Rice Bioactive Peptide Binding with TLR4 To Overcome H2O2

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A rice bioactive peptide binding with TLR4 to overcome H2O2-induced injury in human umbilical vein endothelial cells through NF-#B signaling Ying Liang, Qinlu Lin, Ping Huang, Yuqian Wang, Jiajia Li, Lin Zhang, and Jianzhong Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04036 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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Journal of Agricultural and Food Chemistry

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A rice bioactive peptide binding with TLR4 to overcome H2O2-induced injury in

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human umbilical vein endothelial cells through NF-κB signaling

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Ying Lianga, b, Qinlu Lina, b*, Ping Huanga, b, Yuqian Wanga, b, Jiajia Lib, Lin Zhanga, b,

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Jianzhong Caoa, b

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a. Molecular Nutrition Branch, National Engineering Laboratory for Rice and

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By-product Deep Processing, Central South University of Forestry and Technology,

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Changsha 410004, Hunan, China

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b. College of Food Science and Engineering, Central South University of Forestry and

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Technology, Changsha 410004, Hunan, China

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*Corresponding author: Qinlu Lin, College of Food Science and Engineering, Central

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South University of Forestry and Technology, No. 498 Shaoshan South Road,

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Changsha

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+86-731-85623096. Fax: +86-731-85623038.

410004,

Hunan,

China.

E-mail:

17 18 19 20 21 22

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ACS Paragon Plus Environment

[email protected],

Tel:


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ABSTRACT: Reactive oxygen species (ROS)-induced vessel endothelium injury is

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crucial in cardiovascular diseases progression. Rice-derived bran bioactive peptides

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(RBAP) might exert antioxidant effect through unknown mechanisms. Herein, we

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validated the antioxidant effect and mechanism of RBAP on H2O2-induced oxidative

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injury in human umbilical vein endothelial cells (HUVECs). Here, HUVECs were

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treated with RBAP under H2O2 stimulation; the effects of RBAP on HUVECs

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oxidative injury were evaluated. H2O2 injury-induced cell morphology changes were

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ameliorated by RBAP. The effect of H2O2- on HUVEC apoptosis

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apoptotic cell: 38.00 ± 2.00 in H2O2 group vs. 21.07 ± 2.06 in RBAP + H2O2 group, P

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= 0.0013 compared to H2O2 group), the protein levels of cleaved-Caspase-3 (relative

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protein expression: 2.90 ± 0.10 in H2O2 group vs. 1.82 ± 0.09 in RBAP + H2O2 group,

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P < 0.0001 compared to H2O2 group) and p-p65 (relative protein expression: 1.86 ±

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0.09 in H2O2 group vs. 1.35 ± 0.08 in RBAP + H2O2 group, P < 0.0001 compared to

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H2O2 group) could be attenuated by RBAP. RBAP exerts its protective function

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through binding with Toll-like receptor 4 (TLR4). Taken together, RBAP protects

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HUVECs against H2O2-induced oxidant injury, which provided the theoretical basis

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for the molecular mechanism of rice deep processing and exploitation of functional

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peptides.

(percentage of

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KEYWORDS: Rice-derived bran bioactive peptide (RBAP); Toll-like receptor 4

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(TLR4); Antioxidant; NF-κB (p65); HUVECs

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INTRODUCTION

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Currently, nearly 1500 kinds of peptides with different functions have been

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identified in various food materials. Plant-derived bioactive peptides have been

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extensively

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regulation, cell differentiation and metabolism activity 1. Compared with proteins,

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plant-derived bioactive peptides are more easily digested and absorbed; these peptides

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have the ability to promote immunity 2, lower blood pressure 3, lower cholesterol

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levels 4, decrease blood sugar 5, act as an antioxidant

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peptides, rice-derived bioactive peptides (RBAP) have the advantages of high

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biological potency and low sensitivity; they are the only type of grain protein that

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requires no allergy tests. Therefore, it is of great significance to explore the properties,

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functions and applications of rice bran bioactive peptides.

studied

for

immunomodulation,

neurotransmitter

6

neurohormonal

and so on. Among these

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Vascular endothelial cell (VEC) damage is related to many vascular diseases,

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including cardiovascular disease 7. When a VEC is damaged, its function may lead to

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imbalances

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cerebrovascular diseases 8. Thus, in vitro studies of VECs can help to establish a full

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understanding of the above diseases. Human umbilical vein endothelial cells

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(HUVECs), which are derived from the vein endothelium in

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widely used as an experimental model of endothelial cell function and/or pathogenesis

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9

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model of endothelial cell injury. Wang et al. demonstrated that Interleukin 24 (IL-24)

