research article blackcurrant suppresses...

Research ArticleBlackcurrant Suppresses Metabolic Syndrome Induced byHigh-Fructose Diet in Rats

Ji Hun Park1 Min Chul Kho12 Hye Yoom Kim12 You Mee Ahn12

Yun Jung Lee12 Dae Gill Kang123 and Ho Sub Lee123

1Hanbang Body-Fluid Research Center Wonkwang University Shinyong-dong Iksan Jeonbuk 570-749 Republic of Korea2College of Oriental Medicine and Professional Graduate School of Oriental Medicine Wonkwang University Shinyong-dongIksan Jeonbuk 570-749 Republic of Korea3Brain Korea (BK) 21 Plus Team Professional Graduate School of Oriental Medicine Wonkwang UniversityIksan Jeonbuk 540-749 Republic of Korea

Correspondence should be addressed to Ho Sub Lee hostwkuackr

Received 13 May 2015 Revised 29 June 2015 Accepted 28 July 2015

Academic Editor Abbas A Mahdi

Copyright copy 2015 Ji Hun Park et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increased fructose ingestion has been linked to obesity hyperglycemia dyslipidemia and hypertension associated with metabolicsyndrome Blackcurrant (Ribes nigrum BC) is a horticultural crop in Europe To induce metabolic syndrome Sprague-Dawley ratswere fed 60 high-fructose diet Treatment with BC (100 or 300mgkgday for 8 weeks) significantly suppressed increased liverweight epididymal fatweight C-reactive protein (CRP) total bilirubin leptin and insulin in ratswith inducedmetabolic syndromeBC markedly prevented increased adipocyte size and hepatic triglyceride accumulation in rats with induced metabolic syndromeBC suppressed oral glucose tolerance and protein expression of insulin receptor substrate-1 (IRS-1) and phosphorylated AMP-activated protein kinase (p-AMPK) in muscle BC significantly suppressed plasma total cholesterol triglyceride and LDL contentBC suppressed endothelial dysfunction by inducing downregulation of endothelin-1 and adhesion molecules in the aorta Vascularrelaxation of thoracic aortic rings by sodium nitroprusside and acetylcholine was improved by BC The present study providesevidence of the potential protective effect of BC against metabolic syndrome by demonstrating improvements in dyslipidemiahypertension insulin resistance and obesity in vivo

1 Introduction

Metabolic syndrome is a disease condition characterized byvariable coexistence of obesity hyperuricemia hyperinsu-linemia hypertension and dyslipidemia The pathogenesisof metabolic syndrome includes multiple organs in thecardiorenal system [1] Patients with metabolic syndromeas defined by the National Cholesterol Education ProgramAdult Treatment Panel III (NCEP-ATP III) simultaneouslyexhibit 3 or more of the following traits increased waistcircumference elevated blood pressure reduced high-densitylipoprotein (HDL) level elevated triglyceride level andhyperglycemia [2 3]

Fructose present in added sugars such as sucroseand high-fructose corn syrup has been epidemiologicallylinked with metabolic syndrome Increased consumption of

fructose commonly used in processed food and soft drinks isone of themost important factors contributing to the growingprevalence of metabolic syndrome [4] Recent findings haveshown that dietary fructose accelerates metabolic disordersand induces oxidative damage [5] A high-fructose dietinduces a well-characterized metabolic syndrome generallyresulting in hyperinsulinemia hypertension dyslipidemiaand a low HDL level [6] A recent study suggested thatrenal damage is associated with metabolic syndrome [7]Exposure of the liver to high levels of fructose leads to rapidstimulation of lipogenesis and triglyceride accumulationwhich lead to reduced insulin sensitivity and hepatic insulinresistanceglucose intolerance [8]

Blackcurrant (Ribes nigrum BC) is a valuable horticul-tural crop in Russia Poland German Scandinavia Eng-land New Zealand and several Eastern European nations

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 385976 11 pageshttpdxdoiorg1011552015385976

2 Evidence-Based Complementary and Alternative Medicine

Annual worldwide production of BC is approximately500000 to 600000 tons [9] BC contains numerous physio-logically active components including vitamins carotenoidsand flavonoids which have antiulcer anticonvulsion andantidiarrhea effects as well as protective effects againstcarcinomas diabetes and regressive disease [10 11]

Several studies have reported that BC produces antiox-idative effects Anthocyanins from BC abrogate oxida-tive stress through Nrf2-mediated antioxidant mechanisms[12] In addition BC extract has cytoprotective and anti-inflammatory properties [13] Moreover BC protects againstkidney stones [14] However the therapeutic effects of BCin subjects with metabolic disorder have not been reportedThus this study was designed to identify the effect of BCextract on high-fructose diet-inducedmetabolic syndrome inrats

2 Methods and Materials

21 Plant Material and Preparation of BC Extract BC waspurchased fromGukmin Farm (Jeongeup Korea) A voucherspecimen (number HBF191) was deposited in the Herbariumof the Professional Graduate School of Oriental MedicineWonkwang University (Korea) BC (400 g) was boiled with3 L of distilled water at 100∘C for 2 hThe resulting extract wasfiltered through Whatman No 3 filter paper and centrifugedat 990timesg for 20min at 4∘C The resulting supernatant wasconcentrated using a rotary evaporator after which theresulting extract (6319 g) was lyophilized using a freeze-drierand stored at minus70∘C until required

