping requirements. Cells were incubated with 20 μl MTS (Beyotime, China) for 2 h at 37 ℃ to measure their growth. The absorbance of each well was quantified at 490 nm. There were 6 multiple holes in a single group.

Isolation of RNA and real-time quantitative RT-PCR analysis (qRT-PCR). For analysis of miRNA
and Total RNA were extracted by TRIzol (Invitrogen). The first strand cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa) at 37 ℃, 60 min, 85 ℃, 5 min, stored at 4 ℃. qPCR was performed with SYBR Premix Ex TaqTM II (TaKaRa) according to the manufacturer's instructions. Gene relative quantitative was analyzed by 2 -ΔΔCt method and all analyses were performed in triplicate. The primer sequences used in this study were as follows: miR-34a: TGG CAG TGT CTT AGC TGG TTGT, U6: ATT GGA ACG ATA CAG AGA AGATT.
Western blot analysis. RIPA lysis buffer was added to the collected cells and the concentration was measured with a bicinchoninic acid (BCA) kit (Beyotime, China). Protein samples were separated by SDS-PAGE using a 10% polyacrylamide gel. Then membranes were exposed to anti-SIRT1 (1:1,000, CST), anti-p21 (1:2,000, Proteintech), anti-PAI-1 (1:1,000, CST), and anti-GAPDH (1:5,000, Proteintech) overnight at 4 ℃. The membranes were washed (three times, 10 min each) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and then incubated with the corresponding secondary antibody. Based on the protein band needed, the PVDF membrane was cut prior to antibody hybridization according to the protein size. Band intensity was quantified by Image J software.