Tartary Buckwheat Protein-derived Peptide AFYRW alleviates H2O2 -induced vascular injury via the PI3K/AKT/NF-κ B pathway

Tartary buckwheat protein-derived peptide (Ala-Phe-Tyr-Arg-Trp, AFYRW) is a natural active peptide that hampers the atherosclerosis process, but the underlying role of AFYRW in angiogenesis remains unknown. Here, we present a system-based study to evaluate the effects of AFYRW on H 2 O 2 -induced vascular injury in human umbilical vein endothelial cells (HUVECs). HUVECs were co-incubated with H 2 O 2 for 2 h to the vascular injury model, and AFYRW was added 24 h in advance to investigate the protective mechanism of vascular injury. We identi�ed that AFYRW inhibits oxidative stress, cell migration, cell invasion, and angiogenesis in H 2 O 2 -treated HUVECs. In addition, we found H 2 O 2 -induced upregulation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), phosphorylation of nuclear factor-κ B (NF-κ B) p65 and nuclear translocation of NF-κ B decreased by AFYRW. Taken together, AFYRW attenuated H 2 O 2 - induced vascular injury through the PI3K/AKT/NF-κ B pathway. Thereby, AFYRW may serve as a therapeutic option for vascular injury.


Introduction
The formation of new blood vessels, or angiogenesis, undergoes migration and proliferation of endothelial cells to develop new blood vessels from capillaries []. The vital steps of angiogenesis include the proliferation, migration, differentiation, and tube formation of vascular endothelial cells. Angiogenesis is a complex process that plays important roles in embryonic development, reproduction, menstrual cycle, and wound repair []. Aberrant angiogenesis is observed as a promoter of disease development, especially in tumor development and blindness in diabetes [,]. Despite the fact that inhibition of angiogenesis is an established treatment strategy for many diseases, the present therapies are, for the most part, far from satisfying.
Angiogenesis is usually accompanied by oxidative stress due to reactive oxygen (ROS), which promote angiogenesis by producing active oxidation products []. ROS not only supports angiogenesis by upregulating vascular endothelial growth factor (VEGF), but also acts as a key regulator of angiogenesis to induce endothelial cell migration, proliferation, and tubular structure formation in vivo. It is noteworthy that ROS regulates a variety of cellular signaling pathways, such as PI3K/Akt and NF-κB []. The PI3K/Akt pathway is involved in proliferation, adhesion, migration, invasion, metabolism, survival, and angiogenesis []. PI3K, intracellular phosphatidylinositol kinase, is activated by dimer conformational changes caused by upstream signal factors, and the activated PI3K then drives AKT. AKT regulates downstream targets through phosphorylation, among which NF-κB, as a downstream target of AKT, is involved in angiogenesis [,]. Currently, there are few studies on PI3K/AKT/NF-κB as targets for angiogenesis therapy.
Tartary buckwheat (Fagopyrum tataricum Gaertn.) has high nutritional value due to its richness in protein, vitamins, dietary ber, avonoids, and trace mineral elements [,]. Our previous study successfully isolated and identi ed three novel antioxidant peptides (Gly-Glu-Val-Pro-Trp, GEVPW; Tyr-Met-Glu-Asn-Phe, YMENF; Ala-Phe-Tyr-Arg-Trp, AFYRW) by alkaline protease from Tartary buckwheat albumin; Among these, AFYRW exhibited stability and was non-toxic [,]. Our further research demonstrated that AFYRW hampered the progress of atherosclerosis by reducing the aortic plaque area (data not published). Notably, one therapeutic target for stabilizing atherosclerotic plaques is inhibition of angiogenesis []. However, the relevance of AFYRW and angiogenesis remains unknown.
In the present study, we evaluated the effects of AFYRW on H 2 O 2 -induced vascular injury and explored the underlying mechanism, aiming to identify new potential therapeutic targets that augment the effectiveness of treatments. We found that AFYRW alleviates H 2 O 2 -induced vascular injury via the PI3K/AKT/NF-κB pathway, demonstrating that AFYRW may serve as a therapeutic option in angiogenesis and providing a basis for the further utilization of Tartary buckwheat.

Materials
Tartary buckwheat powder was sold from the Guizhou Weining Tartary Buckwheat Products Co., Ltd.
(Guizhou, China). Our previous study isolated antioxidant peptides from Tartary buckwheat albumin, whose sequence was identi ed by Q-TOF mass spectrometer coupled with an electrospray ionization source as Ala-Phe-Tyr-Arg-Trp (AFYRW) with a molecular mass of 741.85Da (Fig. 1) Co., Ltd., China) was added to each well and incubated for 0.5-1 h at 37˚C in a culture incubator. The OD values were measured at 450 nm wavelength.