22 Animal Experiments and Diet All experimental pro-cedures were carried out in accordance with the NationalInstitute of Health Guide for the Care and Use of LaboratoryAnimals and were approved by the Institutional AnimalCare and Utilization Committee for Medical Science ofWonkwang University (approval number WKU 14-50) Six-week-oldmale Sprague-Dawley (SD) rats were obtained fromSamtako (Osan Korea) and kept in a roomwith automaticallymaintained temperature (23∘C) humidity (50ndash60) andlightdark cycles (12-h each) throughout the experimentsAfter 1 week of acclimatization the rats were randomlydivided into 4 groups (10 rats per group) The control group(Cont) was fed a regular diet The high-fructose (HF) dietgroup was fed a 60 fructose diet (Research Diet USA)The low-dose BC group was fed 60 fructose diet with100mgkgday BC administered orally by Sonde for a periodof 4weeksThehigh-dose BC groupwas fed 60 fructose dietwith 300mgkgday of BC administered orally by Sonde fora period of 4 weeks The regular diet was composed of 50starch 21 protein 4 fat and a standard mix of vitaminsand minerals The high-fructose diet was composed of 60fructose 20protein 10 fat and a standardmix of vitaminsand minerals

23 Measurement of Blood Pressure Systolic blood pres-sure (SBP) was determined using noninvasive tail-cuffplethysmography and recorded with an automatic sphygmo-graph (MK2000 Muromachi Kikai Tokyo Japan) SBP was

measured once per week At least 10 determinations of SBPwere made during every measurement session Values arepresented as the mean plusmn SEM of 8 measurements

24 Estimation of Oral Glucose Tolerance Two oral glucosetolerance tests (OGTT) were performed 2 days apart after8 weeks of treatment Briefly basal blood glucose concen-trations were measured after 10ndash12 h of overnight fastingafter which glucose (2 gkg body weight) was immediatelyadministered via oral gavage Tail vein blood samples werecollected 30 60 90 and 120min after glucose administration

25 Blood and Tissue Sampling At the end of the experi-ments the thoracic aorta and muscle were separated rinsedwith cold saline and frozen Plasma was obtained fromcoagulated blood samples by centrifugation at 3000 rpm for15min at 4∘C and frozen at minus80∘C

26 Blood Parameters Triglyceride levels in plasma weremeasured using commercial kits (AM 157S-K ASAN Korea)Levels of HDL total cholesterol and low-density lipoprotein(LDL) in plasma were measured using HDL and LDL assaykits (E2HL-100 Bio Assay Systems Germany) Levels ofinsulin in plasma were measured using commercial kits (80-INSRT-E01 ALPCOUK) Levels of C-reactive protein (CRP)in plasma were measured using commercial kits (557825BD Biosciences America) Levels of leptin in plasma weremeasured using commercial kits (ab100773 Abcam UK)Levels of T-Bill and BUN in plasma were measured usingcommercial kits (77184 Arkray Japan)

27 Preparation of Aorta and Measurement of Vascular Reac-tivity The thoracic aorta was rapidly and carefully collectedfrom each rat and placed into cold Krebrsquos solution (118mMNaCl 47mMKCl 11mMMgSO

4 12mMKH

2PO4 15mM

CaCl 25mM NaHCO3 10mM glucose and pH 74) Con-

nective tissue and fat were removed from each thoracic aortaEach thoracic aorta was cut into rings of approximately3mm in length Care was taken to protect the endotheliumfrom accidental damage during the dissection procedureThe thoracic aortic rings were suspended in a tissue bathcontaining Krebrsquos solution at 37∘C by means of 2 L-shapedstainless-steel wires inserted into the lumen and aeratedwith 95 O

2and 5 CO

2 The isometric forces of the

rings were measured using a Grass FT 03 force displacementtransducer connected to a Model 7E polygraph recordingsystem (Grass Technologies Quincy MA USA) A passivestretch of 1 g in the thoracic aortic rings was determinedto be the optimal tension for maximal responsiveness tophenylephrine (10minus6M) The preparations were allowed toequilibrate for approximately 1 h with the Krebrsquos solutionreplaced every 10min The relaxant effects of acetylcholine(ACh 10minus9ndash10minus6M) and sodium nitroprusside (SNP 10minus10ndash10minus5M) in the thoracic aorta rings were studied

28 Western Blot Analysis in the Rat Aorta and MuscleThoracic aorta and muscle homogenates were prepared inice-cold buffer containing 250mM sucrose 1mM EDTA

Evidence-Based Complementary and Alternative Medicine 3

01mM phenylmethylsulfonyl fluoride and 20mM potas-sium phosphate buffer (pH 76) The homogenates werecentrifuged at 8000 rpm for 10min at 4∘C after which theresulting supernatants were centrifuged at 13000 rpm for5min at 4∘C to produce a cytosolic fraction for proteinanalysis The recovered proteins were separated by 10SDS-polyacrylamide gel electrophoresis and transferred tonitrocellulose membranes which were blocked with 5bovine serum albumin (BSA) powder in 005 Tween 20-Tris-buffered saline (TBS-T) for 1 h Specific primary anti-bodies against ICAM-1 VCAM-1 E-selectin eNOS ET-1 (in aorta) AMP-activated protein kinase (AMPK) p-AMPK and insulin receptor substrate-1 (IRS-1) (in mus-cle) were purchased from Santa Cruz Biotechnology Inc(Santa Cruz CA USA) The nitrocellulose membranes wereincubated overnight at 4∘C with protein antibodies Theblots were washed several times with TBS-T and incubatedwith horseradish peroxidase-conjugated secondary antibodyfor 1 h after which immunoreactive bands were visualizedusing an enhanced chemiluminescence system (AmershamBuchinghamshire UK) The bands were analyzed densit-ometrically using a Chemi-doc Image Analyzer (Bio-RadHercules CA USA)