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gene could protect HUVEC model against H2O2-induced oxidative injury10b. Herein,

in

atherosclerosis,

hypertension

, including cardiovascular disorders

and

other

cardiovascular

and

the umbilical cord, are

10

. H2O2-induced HUVEC injury is a classic

3



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we established a H2O2-induced oxidative injury cell model in HUVECs and assessed

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the detailed functions of a rice-derived bran bioactive peptide in protecting against

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H2O2-induced oxidative injury. Herein,

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we

first

extracted

and

identified

a

RBAP

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(Lys-His-Asn-Arg-Gly-Asp-Glu-Phe) and assessed the detailed effect of RBAP on

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H2O2-induced injury model of HUVEC. We also investigated the underlying

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mechanism by which RBAP exerted its function and the downstream signaling

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pathway involved. Considering these factors, we provide novel an experimental basis

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for treating H2O2-induced HUVEC injuries using RBAP.

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MATERIALS AND METHODS

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Chemicals

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Description Roswell Park Memorial Institute (RPMI) 1640 and fetal bovine

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serum (FBS) were purchased from Gibco BRL (Gibco-BRL, Carlsbad, CA, USA). An

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HE Staining Kit was obtained from Beijing Biosynthesis Biotechnology Co., Ltd.

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(Beijing, China). A BCA Protein Assay Kit was acquired from CoWin Biosciences

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Co., Ltd. (Beijing, China). A Nuclear and Cytoplasmic Protein Extraction Kit was

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purchased from the Promega Corporation (Madison, WI, USA). Protease inhibitors

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and phosphatase inhibitors were obtained from Roche (Roche Applied Science, USA).

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3-(4,5)-dimethylthiahiazo

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purchased from Roche Diagnostics (Mannheim, Germany).

(-z-y1)-3,5-di- phenytetrazoliumromide

(MTT) were

All other reagents except for the above mentioned used in this study were of

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analytical grade (analytical pure).

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Preparation of the rice bran active peptide

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We used MicroTOF-QII tandem mass spectrometry for the separation of PF3-γ

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purified fractions with a molecular weight of 1002.6 Da comprising of an 8-amino

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acid sequence. The amino acid sequence is as follows (starting from the N-terminal):

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Lys-His-Asn-Arg-Gly-Asp-Glu-Phe. Considering the experimental cell culture system

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and the operation during the special requirements, we synthesized the same amino

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acid sequence of the active peptide-by-peptide synthesizer as an experimental sample

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of RBAP. Measuring the antioxidative activities of RBAP in vitro

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Measuring

99 100

(DPPH)

radical

scavenging

activities The DPPH radical scavenging activity of RBAP was determined according to the

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2,2-diphenyl-1-picrylhydrazyl

methods described by Suda (2000) with some modifications.

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Different volumes of RBAP/Trolox (0.01, 0.025, 0.05, 0.1, 0.2, 0.3. 0.4 and 0.5

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mg/mL) were added into a test tube and diluted to a final volume of 2.0 mL. Then, 2.0

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mL DPPH solution (0.2 mmol/L) was added in each tube. The mixture was kept at

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37 °C for 30 min, and subjected to absorbance values determination at 517 nm. Measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)+

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radical scavenging activities

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Potassium persulfate stock solutions (7.4 mmol/L ABTS+) were stored at 4 °C in

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the dark, and the two stock solutions were mixed in the dark for 12 h to produce

5



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ABTS free radical electrons, which were then diluted 60 times with methanol and

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adjusted if the optical density (OD)

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appropriate extract dilution was added to be 150 µL and 2.85 mL ABTS+ diluent, and

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then the solution was mixed and placed in the dark for 30 min. Then, the OD734 nm

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value was measured, and the standard curve linear 0 ~ 600 mmol/L Trolox was

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established; the results were expressed in micromoles per mole of Trolox Equivalent

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(TE) in the dry basis (µmol TE/g DW) using Vitamin C (Vc)/butylated

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hydroxytoluene (BHT) as the reference compound.

734 nm

value is not 1.1 ± 0.01 range. The

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H2O2 content measurements

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The H2O2 content was determined using an A064-1 kit (purchased from Jian

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Cheng Bioengineering Institute, China). Samples treated with catalase and/or RBAP

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were homogenized in an iced PBS buffer (pH 7.5, 0.1 M) with a sonicator (NingBo

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Scientz Biotechnology Co., Ltd, China), and then centrifuged at 12,000×g for 10 min.

