Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via

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Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-Signaling Pathway Sun Woo Jin, Chul Yung Choi , Yong Pil Hwang, Hyung Gyun Kim , Se Jong Kim, Young Chul Chung, Kyung Jin Lee, Tae Cheon Jeong, and Hye Gwang Jeong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05416 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 22, 2016

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Journal of Agricultural and Food Chemistry

TOC graphic

STO-609 P CaMKK

Tetracaine

Compound C P

L-NAME

RyR Betulinic acid

AMPK

Ca2+

P

CaM W7

LTCC

P

CaMKII

Nifedifine KN-93

ACS Paragon Plus Environment

eNOS

NO


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Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-

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Signaling Pathway

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Short title: Betulinic acid activates eNOS

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Sun Woo Jin †,¶ , Chul Yung Choi ‡,¶, Yong Pil Hwang §, Hyung Gyun Kim †, Se Jong Kim †,

6

Young Chul Chung

∥

, Kyung Jin Lee

⊥

, Tae Cheon Jeong #, *, Hye Gwang Jeong †, *

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†

College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea

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‡

Jeollanamdo Institute of Natural Resources Research, Jeollanamdo 529-851, Republic of

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Korea

11

§

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University of Korea, Jinju 660-759, Republic of Korea

13

⊥

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College of Medicine, Asan Medical Center, Seoul 138-736, Republic of Korea

15

#

Department of Pharmaceutical Engineering,

∥

Department of Food Science, International

Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan

College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea

16 17

¶

The first two authors contributed equally to this work.

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Journal of Agricultural and Food Chemistry 2

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* To whom correspondence should be addressed:

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Hye Gwang Jeong; College of Pharmacy, Chungnam National University, Daejeon 305-764,

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Republic

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[email protected]

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Tae Cheon Jeong: College of Pharmacy, Yeungnam University, Gyeongsan 712-749,

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Republic of Korea, Tel: +82-53-810-2819, E-mail: [email protected]

of

Korea,

Tel:

+82-42-821-5936,

Fax:

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+82-42-823-6566.

E-mail:


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ABSTRACT

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Betulinic acid (BA) is a naturally occurring pentacyclic triterpene that attenuates vascular

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diseases and atherosclerosis, but the mechanism by which it stimulates endothelial nitric

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oxide synthase (eNOS) is unclear. eNOS is the key regulatory enzyme in the vascular

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endothelium. This study examined the intracellular pathways underlying the effects of BA on

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eNOS activity and endothelial nitric oxide (NO) production in endothelial cells. BA treatment

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induced both eNOS phosphorylation at Ser1177 and NO production. It also increased the

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level of intracellular Ca2+ and phosphorylation of Ca2+/calmodulin-dependent kinase IIα

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(CaMKIIα) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Inhibition of

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the L-type Ca2+ channel (LTCC) and the ryanodine receptor (RyR) abolished BA-induced

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intracellular levels of Ca2+ and eNOS phosphorylation. Treatment with W7 (a CaM

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antagonist), KN-93 (a selective inhibitor of CaMKII), and STO 609 (a selective inhibitor of

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CaMKK) suppressed eNOS phosphorylation and NO production. Moreover, AMP-activated

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protein kinase (AMPK) was induced by BA, and BA-induced eNOS phosphorylation was

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inhibited by compound C, an AMPK inhibitor. Taken together, these results indicate that BA

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activates

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Ca2+/CaMKK/AMPK pathways. These findings provide further insight into the eNOS

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signaling pathways involved in the anti-atherosclerosis effects of BA.

eNOS

phosphorylation

and

NO

synthesis

via

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Keywords: Betulinic acid; eNOS; AMPK; CaMKII; CaMKK

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the

Ca2+/CaMKII

and

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INTRODUCTION

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Endothelial activation and dysfunction play an important role in the maintenance of vascular

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integrity and homeostasis by regulating the bioavailability of endothelial nitric oxide (NO).1,2

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In the vascular endothelium, NO bioavailability is regulated mainly by endothelial NO

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synthase (eNOS) upon the conversion of l-arginine to l-citrulline, and it plays a protective

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physiological role.3 Therefore, activation of eNOS and subsequent NO production is

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considered a promising therapeutic approach for vascular diseases, including atherosclerosis.4

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Increases in intracellular free Ca2+ and calmodulin concentrations lead to eNOS activation

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and NO production, which requires the ubiquitous Ca2+-binding protein calmodulin.5,6 As a

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result of Ca2+-dependent eNOS activation, eNOS phosphorylation at Ser-1177 is regulated

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mainly by Ca2+/calmodulin-dependent kinase II (CaMK II).7 In addition, AMP-activated

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protein kinase (AMPK) has been reported to have an anti-atherosclerosis effect by promoting

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eNOS phosphorylation at Ser-1177, which is associated with activation and AMPK-induced

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phosphorylation stimulates NO release by endothelial cells.8,9,10 As a family of upstream

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AMPK kinases, AMPK phosphorylation is regulated mainly by Ca2+/calmodulin-dependent

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protein kinase kinase β (CaMKKβ) and liver kinase B1 (LKB1).11,12

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Betulinic acid (3 beta-hydroxylup-20(29)-en-28-oic acid) is a pentacyclic triterpene prepared

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from betulin obtained from white-barked birch trees.13,14,15 BA has been reported to show

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various types of pharmacological properties, including anti-inflammation,16 anticancer,17,18

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antimalarial,19 antiAIDS,20 antifatty liver,21 antidiabetes,22,23 antidepression,24 and antiplatelet

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activities. Also, BA showed potential effects on vasorelaxation, 26,27 and eNOS expression,

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involved in anti-atherosclerosis. However, the effect of BA on eNOS activity and its

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mechanism are unclear yet. In this study, we report the signaling pathways underlying the

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effects of BA on eNOS activity using EA.hy926 human endothelial cells. We found that BA

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induces eNOS phosphorylation on Ser1177 and NO production. Then, by investigating the



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effects of BA in the presence of various inhibitors of signaling pathways, we found that

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CaMK II and AMPK are involved in eNOS activation by BA. To the best of our knowledge,

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this is the first report describing calcium dependent eNOS phosphorylation by BA.