29 Hematoxylin and Eosin (H amp E) and Oil Red O Stainingof Aorta Epididymal Fat and the Liver Thoracic aorta tissuefrom 5 random subjects from each group was fixed in 10(vv) formalin in 001Mphosphate-buffered saline (PBS) for 2days with the formalin solution changed every day to removetraces of blood The aortic tissue samples were dehydratedand embedded in paraffin sectioned (6 120583m) and stainedwithH amp E Epididymal fat (from 5 random subjects from eachgroup) and liver tissue (from 5 random subjects from eachgroup) samples were fixed in 4 paraformaldehyde for 48 hat 4∘C and incubated with 30 sucrose for 2 days Each fatand liver sample was embedded in OCT compound (Tissue-Tek Sakura Finetek Torrance CA USA) frozen in liquidnitrogen and stored at minus80∘C Frozen sections were cut witha Shandon Cryotome SME (Thermo Electron CorporationPittsburg PA USA) and mounted on poly-l-lysine-coatedslides Epididymal fat sections were stained with H amp Ewhereas liver sections were stained with Oil Red O Forquantitative histopathological comparisons each section wasanalyzed using Axiovision 4 ImagingArchiving software

210 Immunohistochemistry of Thoracic Aorta Tissue Priorto immunohistochemical staining tissue sections in paraffinwere mounted on poly-l-lysine-coated slides (Fisher Scien-tific Pittsburgh PA USA) The tissue on each slide wasimmunostained using Invitrogen HISOTO-STAIN-SP kitswith the labeled streptavidin-biotin (LAB-SA) method Afterantigen retrieval slides were immersed in 3 hydrogen per-oxide for 10min at room temperature to block endogenousperoxidase activity and rinsed with PBS Next slides wereincubated with 10 nonimmune goat serum for 10min atroom temperature and incubated with primary antibodiesagainst ICAM-1 VCAM-1 ET-1 and eNOS (1 200 SantaCruz CA USA) in humidified chambers overnight at 4∘CNext slides were incubated with biotinylated secondary

antibodies for 20min at room temperature followed by incu-bation with horseradish peroxidase-conjugated streptavidinfor 20min at room temperature Peroxidase activity wasvisualized using a 331015840-diaminobenzidine (DAB Novex CA)substrate-chromogen systemwith hematoxylin counterstain-ing (Zymed CA USA) For quantitative analysis the averagescore of 10ndash20 randomly selected areas was calculated usingNIH Image Analysis Software ImageJ (NIH Bethesda MDUSA)

211 Statistical Analysis All experiments were repeated atleast 3 times Results are expressed as mean plusmn SD or mean plusmnSEM Data were analyzed using Sigmaplot 100 softwareStudentrsquos 119905-test was used to determine significant differencesbetween groups Results of 119875 lt 005 were considered statis-tically significant

3 Results

31 Effects of BC on Body Weight and Tissue Weights Dur-ing the experimental period all groups showed significantincreases in body weight The body weight of the HF dietgroup was not significantly different from that of the controlgroup However the BC1 and BC2 groups showed signifi-cantly decreased body weight in comparison with the HF dietgroup during the final week of the experiment

In comparison with the control group the HF diet groupshowed significantly increased liver weight liver weight as apercentage of BW epididymal fat pad weight and epididymalfat pad weight as a percentage of BW However the BC1 andBC2 groups showed significantly reduced liver weight liverweight as a percentage of BW epididymal fat pad weight andepididymal fat pad weight as a percentage of BW (Table 1)

32 Effects of BC on Plasma Parameters The HF diet groupshowed significantly increased CRP T-Bil and insulin levelsin comparison with those of the control group Howeverthe BC2 group showed significantly decreased CRP T-Biland insulin in comparison with those of the HF diet groupThe HF diet group showed significantly decreased leptin incomparison with that of the control group The BC2 groupshowed significantly increased leptin in comparisonwith thatof the HF diet group (Table 2)

33 Effects of BC on Oral Glucose Tolerance and Expressionof IRS-1 and p-AMPK in Muscle Each rat was subjected tothe OGTT to measure insulin resistance The HF diet groupshowed significantly increased blood glucose content 90 and120min after glucose administration However the bloodglucose content of the BC2 group significantly reduced incomparison with that of the HF diet group 90 and 120minafter glucose administration

The IRS-1 and p-AMPK protein expression levels of theHF diet group significantly decreased in comparison withthose of the control group However the IRS-1 and p-AMPKprotein expression levels in muscle tissue from the BC1 andBC2 groups reduced in comparison with those of the HF dietgroup (Figure 1)


(min)

Glu

cose

(mg

mL)

60

90

120

150

180

0 30 60 90 120

lowast

lowast

ContHF

BC1BC2

(a)

IRS-1

p-AMPK

AMPK

120573-actin

Cont HF BC1 BC2

(b)

Figure 1 Effects of BC on oral glucose tolerance test (a) and the expression of IRS-1 and p-AMPK in the muscle (b) Values were expressedas mean plusmn SE (119899 = 10) lowast119875 lt 005 versus Cont 119875 lt 005 versus HF Representative western blots of IRS-1 and p-AMPK protein levels areshown (119899 = 4) IRS-1 insulin receptor substrate 1 Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2 blackcurrant300mgkgday