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The supernatant was then subjected to H2O2 measurement following the kit’s protocol. Cell culture and drug treatment

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HUVECs were purchased from the Institute of Cell Resource Center, Chinese

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Academy of Science (Shanghai China); the cell line originated from ATCC (American

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Type Culture Collection, USA). HUVECs were cultured in a RPMI Medium 1640

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supplemented with 10% dialyzed heat-inactivated FBS at 37 °C inCO2. To assess the

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effect of RBAP on H2O2-induced HUVEC injury, the isolated HUVECs were treated

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with H2O2 or RBAP. Cells are grouped as follows: the control group, the RBAP group

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(0.1 mM RBAP), the H2O2 group (100 µM H2O2), and the protection group (0.1 mM

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RBAP + 100 µM H2O2). Cells at passages 3-5 were used for all experiments. Cell viability assay

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The cell viability analysis was performed using a modified MTT assay according

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to Mosmann 11. HUVECs were placed within a 96-well plate (6×103 cells /well). After

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6 h, RBAP was added and incubated with the target cells for 12 h; then, 25-400 µM

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H2O2 (25, 50, 100, 200, 400 µM) was added and incubated for 24 h. After discarding

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the supernatant, the above cells were cultured with 100 µL of 0.5 mg/mL MTT

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dissolved in RPMI 1640, and then filtered through a 0.22 µm membrane at 37 °C. 4 h

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later, the formazan crystals were dissolved in 150 µL dimethyl sulfoxide (DMSO); the

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absorption values were determined using an automated microplate reader at 490 nm.

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The cell viability without any treatment was defined as 100%, and the viability of

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other groups was calculated separately by comparing them to the control group.

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Cell apoptosis analysis

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Flow cytometer assays were performed to examine the apoptotic cells by using

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an Annexin V-FITC apoptosis detection kit (Keygen, China). HUVEC were seeded in

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6-well plates at 2×104 cells/well and cultured overnight. After above described

149

treatment, the target cells were harvested, washed with iced PBS, and re-suspended.

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Target cells were then incubated with 5 µl Annexin V-FITC-specific antibodies and 5

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µl propidium iodide (PI) for 15-20 min in dark, and then subjected to cell apoptosis

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analysis by using BD Accuri C6 flow cytometer (BD, USA) as previously described

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. HE staining

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Treated cells were placed on the 6-well plate (5 × 104 cells/well) and washed

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twice with 0.1 mol/L PBS (pH 7.4) and fixed with Bouin’s fixative for 60 min at

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37 °C. The cells were then stained with hematoxylin-eosin and dehydrated in a graded

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series of alcohol while being observed with a microscope. Hoechst staining

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The cells were placed within 6-well plate (5×104 cells/well). The culture medium

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was discarded 24 h later, and the cells were fixed with paraformaldehyde (1 mL) at

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4 °C for 30 min. After rinsing with PBS, each group was stained with Hoechst33258

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at 37 °C for 20 min. Morphological changes of apoptosis in each group were observed

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with an inverted fluorescence microscope (OLYMPUS IX71). Western blotting

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Target cells were placed on 96-well plates (2 × 105 cells/well) and then exposed

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to different treatments, as indicated. Immunoblotting assays were employed with the

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following antibodies: anti-Caspase-3 (caspase-3 p17, sc-271028, Santa Cruz, USA),

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anti-NF-κB (p-p65) (ab86299 (phospho S536), Abcam, USA), anti-TLR4 (ab 13556,

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Abcam), anti-TIR-domain-containing adapter-inducing interferon-β (TRIF) (ab13810,

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Abcam) and anti-IKKβ (Cat# Y466, Abcam) to determine their protein levels.. Target

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cells were lysed in RIPA buffer with 1% PMSF. Extracted proteins were loaded onto a

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SDS-PAGE minigel, transferred onto PVDF membrane, and then probed with the

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above antibodies. The membranes were washed and incubated with HRP-conjugated

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secondary goat anti-rabbit for 1 h at 37 °C. ECL Plus™ western blot detection system

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(Pierce, Rockford, USA) was used to visualize the signals. A Gel Imaging System

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(ChemiDoc™ XRS+, BIO-RAD) was used for imaging.

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For IKKβ inhibition, 5 µM IMD-0354, an IKKβ inhibitor, was added with or

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without RBAP treatment upon H2O2 stimulation. The protein levels of TLR4, TRIF

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and IKKβ were then determined using the indicated assays. Vector construction for GST pull-down and Co-Immunoprecipitation

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(Co-IP)

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The vectors used in these two assays were constructed as previously described 12.