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MATERIALS AND METHODS

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Chemicals

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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were

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purchased from Hyclone (Logan, UT), and 4,5-diaminofluorescein diacetate (DAF-2 DA)

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and fluo-4 acetoxymethyl ester (Fluo-4-AM) were purchased from Invitrogen (Carlsbad, CA).

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L-NAME, compound C, LY294002, PD98059, SB203580, SP600125, and N-[6-

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aminohexyl]-5-chloro-1-napthalenesulfonamide hydrochloride (W7) were purchased from

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Calbiochem (La Jolla, CA). STO-609 and KN-93 were purchased from Sigma-Aldrich (St.

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Louis, MO). Antibodies against p-eNOS, eNOS, p-AMPK, AMPK, p-CaMKIIα, p-CaMKKβ,

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and CaMKKβ as well as horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG

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antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin

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antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 3-(4,5-

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Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from USB

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Corporation (Cleveland, OH). The cytotoxicity detection kit used to measure lactate

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dehydrogenase (LDH) release was purchased from Roche Applied Science (Indianapolis, IN).

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All other chemicals were of the highest grade commercially available.

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Cell culture

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EA.hy926 cells were obtained from the American Type Culture Collection (Bethesda, MD).

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The cells were grown in DMEM containing 10% FBS at 37°C in a humidified incubator with

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5% CO2. BA was dissolved in dimethylsulfoxide (DMSO) and stored at -20°C until use. The


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stock solution was prepared immediately before use. Control cells were added with DMSO

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alone and the final concentration of DMSO did not exceed 0.1%.

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Cytotoxicity of BA in endothelial cells

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Conventional MTT reduction and LDH assays were used to determine the toxicity of BA to

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EA.hy926. Cells were seeded in 48-well plates and incubated at 37°C for 24 h. Wells were

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treated with various concentration of BA and the plates were incubated at 37°C for 24 h.

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MTT solution was added, followed by incubation for 30 min, and formazan crystals were

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solubilized by adding DMSO. The absorbance at 550 nm was measured using a

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BioTek Synergy HT microplate reader (BioTek Instruments, Winooski). The media were

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assayed using an LDH assay at 490 nm with a BioTek Synergy HT microplate reader

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(BioTek Instruments, Winooski). Calculations of cell viability (%) and cytotoxicity (fold-

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change) were based on the absorbance of treated cells relative to that of cells exposed to

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DMSO alone.

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Western blot analysis

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After treatment, EA.hy926 cells were lysed in lysis buffer (120 mM NaCl, 40 mM Tris [pH

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8], and 0.1% NP40 [Nonidet P-40]) on ice for 30 min and centrifuged at 12,000 rpm for 20

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min. Supernatants were collected and protein concentrations were measured using a protein

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assay kit (Pro-Measure, Intron biotechnology). Aliquots of the lysates (50 µg protein) were

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boiled for 5 min and then electrophoresed in 10% SDS-PAGE gels. Then the proteins were

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transferred to PVDF membranes, which were incubated with primary antibodies. Then the

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membranes were incubated with secondary anti-mouse or anti-rabbit antibody. Finally, the

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protein bands were detected using an enhanced chemiluminescence Western blotting

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detection kit (Biofact). The integrated optical density for the protein band was calculated by



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Image-J software, and then the values were normalized to an internal control (β-actin level

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and/or total forms).

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Measurement of NO production

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The production of NO was measured using the NO-specific fluorescent dye DAF-2 DA

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(Calbiochem) as described previously.29 In brief, EA.hy926 cells were grown to 95%

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confluence in 96-well plates and serum-starved overnight. The cells were loaded with DAF-2

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DA (final concentration, 2 µM) for 30 min at 37°C, rinsed three times with DMEM. Then the

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cells were either treated with BA or not, as indicated in the figure legends. In some

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experiments, l-NAME (100 µM), compound C (10 µM), W7 (10 µM), or KN-93 (10 µM)

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was added 30 min before loading with DAF-2 DA. Then the absorbance of culture media was

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measured at 495/515 nm using a BioTek Synergy HT microplate reader (BioTek Instruments,

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Winooski). The cells were fixed in 5% paraformaldehyde for 5 min at 4°C and then

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visualized using an EVOS fluorescence microscope (Life Technologies).

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Ca2+ measurement

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Intracellular levels of Ca2+ were measured using Fluo-4AM according to the manufacturer’s

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instructions. Briefly, cells were plated in 96-well plates, loaded with 5 µM Fluo-4AM for 30

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min at 37°C, and then incubated in the dark for 30 min at 25°C. The cells were treated with

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BA as indicated in the figure legends. Fluo-4AM was excited at a wavelength of 488 nm and

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emissions were monitored at 512 nm. All captured images were analyzed as described

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previously.30 In brief, fluorescence images of the selected cells were captured using an

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EVOS fluorescence microscope (Life Technologies). All quantifications were performed

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using the Image J software.

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Statistical analysis

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All experiments were repeated at least three times. Results are reported as means ± SD.

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The significance of differences between mean values was analyzed using the Newman–Keuls

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test for multi-group comparisons. Statistical significance was accepted for p-values

Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via

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