Table 1 Effects of BC on body weight liver weight liver weight of BW epididymal fat pads weight and epididymal fat pads weight ofBW

Cont HF BC1 BC2Body weight (g)

Start 22762 plusmn 196 22175 plusmn 264 23379 plusmn 199 22427 plusmn 231Final 46615 plusmn 1075 45514 plusmn 815 42714 plusmn 1097 41973 plusmn 66

Liver weight (g) 109 plusmn 052 132 plusmn 076lowast 1118 plusmn 021 108 plusmn 023

Liver weight of BW 249 plusmn 007 28 plusmn 003lowast 252 plusmn 003 254 plusmn 004

Epididymal fat pads weigh (g) 664 plusmn 04 747 plusmn 046lowast 642 plusmn 055 515 plusmn 042

Epididymal fat pads weight of BW 145 plusmn 005 174 plusmn 006lowast 138 plusmn 014 119 plusmn 008

Values were expressed as mean plusmn SE (119899 = 10) lowast119875 lt 005 versus Cont 119875 lt 005 and 119875 lt 001 versus HF BW body weight Cont control HF high

fructose BC1 blackcurrant 100mgkgday BC2 blackcurrant 300mgkgday

Table 2 Effects of BC on CRP T-Bil leptin and insulin

Cont HF BC1 BC2CRP(ngmL) 19562 plusmn 132 20882 plusmn 56lowast 1941 plusmn 252 19367 plusmn 239

T-Bil(mgdL) 06 plusmn 003 072 plusmn 005lowast 063 plusmn 004 057 plusmn 002

Leptin(pgdL) 062 plusmn 003 045 plusmn 003lowast 053 plusmn 001 083 plusmn 01

Insulin(mgdL) 064 plusmn 003 115 plusmn 019lowast 064 plusmn 006 062 plusmn 005

Values were expressed as mean plusmn SE (119899 = 10) lowast119875 lt 005 versus Cont 119875 lt005 and

119875 lt 001 versusHF CRPC-reactive protein T-Bil total bilirubinCont control HF high fructose BC1 blackcurrant 100mgkgday BC2blackcurrant 300mgkgday

34 Effect of BC on the Morphology of Epididymal FatPads The HF diet induced fat hypertrophy in this study

However the adipocytes of the BC1 and BC2 groups showedsignificantly reduced hypertrophy in comparison with thoseof the HF diet group (Figure 2)

35 Effect of BC on Hepatic Steatosis To investigate fataccumulation in the liver we prepared frozen liver sec-tions which were stained with Oil Red O Lipid dropletswere detected in the HF diet groups However significantlyfewer lipid droplets were counted in the BC1 and BC2groups compared to those counted in the HF diet group(Figure 3)

36 Effect of BC on Lipid Metabolism Biomarker LevelsTotal cholesterol triglyceride LDL and HDL plasma levelswere measured as biomarkers of lipid metabolism The totalcholesterol triglyceride and LDL levels of the HF dietgroup significantly increased in comparison with those ofthe control group However the total cholesterol triglyceride


HFCont

Cont HF BC1 BC2

BC1 BC2

0

2000

4000

6000

8000

10000

lowastlowast

Size

of a

dipo

se ce

lls (120583

m2)

Figure 2 Effects of BC on epididymal fat pads morphology Representative microscopic photographs in epididymal fat pads of SD rats withcontrol diet and HF diet were stained with hematoxylin and eosin Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont119875 lt 001 versus HF Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2 blackcurrant 300mgkgday

and LDL levels of the BC1 and BC2 groups significantlydecreased in comparison with those of the HF group Therewas no significant difference in theHDL level of either theHFgroup or the BC group (Table 3)

37 Effect of BC on Systolic Blood Pressure and Vascu-lar Tension At the beginning of the experimental feedingperiod systolic blood pressure was measured by the tail-cuff technique After 4 weeks the systolic blood pressureof the HF diet group was significantly higher than that ofthe control group The systolic blood pressure of the BC1group was significantly lower than that of the HF diet group

The systolic blood pressure of the BC2 groupwas significantlylower than that of the HF diet group

Vascular responses to SNP (1 times 10minus10 to 1 times 10minus7M) andACh (1 times 10minus9 to 1 times 10minus6M) were measured in the thoracicaorta ACh-induced relaxation of thoracic aorta rings wassignificantly impaired in the HF diet group in comparisonwith that of the other groups SNP-induced relaxation ofthoracic aorta rings was significantly impaired in the HF dietgroup in comparison with that of the other groups (Figure 4)

38 Effect of BC on the Morphology of Aorta The tunicaintima-media layer of the HF diet group showed significantly


BC1 BC2

Cont HF

Figure 3 Effects of BC on liver morphology Representative microscopic photographs in liver of SD rats with control diet and HF diet werestained with Oil red O (119899 = 5) Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2 blackcurrant 300mgkgday

Table 3 Lipid profile in SD rats fed HF andor BC for 8 weeks

Cont HF BC1 BC2T-Cho (mgdL) 5536 plusmn 165 7366 plusmn 362lowastlowast 5993 plusmn 307 5836 plusmn 376

TG (mgdL) 9813 plusmn 1343 22966 plusmn 1529lowastlowast 13732 plusmn 1442 10938 plusmn 1336

LDL-c (mgdL) 3137 plusmn 373 4676 plusmn 885lowast 3332 plusmn 497 3435 plusmn 263

HDL-c (mgdL) 3037 plusmn 48 2749 plusmn 572 3264 plusmn 52 351 plusmn 762Values were expressed as mean plusmn SE (119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001 versus Cont 119875 lt 005 and

