Vasorelaxant and Antihypertensive Effects That Are Dependent on the

Jan 4, 2019 - The LRA-induced vasorelaxant activity was inhibited by N(G)-nitro-l-arginine methyl ester (L-NAME; NO synthase [NOS] inhibitor) but not ...
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Bioactive Constituents, Metabolites, and Functions

Rice bran-derived tripeptide exhibits vasorelaxant and antihypertensive effects dependent on the endothelial NO system naohisa shobako, Atsushi Ishikado, Yutaro Ogawa, Yoko Sono, Takashi Kusakari, Makoto Suwa, Motonobu Matsumoto, and Kousaku Ohinata J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06341 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Journal of Agricultural and Food Chemistry

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Rice bran-derived tripeptide exhibits vasorelaxant and anti-hypertensive effects

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dependent on the endothelial NO system

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Naohisa Shobako1) 2), Atsushi, Ishikado1), Yutaro Ogawa1), Yoko Sono1), Takashi Kusakari1),

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Makoto Suwa1), Motonobu Matsumoto1), Kousaku Ohinata2)*

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1)

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2)

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University, Uji, Kyoto, 611-0011, Japan

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*Corresponding author

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E-mail: [email protected]

Health Care R&D, SUNSTAR, Takatsuki, Osaka, 569-1044, Japan Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto

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Tel: +81-774-38-3733

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Fax: +81-774-38-3774

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Abstract

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We recently identified a novel, potent anti-hypertensive peptide, Leu-Arg-Ala (LRA;

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minimum effective dose = 0.25 mg/kg), from rice bran protein. In this study, we found that

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LRA potently relaxed mesenteric arteries isolated from spontaneously hypertensive rats

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(SHRs) (EC50 = 0.1 µM). In contrast, the vasorelaxant activity of each amino acid that

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constitutes the LRA tripeptide was remarkably attenuated. The LRA-induced vasorelaxant

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activity was inhibited by N(G)-Nitro-L-arginine methyl ester (L-NAME; NO synthase [NOS]

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inhibitor) but not by an antagonist of bradykinin B2 and Mas receptors or by a

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phosphoinositide 3-kinase inhibitor. The anti-hypertensive effect induced after the oral

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administration of LRA was inhibited by L-NAME. LRA also induced the phosphorylation of

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endothelial NOS in human umbilical vein endothelial cells. Taken together, LRA may exhibit

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anti-hypertensive effects via NO-mediated vasorelaxation. LRA is the first example of a

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NO-dependent vasorelaxant peptide identified from rice bran protein.

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Keywords:

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hypertensive rats, vasorelaxation

NO

synthesis, anti-hypertensive

effect,

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novel

peptide, spontaneously

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Journal of Agricultural and Food Chemistry

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1. Introduction Rice (Oryza sativa), one of the world’s three major grains, serves as the staple food

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for almost half of the global population, usually in the form of polished white rice. Although

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rice bran, a byproduct of white rice polishing, is rich in protein and has a high protein

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efficiency ratio, it is not well used in the food industry.1–3 We investigated its further

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processing and identified that thermolysin-digested rice bran has anti-hypertensive effects.4

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We also identified an anti-hypertensive peptide, Leu-Arg-Ala (LRA), from the digest, based

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on angiotensin I-converting enzyme (ACE) inhibitory activity. LRA exhibited potent

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anti-hypertensive effects after oral administration at a low dose (0.25 mg/kg body weight),

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comparable to pharmaceuticals; however, its ACE inhibitory activity was not sufficiently

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high to explain the potent anti-hypertensive effect.

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The renin–angiotensin system (RAS) is thought to be critical for blood pressure

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regulation.5 Although ACE is an important target for hypertension treatment, other

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mechanisms, including vasorelaxation, have also been the subject of recent research. For

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example, activation of the protective axis of RAS by angiotensin (1-7) [ang (1-7)] induced an

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anti-hypertensive effect, which was mediated by vasorelaxation.6,7 To date, a number of

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anti-hypertensive peptides have been identified from the digests of food proteins; however,

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most of the related anti-hypertensive mechanisms were coupled to the ACE inhibitory effect,

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and few vasorelaxant peptides have been reported.8,9 Few studies have reported on the

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vasorelaxant mechanisms of food-derived peptides. For example, milk casein-derived

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Ile-Pro-Pro (IPP)/Val-Pro-Pro (VPP) and rapeseed-derived rapakinin relaxed blood vessels

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via the nitric oxide (NO) and CCK/PGI2 pathways, respectively.10,11

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In the present study, we demonstrated the potent vasorelaxant activity of LRA and

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investigated the mechanisms underlying the anti-hypertensive effect using ex vivo, in vitro, 3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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and in vivo studies.

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2. Materials and methods

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2.1. Materials

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We isolated a tripeptide LRA from the thermolysin digest and demonstrated that

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properties of the LRA were consistent with those of chemosynthesized LRA4. Thus, we used

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LRA, which was chemosynthesized by Fmoc method at Kokusan Chemical Co. Ltd (Tokyo,

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Japan) (purity: 98.96%). N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) was

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obtained from DOJINDO (Kumamoto, Japan). L-leucine and L-α-alanine were obtained from

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Nacalai Tesque (Kyoto, Japan). L (+) arginine was obtained from Wako (Osaka, Japan).

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Indomethacin, HOE140, (R)-(-)-phenylephrine hydrochloride, lorglumide, 1H-[1,2,4]

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oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), and phosphate-buffered saline were obtained

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from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). A779 was obtained from LKT Labs (St.