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TLR4 encoding-sequence was cloned into pGEX-6P-1 vector and Glutathione

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S-transferases (GST) tagged, named GST-TLR4. Peptide-K-encoding sequence was

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cloned into pET22b (+) vector and His-tagged, named His-Peptide-K. For Co-IP

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assay, TLR4-encoding sequence was cloned into pcDNA-Flag vector, named

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Flag-TLR4. Recombinant protein expression and purification

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GST-TLR4 and His-Peptide-K were transformed into Escherichia coli BL21

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(DE3). The recombinant protein expression determination and purification were then

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performed as previously described 13. GST pull-down analysis

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GST pull-down analysis was performed to validate the interactions of TLR4 and

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14

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Peptide-K as described

. Recombined GST-fused proteins were incubated with

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glutathione sepharose beads (GE Health, Glutathione Sepharose 4B, 17-0756-01) on

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rotating incubator at 37 °C for 0.5 h. Incubated beads were collected, washed, and

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incubated with 0.1 mg/mL input proteins for 0.5 h. The beads were washed with the

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reaction buffer four times after the supernatant was discarded. After washing down

200

with 10% SDS the target proteins were analyzed by SDS-PAGE and Immunoblotting

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assays. Co-IP assay

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Co-IP assays were performed as previously described

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12

. Flag-TLR4 and

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Peptide-K were co-transfected into HUVECs. 36 h later, proteins were extracted and

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subjected to IP testing by using Flag monoclonal antibodies and Immunoblotting

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assays using Flag and Myc antibodies. DNase and RNase (5 mg/mL) was used,

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respectively, to avoid the extra effects of DNase and RNase . Website of predicting the interaction between TLR4 and Peptide-K

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PEP-FOLD3 website 15 was used to predict the interaction and structure of TLR4

209 210

protein

and

Peptide-K

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(http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3).

(KHNRGDEF)

Statistical analysis

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All data were processed using SPSS 17.0 statistical analysis software (Armonk,

214

NY, USA). Results were expressed as mean ± SD of at least three independent

215

experiments. Differences among groups were analyzed using a one-way ANOVA test

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followed by multiple post hoc comparisons (SNK-q). The P values < 0.05 were

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considered statistically significant.

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RESULTS RBAP product sequence and purity identification

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The RBAP product sequence was verified and exhibited in Figure 1A as follows

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(three letter codes): Lys-His-Asn-Arg-Gly-Asp-Glu-Phe. Using the HPLC analysis,

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the product purity was up to 99.62% (Figure 1B). Electrospray ionization mass

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spectrometry (ESI-MS) was used to identify the products (Figure 1C). Free radical scavenging activities of RBAP

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Initially, we determined the free radical (ABTS and DPPH) scavenging activities

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of different doses of RBAP, compared with Trolox. The experimental results show

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that the scavenging capacity of RBAP and Trolox on ABTS and DPPH gradually

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increased in a dose-dependent manner; although, the radical scavenging capacity of

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RBAP on ABTS and DPPH was lower than Trolox. Therefore, high doses of RBAP

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could significantly scavenge both ABTS and DPPH, compared low doses of RBAP

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(Figure 2A-D).

233

To ensure that RBAP exerts its effect by scavenging free radicals rather than

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resolving H2O2, we determined the H2O2 content in response to the co-treatment of

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catalase and RBAP. The results showed that H2O2 content could be significantly

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reduced by catalase but not by RBAP (Figure 2E), indicating that RBAP scavenges

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free radicals but does not resolve H2O2. Effect of RBAP on H2O2-induced HUVEC oxidative injury

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We determined the free radical scavenging activity of RBAP and validated the

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effect of RBAP on H2O2-induced HUVEC oxidative injury. HUVECs were treated

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with a series of doses of H2O2 (25, 50, 100, 200 and 400 µM) 10b, 16; the cell viability

242

was examined using MTT assays. As shown in Figure 3A, the cell viability in the

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control 25 and 50 µM H2O2 groups was positively correlated with the incubation time;

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H2O2-induced HUVEC oxidative injury was obvious in the 100, 200 and 400 µM

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H2O2 groups within 6 h of incubation and gradually restored afterwards. The cell

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viability in the 100 µM H2O2 group was restored to approximately 50% of the control

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group and regarded as a successful H2O2-induced HUVEC injury model.

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Then, a series of doses of RBAP (0.01, 0.05, 0.1 and 0.2 mM) was added into the

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100 µM H2O2-induced HUVEC oxidative damage model, and the cell viability was

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examined by using MTT assays in order to evaluate the effect of RBAP on

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H2O2-induced HUVEC oxidative injury, compared with the control and 100 µM H2O2

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groups. As shown in Figure 3B, the cell viability of the control group was correlated

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with the culture time, while the cell viability of the RBAP groups was affected by

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H2O2 in 0-6 h. The cell viability of HUVECs with oxidative injury increased with the

255

increase of RBAP concentration, and the protective effect of RBAP gradually

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appeared within 6-12 h. When the concentration of RBAP was increased to 0.1 mM,

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the cell viability was significantly promoted compared with the 100 µM H2O2 group

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(P

Rice Bioactive Peptide Binding with TLR4 To Overcome H2O2

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