119875 lt 001 versus HF T-Cho total cholesterolTG triglyceride LDL-c low-density lipoprotein HDL-c high-density lipoprotein Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2blackcurrant 300mgkgday

increased thickness in comparison with that of the controlgroup However the tunica intima-media layer of the BC1and BC2 groups showed significantly decreased thickness incomparison with that of the HF diet group (Figure 5)

39 Effects of BC on Expressions Levels of Adhesion MoleculeseNOS and ET-1 in Aortic Tissue Protein expression levelsof adhesion molecules (VCAM-1 ICAM-1 and E-selectin)ET-1 and eNOS in aortic tissue were determined by westernblotting Protein levels of adhesion molecules and ET-1increased in the HF diet group in comparison with those ofthe control group However the BC1 and BC2 groups showeddecreased protein levels of adhesion molecules and ET-1 incomparison with those of the HF diet group In additioneNOS protein expression in the HF diet group decreased incomparison with that of the control group However the BC1

and BC2 groups showed increased eNOS protein expressionin comparison with that of the HF group (Figure 6)

310 Effects of BC on Immunoreactivity of AdhesionMoleculeseNOS and ET-1 in Aortic Tissues Immunohistochemistrywas used to evaluate expression of ET-1 ICAM-1 VCAM-1and e-NOS in the aortic wall ET-1 ICAM-1 and VCAM-1protein expression levels increased in the HF diet group incomparison with those of the control group However theET-1 ICAM-1 and VCAM-1 protein expression levels of theBC1 and BC2 groups decreased in comparison with those ofthe HF diet group eNOS expression decreased in the HF dietgroup in comparisonwith that of the control group Howeverthe groups treatedwith BC1 andBC2 showed increased eNOSexpression in comparison with that of the HF diet group(Figure 7)


Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast

lowastlowastlowastlowast

ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)

Figure 4 Effects of BC on systolic blood pressure (a) vascular tone in thoracic aorta effect of a BC on sodium nitroprusside-inducedcontraction in thoracic aorta (b) and effect of a BC on acetylcholine-induced relaxation in thoracic aorta (c) Values were expressed asmean plusmn SE (119899 = 10) lowast119875 lt 005 and lowastlowast119875 lt 001 versus Cont 119875 lt 005 and

119875 lt 001 versus HF SNP sodium nitroprusside AChacetylcholine Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2 blackcurrant 300mgkgday

4 Discussion

The results of this study demonstrate that theHF diet inducedmetabolic syndrome with increased epididymal fat padweight resulting from increased plasma levels of triglyceridesand LDL Treatment with BC lowered epididymal fat padweight triglyceride levels and LDL levels whereas it elevatedlevels of HDL which enhances lipid metabolism Thus BCimproves lipid metabolism by decreasing plasma levels oftriglycerides and LDL Although epididymal fat pad weightincreased in response to the HF diet the body weight of thecontrol diet and HF diet groups was similar [15] HF hasbeen shown to increase hepatic lipase activity and epididymalfat hypertrophy [7 16] Experiments intended to measure

increased body weight should be longer than the period of8 weeks used in the present study [17] BC reduces obesity inHF diet rats because BC significantly decreased the HF diet-induced increase in bodyweight in this study In addition BCsuppressed insulin and leptin levels [18 19]

Because HF impairs glucose tolerance and induces obe-sity dyslipidemia and fatty liver this study focused onthe expression of AMPK in the liver and muscle [20] HFdecreased expression of IRS-1 IRS-1 deficiency causes insulinresistance In addition IRS-1 plays a very important role insecretion of insulin from pancreatic 120573-cells in the liver andmuscle [21] The BC group showed reduced IRS-1 expressionin comparison with that of the HF group


HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast

Figure 5 Effects of BC on aorta morphology Representative microscopic photographs in aorta of SD rats with control diet and HF diet werestained with hematoxylin and eosin Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 versus HF Contcontrol HF high fructose BC1 blackcurrant 100mgkgday BC2 blackcurrant 300mgkgday

In the present study HF increased circulating levels ofinflammatory marker CRP Increased CRP is an independentrisk factor for coronary artery disease [22] Altered lipid levelsinduced by the HF diet were associated with aortic lesionsHistological analysis demonstrated that the endothelial layerswere rougher in aortic sections from HF diet rats which wasassociated with a trend towards increased development ofatherosclerosis Intima-media thickness of the thoracic aortahas been shown to correlate with prognosis and the degreeof coronary artery disease [23] BC treatment maintainedsmooth and soft intima endothelial layers and decreasedintima-media thickness in the aortic sections of HF diet rats

Endothelial dysfunction plays an important role in hyper-tension vascular inflammation other cardiovascular dis-eases and metabolic syndrome [24 25] In this experimentalmodel expression of ET-1 and inducible adhesion moleculessuch as ICAM-1 and VCAM-1 in the arterial wall representeda key event in the development of atherosclerosis BC ame-liorated vascular inflammation by downregulation of ET-1ICAM-1 VCAM-1 and E-selectin expression in the thoracicaorta Several studies have shown that reduced blood pressureand endothelial function are related to increased eNOSreactivity which results in increased production of NOa strong vasodilator [26 27] In the present study BC


Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin

Figure 6 Effect of BC on the expression of adhesion molecules ET-1 and eNOS in the aorta Representative western blots of adhesionmolecules eNOS and ET-1 protein levels are shown (119899 = 5) Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2blackcurrant 300mgkgday

BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)