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Paul, MN, USA). Wortmannin was obtained from Abcam (Cambridge, UK). LR and

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angiotensin(1-7) were obtained from Bachem AG (Bubendorf, Switzerland). LKA and VPP

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were chemically synthesized by the F-moc method. Acetylcholine chloride was obtained

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from Tokyo Kasei Kogyo (Tokyo, Japan) and MCDB 131 medium and L-glutamine were

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obtained from Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained

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from Biowest (Miami, FL, USA). Basic fibroblast growth factor (FGF) was obtained from

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Kaken Pharmaceutical (Tokyo, Japan). Physiological saline was obtained from Otsuka

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(Tokyo, Japan). 2×Laemmli buffer was obtained from Bio-Rad (Hercules, CA, USA).

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Antibodies against eNOS, β-actin, Akt and p-Akt were purchased from Cell Signaling

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Technology (Danvers, MA, USA). Antibody against p-eNOS was purchased from Becton,

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Dickinson and Co. (Franklin Lakes, NJ, USA). 4 ACS Paragon Plus Environment

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2.2. Animals

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Male SHR/Izm rats (12 weeks old) were obtained from SLC (Shizuoka, Japan) and

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kept in a temperature-controlled room (23C) on a daily 12-hr light: 12-hr dark cycle. These

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spontaneously hypertensive rats (SHRs) were fed SP pellets (Funabashi Farm, Chiba, Japan)

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with free access to water. All experiments were approved by the Kyoto University Ethics

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Committee for Animal Research Use (permission no. 29-67).

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2.3. Vasorelaxation assay

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Determination of the vascular relaxation response was performed as described

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previously11 with some modifications: the small mesenteric artery was isolated from SHRs,

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150–200 mm in diameter, was cut into helical strips. The strips were suspended in a bathing

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medium (Krebs–Henseleit solution containing 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2,

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1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM glucose) maintained at

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37C, and continuously aerated with O2/CO2 (95%:5%). The resting tension was adjusted to

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0.3 g. The artery was preconstricted with 0.5 µM phenylephrine. Samples were applied 10

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min after the application of phenylephrine. Relaxing activity was assayed in the absence and

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presence of inhibitors and antagonists applied 10 min before the addition of phenylephrine.

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To determine the complete relaxation point, 100 µM papaverine was added at the end of each

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experiment. The endothelium was mechanically removed by rubbing with stainless steel

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needle as described previously.11 The removal of endothelium was confirmed by the

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disappearance of 100 µM acetylcholine (Ach)-induced vasorelaxation in the 60 mM

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KCl-contracted mesenteric artery.

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2.4. Blood pressure measurement

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The systolic blood pressure (SBP) of SHRs was measured using the method

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described previously.4 The animals were trained to undergo measurements using the tail-cuff

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method for 3 weeks with an MK-2000ST (Muromachikikai, Kyoto, Japan). Samples

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dissolved in saline were administered orally after measuring the basal SBP. Control rats were

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given the same volume of physiological saline. SBP was measured 6 times and mean values

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were calculated.

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2.5. Cell culture

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Human umbilical vein endothelial cells (HUVECs) are an endothelial cell line

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commonly used as a model of the endothelial layer.10,12–14 They were cultured in accordance

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with a previously reported method with slight modifications.15 Cells were cultured at 37C

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with 5% CO2 in MCDB131 supplemented with 10% FBS, 10 ng/ml FGF, and 10 mM

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glutamine on a type I collagen-coated plate (Sumitomo Bakelite, Tokyo, Japan). HUVECs at

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the 4th passage were seeded onto a type I collagen-coated plate. When cells reached

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confluence, they were starved overnight in medium containing 1% FBS without FGF before

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use. Peptides were dissolved in ultra-pure water and further dissolved in serum-containing

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medium at the desired final concentration. After the starvation phase, cells were exposed to

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peptide solution for 3 min.

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2.6. Western blotting analysis

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Western blotting analysis was performed as described previously with some

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modifications.16 Whole cell lysate from HUVECs was prepared in Laemmli buffer (Bio-Rad,

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Hercules, CA), denatured by boiling, resolved by SDS-polyacrylamide gel electrophoresis 6 ACS Paragon Plus Environment

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(PAGE), and then transferred to a nitrocellulose membrane by electroblotting. Blots were

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then incubated with a rabbit anti-endothelial nitric oxide synthase (eNOS) primary antibody

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(1:1,000), a mouse anti-phospho eNOS (Ser1177) primary antibody (1:1,000), a rabbit anti-Akt

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primary antibody (1:1,000), a rabbit anti-phospho Akt primary antibody (1:1,000) or a mouse

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anti-β-actin primary antibody (1:1,000) plus a horseradish peroxidase-linked secondary

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antibody, and detected by chemiluminescence using an Image Quant LAS 4000 mini system

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(GE Healthcare, Japan).

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Capillary electrophoresis western analysis (Wes; ProteinSimple, San Jose, CA,

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USA) was also performed to verify the result of western blotting analysis as described

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previously with some modifications17. Briefly, the samples, blocking reagent, wash buffer,

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eNOS

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chemiluminescent substrate were dispensed into the micro plate by the manufacturer.

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Electrophoretic separation and immunodetection were performed automatically using the

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default settings. The data were analyzed with the installed Compass software (Protein

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Simple). Electropherograms are represented as pseudo blots generated using Compass

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software in the figures.

(1:50)

and

phospho-eNOS

antibodies

(1:50),

secondary

antibody

and

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2.7. Data analysis

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All experiments are expressed as the mean ±SEM. Dunnett’s multiple comparison

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was used to compare the vasorelaxation effect in the ex vivo studies. One-way analysis of

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variance (ANOVA) followed by Tukey–Kramer multiple-comparison test was used to assess

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differences among the four groups in the in vivo study. Student’s t-test was used to compare

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two groups. P