Plate Colony Assay
HUVECs in the logarithmic growth phase were planted in the 6-well plate (LABSELECT, China), and then cultured for 5 days. After treatment with H 2 O 2 and AFYRW, HUVECs were xed in 4% paraformaldehyde (Leagene Biotechnology, China) for 20 min at room temperature. Then, stained with 0.1% crystal (Solarbio, China) violet solution for 1 h. Finally, the number of clones was counted after washing and drying.

Ros Assay
HUVECs were cultured in serum-free medium containing 10 µM DCFH-DA (Dalian Meilun Biotechnology Co., Ltd., China) at 37°C in a 5% CO 2 cell incubator for 1 h. After washing, 200 µL PBS was added, and the uorescence intensity was observed by an inverted uorescence microscope (Nikon, Japan) and the images were collected.

Sa-β-gal Staining
Degree of HUVECs senescence was determined using SA-β-gal staining kits (Solarbio, China), according to the manufacture's protocols. HUVECs were xed with β-Gal xative for 15 min at room temperature, and then incubated with staining solution at 37°C overnight. In senescent cells, the SA-β-gal activity was shown by blue staining. The number of senescent cells was observed by a microscope.

No And Mda Testing
After treatment with AFYRW and H 2 O 2 , HUVECs were washed twice with PBS buffer and digested with trypsin. Then, the cells were collected and centrifuged at 1000 rpm for 3 min. Finally, NO and MDA of HUVECs were tested by kits (Nanjing Jiancheng Bioengineering Research Institute, China), according to the manufacture's protocols.

Matrigel Assay
Precooled matrigel gel (CORNING, U.S.A) was slowly added to the 96-well plate and incubated at 37°C for 30 min. After digestion, the cell suspension was adjusted to 1×10 5 /mL. 50 µL of the cell suspension was processed and cultured at 37°C in a 5% CO 2 cell incubator for 3 h. The formation of lumen-like structures was observed by an inverted microscope (Nikon, Japan).

Real-time Pcr
Total RNA was extracted and quanti ed by Nanodrop 2000 (Implen, German). 1 µg of RNA was reverse transcribed to cDNA. Transcripts were quanti ed by real-time PCR using CFX96 Real-Time Detection System (Applied Biosystems, U.S.A). Gene-speci c primers were shown as follows.

Scratch Assay
After drawing vertical lines with the same breadth and narrowness in each well, the cells were rinsed 3 times with PBS to get rid of the oating cells. The medium was replaced with serum-free medium and pretreated with AFYRW at 37°C in a 5% CO 2 cell incubator for 24 h, and then photographed under the microscope. Successively, cells were incubated with H 2 O 2 for 12 h, and the images were acquired by an inverted microscope at the same position. The changes of scratch healing distance were analyzed by Image J software.
Mononuclear/endothelial Cell Binding THP-1 cells were centrifuged at 1000 rpm for 3 min, resuspended in RPMI 1640 medium and labeled with BCECF-AM (5 µmol/L). After 30 min, the cells were washed 3 times with PBS to remove excess BCECF-AM (Dalian Meilun Biotechnology Co., Ltd., China), and then THP-1 were co-cultured with HUVECs for 1 h. The non-adherent cells were washed with PBS, and adherent cells were photographed by an inverted uorescence microscope.

Immuno uorescence
Cells were xed with 4% paraformaldehyde on ice for 20 min. 5% bovine serum albumin and 0.2% Triton X-100 (Solarbio, China) were used to block nonspeci c staining at 4°C for 1 h. Cells were incubated sequentially with primary antibody and uorescence-labeled secondary antibody (Proteintech, China). Finally, cells were stained with 10 µg/ml of DAPI (Solarbio, China) for 10 min. The images were obtained by laser scanning confocal microscope (Olympus, Japan).

Data analysis
All experiments were repeated at least 3 times. The experimental data of each group was shown as mean ± standard and analyzed by SPSS software. The homogeneity of variance test and OneWay ANOVA were used to compare multiple groups. P < 0.05 was considered statistically signi cant.