Figure 7 Effects of BC on ET-1 (a) ICAM-1 (b) VCAM-1 (c) and eNOS (d) immunoreactivity in aortic tissues Values were expressed asmean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 versus HF Cont control HF high fructose BC1 blackcurrant 100mgkgday BC2blackcurrant 300mgkgday


upregulated eNOS levels in the aorta and recovered HF diet-induced impairment of endothelium-dependent vasorelax-ation These results suggest that the hypotensive effect of BCismediated by endothelium-dependentNOcGMP signalingThe histological study revealed that BC suppressed vascularinflammation compatible with the processes of atherosclero-sis In fact endothelial dysfunction was initially identified asimpaired vasodilation in response to specific stimuli such asACh and bradykinin therefore improvement of endothelialfunction is predicted to regulate lipid homeostasis [28]

5 Conclusion

BC improved reduced plasma levels of biomarkers of dyslipi-demia including total cholesterol triglycerides and LDL BCenhanced SNP- and ACh-induced relaxation and suppressedexpression of adhesion molecules in the thoracic aortareduced systolic blood pressure and reduced C-reactive pro-tein levels In addition BC ameliorated insulin resistance bydecreasing insulin release improving glucose tolerance andrestoring insulin signaling by recovering IRS-1 expressionin skeletal muscle tissue In addition BC improved obesityparameters such as leptin and adipocyte size These resultssuggest that BC ameliorated dyslipidemia hypertensioninsulin resistance and obesity in rats with HF-inducedmetabolic syndrome Taken together the results of this studydemonstrate that BC may be used as a new therapeuticapproach for metabolic syndrome

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF) grant funded by the Korea government(MSIP) (2008-0062484) (NRF-2014R1A2A2A01005101)

References

[1] H M El-Bassossy and H A Shaltout ldquoAllopurinol alleviateshypertension and proteinuria in high fructose high salt andhigh fat induced model of metabolic syndromerdquo TranslationalResearch vol 165 no 5 pp 621ndash630 2015

[2] ldquoExecutive summary of the third report of the National Choles-terol Education Program (NCEP) expert panel on detectionevaluation and treatment of high blood cholesterol in adults(Adult Treatment Panel III)rdquo The Journal of the AmericanMedical Association vol 285 pp 2486ndash2497 2001

[3] S M Grundy H B Brewer Jr J I Cleeman S C Smith Jrand C Lenfant ldquoDefinition of metabolic syndrome report ofthe National Heart Lung and Blood InstituteAmerican HeartAssociation conference on scientific issues related to definitionrdquoCirculation vol 109 no 3 pp 433ndash438 2004

[4] R Kumamoto H Uto K Oda et al ldquoDietary fructose enhancesthe incidence of precancerous hepatocytes induced by admin-istration of diethylnitrosamine in ratrdquo European Journal ofMedical Research vol 18 no 1 article 54 2013

[5] P J Havel ldquoDietary fructose implications for dysregulationof energy homeostasis and lipidcarbohydrate metabolismrdquoNutrition Reviews vol 63 no 5 pp 133ndash137 2005

[6] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition amp Metabolismvol 2 article 5 2005

[7] T Ishimoto M A Lanaspa M T Le et al ldquoOpposing effects offructokinase C and A isoforms on fructose-induced metabolicsyndrome in micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 109 no 11 pp 4320ndash4325 2012

[8] B J An J H Kim and O J Kwon ldquoBiological activity of Omija(Schizandra chinensis Baillon) extractsrdquo Journal of the KoreanSociety for Applied Biological Chemistry vol 50 pp 198ndash2032007

[9] D B Shin D W Lee R Yang and J A Kim ldquoAntioxidativeproperties and flavonoids contents of matured Citrus peelextractsrdquo Food Science and Biotechnology vol 15 pp 357ndash3622006

[10] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006

[11] A Bishayee T Mbimba R J Thoppil et al ldquoAnthocyanin-rich black currant (Ribes nigrum L) extract affords chemo-prevention against diethylnitrosamine-induced hepatocellularcarcinogenesis in ratsrdquoThe Journal of Nutritional Biochemistryvol 22 no 11 pp 1035ndash1046 2011

[12] R J Thoppil D Bhatia K F Barnes et al ldquoBlack currantanthocyanins abrogate oxidative stress through Nrf2-mediatedantioxidant mechanisms in a rat model of hepatocellular car-cinomardquo Current Cancer Drug Targets vol 12 no 9 pp 1244ndash1257 2012

[13] EAmbrozewicz AAugustyniakAGęgotek K Bielawska andE Skrzydlewska ldquoBlack-currant protection against oxidativestress formationrdquo Journal of Toxicology and EnvironmentalHealth Part A Current Issues vol 76 no 23 pp 1293ndash1306 2013

[14] T Keszligler B Jansen and A Hesse ldquoEffect of blackcurrant- cranberry- and plum juice consumption on risk factorsassociated with kidney stone formationrdquo European Journal ofClinical Nutrition vol 56 no 10 pp 1020ndash1023 2002

[15] K G M M Alberti R H Eckel S M Grundy et al ldquoHar-monizing the metabolic syndrome a joint interim statement ofthe international diabetes federation task force on epidemiologyand prevention National heart lung and blood instituteAmerican heart association World heart federation Interna-tional atherosclerosis society And international association forthe study of obesityrdquo Circulation vol 120 no 16 pp 1640ndash16452009

[16] M Giris S Dogru-Abbasoglu A Kumral V Olgac N Kocak-Toker and M Uysal ldquoEffect of carnosine alone or combinedwith 120572-tocopherol on hepatic steatosis and oxidative stress infructose-induced insulin-resistant ratsrdquo Journal of Physiologyand Biochemistry vol 70 no 2 pp 385ndash395 2014

[17] S M Grundy J I Cleeman S R Daniels et al ldquoDiagnosis andmanagement of the metabolic syndrome an American HeartAssociationNationalHeart Lung andBlood Institute scientificstatementrdquo Current Opinion in Cardiology vol 21 no 1 pp 1ndash62006

[18] G M Reaven ldquoPathophysiology of insulin resistance in humandiseaserdquo Physiological Reviews vol 75 no 3 pp 473ndash486 1995


[19] Y Zhang R Proenca M Maffei M Barone L Leopold and JM Friedman ldquoPositional cloning of the mouse obese gene andits human homologuerdquo Nature vol 372 no 6505 pp 425ndash4321994

[20] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007

[21] JOClausen THansenC S Bjorbaek et al ldquoInsulin resistanceinteractions between obesity and a common variant of insulinreceptor substrate-1rdquoTheLancet vol 346 no 8972 pp 397ndash4021995

[22] J Danesh J Muir Y-K Wong M Ward J R Gallimore andM B Pepys ldquoRisk factors for coronary heart disease and acute-phase proteins A population-based studyrdquo European HeartJournal vol 20 no 13 pp 954ndash959 1999

[23] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009

[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010

[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009

[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012

[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004

[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Annual worldwide production of BC is approximately500000 to 600000 tons [9] BC contains numerous physio-logically active components including vitamins carotenoidsand flavonoids which have antiulcer anticonvulsion andantidiarrhea effects as well as protective effects againstcarcinomas diabetes and regressive disease [10 11]

Several studies have reported that BC produces antiox-idative effects Anthocyanins from BC abrogate oxida-tive stress through Nrf2-mediated antioxidant mechanisms[12] In addition BC extract has cytoprotective and anti-inflammatory properties [13] Moreover BC protects againstkidney stones [14] However the therapeutic effects of BCin subjects with metabolic disorder have not been reportedThus this study was designed to identify the effect of BCextract on high-fructose diet-inducedmetabolic syndrome inrats

2 Methods and Materials

21 Plant Material and Preparation of BC Extract BC waspurchased fromGukmin Farm (Jeongeup Korea) A voucherspecimen (number HBF191) was deposited in the Herbariumof the Professional Graduate School of Oriental MedicineWonkwang University (Korea) BC (400 g) was boiled with3 L of distilled water at 100∘C for 2 hThe resulting extract wasfiltered through Whatman No 3 filter paper and centrifugedat 990timesg for 20min at 4∘C The resulting supernatant wasconcentrated using a rotary evaporator after which theresulting extract (6319 g) was lyophilized using a freeze-drierand stored at minus70∘C until required

22 Animal Experiments and Diet All experimental pro-cedures were carried out in accordance with the NationalInstitute of Health Guide for the Care and Use of LaboratoryAnimals and were approved by the Institutional AnimalCare and Utilization Committee for Medical Science ofWonkwang University (approval number WKU 14-50) Six-week-oldmale Sprague-Dawley (SD) rats were obtained fromSamtako (Osan Korea) and kept in a roomwith automaticallymaintained temperature (23∘C) humidity (50ndash60) andlightdark cycles (12-h each) throughout the experimentsAfter 1 week of acclimatization the rats were randomlydivided into 4 groups (10 rats per group) The control group(Cont) was fed a regular diet The high-fructose (HF) dietgroup was fed a 60 fructose diet (Research Diet USA)The low-dose BC group was fed 60 fructose diet with100mgkgday BC administered orally by Sonde for a periodof 4weeksThehigh-dose BC groupwas fed 60 fructose dietwith 300mgkgday of BC administered orally by Sonde fora period of 4 weeks The regular diet was composed of 50starch 21 protein 4 fat and a standard mix of vitaminsand minerals The high-fructose diet was composed of 60fructose 20protein 10 fat and a standardmix of vitaminsand minerals

23 Measurement of Blood Pressure Systolic blood pres-sure (SBP) was determined using noninvasive tail-cuffplethysmography and recorded with an automatic sphygmo-graph (MK2000 Muromachi Kikai Tokyo Japan) SBP was

measured once per week At least 10 determinations of SBPwere made during every measurement session Values arepresented as the mean plusmn SEM of 8 measurements

24 Estimation of Oral Glucose Tolerance Two oral glucosetolerance tests (OGTT) were performed 2 days apart after8 weeks of treatment Briefly basal blood glucose concen-trations were measured after 10ndash12 h of overnight fastingafter which glucose (2 gkg body weight) was immediatelyadministered via oral gavage Tail vein blood samples werecollected 30 60 90 and 120min after glucose administration

25 Blood and Tissue Sampling At the end of the experi-ments the thoracic aorta and muscle were separated rinsedwith cold saline and frozen Plasma was obtained fromcoagulated blood samples by centrifugation at 3000 rpm for15min at 4∘C and frozen at minus80∘C

26 Blood Parameters Triglyceride levels in plasma weremeasured using commercial kits (AM 157S-K ASAN Korea)Levels of HDL total cholesterol and low-density lipoprotein(LDL) in plasma were measured using HDL and LDL assaykits (E2HL-100 Bio Assay Systems Germany) Levels ofinsulin in plasma were measured using commercial kits (80-INSRT-E01 ALPCOUK) Levels of C-reactive protein (CRP)in plasma were measured using commercial kits (557825BD Biosciences America) Levels of leptin in plasma weremeasured using commercial kits (ab100773 Abcam UK)Levels of T-Bill and BUN in plasma were measured usingcommercial kits (77184 Arkray Japan)