AFYRW increased H 2 O 2 -induced cell viability
To determine whether AFYRW is involved in regulating cell viability of H 2 O 2 -induced HUVECs, we used the CCK8 assay. The results showed that 5-100 µg/mL AFYRW for 24 h had no impact on cell viability, indicating that AFYRW is not cytotoxic (Fig. 2A). In addition, cell viability was unaffected by 50µmol/L H 2 O 2 , but was substantially reduced by 100-200 µmol/L H 2 O 2 for 2 h (Fig. 2B). We next pretreated HUVECs with 5-100 µg/mL AFYRW for 24 h and then stimulated HUVECs with 100 µmol/L H 2 O 2 for 2 h.
As expected, AFYRW, also signi cantly improved the cell viability during induction with H 2 O 2 (Fig. 2C)

Effects Of Afyrw On The Ros Level, Oxidative Stress, And Aging In Hotreated Huvecs
In addition to being an important regulator of angiogenesis, ROS also plays a role in the control of the PI3K/Akt cell signaling pathway []. Therefore, we sought to determine the effects of AFYRW on ROS levels and cell senescence. AFYRW (10 or 60 µg/mL, 24 h) signi cantly hampered the H 2 O 2 -induced (100 µmol/L, 2 h) up-regulation of ROS levels in HUVECs (Fig. 4A-B). Next, we utilized β-galactosidase (SA-βgal) staining to detect the degree of cellular senescence. The result showed that AFYRW seriously inhibited H 2 O 2 -induced cell senescence (Fig. 4C-D Fig. 5A-B). Consistent with our observation of angiogenesis, AFYRW hampered H 2 O 2 -induced VEGF expression in HUVECs (Fig. 5C-E). Overall, AFYRW restrained VEGF expression and angiogenesis.

Afyrw Hampered The Cell Migration And Invasion In Huvecs
The key processes in the initiation of angiogenesis are the proliferation, migration, differentiation, and tube formation of vascular endothelial cells [4]. To determine whether AFYRW was involved in the regulation of cell migration and invasion in HUVECs, we examined the effect of AFYRW on cell migration and invasion. This result suggested that H 2 O 2 (100 µmol/L, 2 h) signi cantly enhanced cell migration, while H 2 O 2 plus AFYRW (10 or 60 µg/mL, 24 h) suppressed cell migration greatly compared with H 2 O 2 alone (Fig. 6A-B). In another similarly designed experiment, we found that AFYRW suppressed the cell invasion in H 2 O 2 -induced HUVECs (Fig. 6C-D). In summary, AFYRW hampered the migration and invasion of HUVECs.

Impacts Of Afyrw On The Nuclear Translocation Of Nf-κb, Adhesion Molecules And In ammatory Factors Expression
NF-κB, as a downstream of PI3K/Akt, plays a key role in in ammatory responses. After NF-κB is activated, p65 translocation enters the nucleus and participates in the transcription of target genes [].
However, the relevance between AFYRW and NF-κB expression in H 2 O 2 -induced HUVECs remains unknown. To test their association, we utilized western blotting and immuno uorescence to measure the expression of p65 and NF-κB respectively. Compared with controls, H 2 O 2 (100 µmol/L, 2 h) promoted p65 phosphorylation, which was signi cantly inhibited by AFYRW (10 or 60 µg/mL, 24 h; Fig. 7A-B). The results of immuno uorescence showed that H 2 O 2 also promoted the nuclear translocation of NF-κB, while AFYRW restored that (Fig. 7C).
After activation of NF-κB, a variety of adhesion molecules and pro-in ammatory cytokines are secreted, which attract monocytes to adhere to the vascular endothelium. Next, we examined the related in ammatory factors expression and cell adhesion ability in HUVECs. The expression levels of TNF-α, IL-6 and VCAM-1 were signi cantly increased after H 2 O 2 (100 µmol/L, 2 h) treatment, while AFYRW (10 or 60 µg/mL, 24 h) decreased the H 2 O 2 -induced up-regulation of in ammatory factors (Fig. 8A-B). Similarly, As a nuclear transcription factor, NF-κB plays vital roles in adjusting the expression of in ammatory factors, growth factors, and adhesion molecules [-]. Notably, NF-κB is involved in angiogenesis as a downstream target of Akt. Our analysis revealed that H2O2 stimulated nuclear translocation of NF-κB and p65 phosphorylation in HUVECs, which was signi cantly inhibited by AFYRW. Moreover, AFYRW largely down-regulated the H2O2-induced increase of VCAM-1, TNF-α, and IL-6 levels in HUVECs. Overall, AFYRW reduced the expression levels of adhesion molecules and in ammatory factors by inhibiting the PI3K/AKT/NF-κB signaling, thus slowing the in ammatory response.

Conclusion
Together our data point toward the conclusion that H 2 O 2 -induced angiogenesis impairment and the in ammatory response are ameliorated by AFYRW through the PI3K/AKT/NF-κB pathway, and a proposed molecular mechanism is shown in Fig. 9. A deep insight into the protective mechanism of AFYRW in angiogenesis may provide data support for the further development and utilization of Tartary