27 Preparation of Aorta and Measurement of Vascular Reac-tivity The thoracic aorta was rapidly and carefully collectedfrom each rat and placed into cold Krebrsquos solution (118mMNaCl 47mMKCl 11mMMgSO

4 12mMKH

2PO4 15mM

CaCl 25mM NaHCO3 10mM glucose and pH 74) Con-

nective tissue and fat were removed from each thoracic aortaEach thoracic aorta was cut into rings of approximately3mm in length Care was taken to protect the endotheliumfrom accidental damage during the dissection procedureThe thoracic aortic rings were suspended in a tissue bathcontaining Krebrsquos solution at 37∘C by means of 2 L-shapedstainless-steel wires inserted into the lumen and aeratedwith 95 O

2and 5 CO

2 The isometric forces of the

rings were measured using a Grass FT 03 force displacementtransducer connected to a Model 7E polygraph recordingsystem (Grass Technologies Quincy MA USA) A passivestretch of 1 g in the thoracic aortic rings was determinedto be the optimal tension for maximal responsiveness tophenylephrine (10minus6M) The preparations were allowed toequilibrate for approximately 1 h with the Krebrsquos solutionreplaced every 10min The relaxant effects of acetylcholine(ACh 10minus9ndash10minus6M) and sodium nitroprusside (SNP 10minus10ndash10minus5M) in the thoracic aorta rings were studied

28 Western Blot Analysis in the Rat Aorta and MuscleThoracic aorta and muscle homogenates were prepared inice-cold buffer containing 250mM sucrose 1mM EDTA







3 Results







(min)

Glu

cose

(mg

mL)

60

90

120

150

180

0 30 60 90 120

lowast

lowast

ContHF

BC1BC2

(a)

IRS-1

p-AMPK

AMPK

120573-actin

Cont HF BC1 BC2

(b)























HFCont

Cont HF BC1 BC2

BC1 BC2

0

2000

4000

6000

8000

10000

lowastlowast

Size

of a

dipo

se ce

lls (120583

m2)








BC1 BC2

Cont HF













Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast


ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)



4 Discussion





HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results







(min)

Glu

cose

(mg

mL)

60

90

120

150

180

0 30 60 90 120

lowast

lowast

ContHF

BC1BC2

(a)

IRS-1

p-AMPK

AMPK

120573-actin

Cont HF BC1 BC2

(b)























HFCont

Cont HF BC1 BC2

BC1 BC2

0

2000

4000

6000

8000

10000

lowastlowast

Size

of a

dipo

se ce

lls (120583

m2)








BC1 BC2

Cont HF













Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast


ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)



4 Discussion





HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(min)

Glu

cose

(mg

mL)

60

90

120

150

180

0 30 60 90 120

lowast

lowast

ContHF

BC1BC2

(a)

IRS-1

p-AMPK

AMPK

120573-actin

Cont HF BC1 BC2

(b)























HFCont

Cont HF BC1 BC2

BC1 BC2

0

2000

4000

6000

8000

10000

lowastlowast

Size

of a

dipo

se ce

lls (120583

m2)








BC1 BC2

Cont HF













Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast


ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)



4 Discussion





HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HFCont

Cont HF BC1 BC2

BC1 BC2

0

2000

4000

6000

8000

10000

lowastlowast

Size

of a

dipo

se ce

lls (120583

m2)








BC1 BC2

Cont HF













Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast


ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)



4 Discussion





HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BC1 BC2

Cont HF













Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast


ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)



4 Discussion





HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Weeks0 1 2 3 4 5 6 7 8

Systo

lic b

lood

pre

ssur

e (m

mH

g)

60

80

100

120

140

160

lowast

lowastlowastlowast


ContHF

BC1BC2

(a)

minuslog[SNP] (M)

Relat

ive r

elax

atio

n (

)

minus20

0

20

40

60

80

100100 707580859095

lowastlowast

lowastlowast

lowast

ContHF

BC1BC2

(b)

ContHF

BC1BC2

minuslog[ACh] (M)

Rela

tive r

elax

atio

n (

)

minus20

0

20

40

60

80

10090 606570758085

lowastlowastlowast

(c)



4 Discussion





HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HFCont

BC1 BC2

Cont HF BC1 BC2

The l

engt

h of

tuni

ca in

tima-

med

ia (120583

m)

(fold

of c

ontro

l)

0

20

40

60

80

100

120

lowastlowast





Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cont HF BC1 BC2

VCAM-1

ICAM-1

E-selectin

ET-1

eNOS

120573-actin


BC1 BC2HFCont

Dig

itize

an im

agemdash

ET-1

(fold

of c

ontro

l)

0

1

2

3

4

Cont HF BC1 BC2

(a)

Dig

itize

an im

agemdash

ICA

M-1

(fold

of c

ontro

l)

0

2

4

6

8

10

lowastlowast

Cont HF BC1 BC2

(b)

Dig

itize

an im

agemdash

VCA

M-1

(fold

of c

ontro

l)

0123456

lowastlowast

Cont HF BC1 BC2

(c)

Cont HF BC1 BC2

Dig

itize

an im

agemdash

eNO

S(fo

ld o

f con

trol)

00020406081012

lowastlowast

(d)




5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion




Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article blackcurrant suppresses...

Documents