Catechin Averts Experimental Diabetes Mellitus-Induced Vascular Endothelial Structural and Functional Abnormalities

Catechin Averts Experimental Diabetes Mellitus-InducedVascular Endothelial Structural and Functional Abnormalities

Pooja Bhardwaj • Deepa Khanna • Pitchai Balakumar

Published online: 19 September 2013

� Springer Science+Business Media New York 2013

Abstract Diabetes mellitus is associated with an induc-

tion of vascular endothelial dysfunction (VED), an initial

event that could lead to the pathogenesis of atherosclerosis

and hypertension. Previous studies showed that catechin, a

key component of green tea, possesses vascular beneficial

effects. We investigated the effect of catechin hydrate in

diabetes mellitus-induced experimental vascular endothe-

lial abnormalities (VEA). Streptozotocin (50 mg/kg, i.p.,

once) administration to rats produced diabetes mellitus,

which subsequently induced VEA in 8 weeks by markedly

attenuating acetylcholine-induced endothelium-dependent

relaxation in the isolated aortic ring preparation, decreasing

aortic and serum nitrite/nitrate concentrations and impair-

ing aortic endothelial integrity. These abnormalities in

diabetic rats were accompanied with elevated aortic

superoxide anion generation and serum lipid peroxidation

in addition to hyperglycemia. Catechin hydrate treatment

(50 mg/kg/day p.o., 3 weeks) markedly prevented diabetes

mellitus-induced VEA and vascular oxidative stress.

Intriguingly, in vitro incubation of L-NAME (100 lM), an

inhibitor of nitric oxide synthase, or Wortmannin

(100 nM), a selective inhibitor of phosphatidylinositol

3-kinase (PI3K), markedly prevented catechin hydrate-

induced improvement in acetylcholine-provoked endothe-

lium-dependent relaxation in the diabetic rat aorta. More-

over, catechin hydrate treatment considerably reduced the

elevated level of serum glucose in diabetic rats. In con-

clusion, catechin hydrate treatment prevents diabetes mel-

litus-induced VED through the activation of endothelial

PI3K signal and subsequent activation of eNOS and gen-

eration of nitric oxide. In addition, reduction in high glu-

cose, vascular oxidative stress, and lipid peroxidation

might additionally contribute to catechin hydrate-associ-

ated prevention of diabetic VEA.

Keywords Diabetes mellitus � Vascular endothelial

abnormalities � Catechin hydrate � PI3K � eNOS �Nitric oxide � Oxidative stress

Abbreviations

Ach Acetylcholine

ANOVA Analysis of variance

CPCSEA Committee for the purpose of control and

supervision of experiments on animals

DMSO Dimethyl sulfoxide

DTPA Diethylene triamine pentaacetic acid

eNOS Endothelial nitric oxide synthase

HDL High density lipoprotein

L-NAME N (omega)-nitro-L-arginine methyl ester

NBT Nitroblute trazolium

NO Nitric oxide

PI3K Phosphatidylinositol 3-kinase

SNP Sodium nitroprusside

STZ Streptozotocin

TBARS Thiobarbituric acid reactive substances

VED Vascular endothelial dysfunction

VEA Vascular endothelial abnormalities

P. Bhardwaj � D. Khanna (&)

Cardiovascular Pharmacology Division, Department of

Pharmacology, Institute of Pharmacy, Rajendra Institute of

Technology and Sciences (RITS), Sirsa 125 055, Haryana, India

e-mail: [email protected]

P. Balakumar

Pharmacology Unit, Faculty of Pharmacy, AIMST University,

Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia

123

Cardiovasc Toxicol (2014) 14:41–51

DOI 10.1007/s12012-013-9226-y

Introduction

Diabetes mellitus is one of insidious conditions, and the

morbidity and mortality due to this disorder remain high

worldwide. As per the 2011 report of the International

Diabetes Federation (IDF), about 366 million people have

been affected by diabetes mellitus worldwide, and this range

is anticipated to reach 552 million by the year 2030 [1]. It

was also suggested by the IDF that 1 per 10 adults will have

diabetes mellitus by 2030 [1]. Diabetes mellitus is a group of

metabolic disorders characterized by chronic hyperglyce-

mia, which occurs as result of either lack of insulin (type 1

diabetes mellitus) or deficient secretion and release of

insulin and or insulin resistance (type 2 diabetes mellitus).

An inadequacy in controlling diabetes mellitus often leads to

various lethal disorders, including vascular complications

[2–4]. Vascular endothelial dysfunction (VED) is an initial

event that accounts for diabetic vasculopathy [5, 6].

Vascular endothelium is a simple monolayer of inner

blood vessels that regulates vascular tone [7]. The endo-

thelium-derived relaxing factor (EDRF) was first demon-

strated in the experiments of Furchgott and Zawadzki [8, 9]

that was subsequently shown to be nothing but nitric oxide

(NO). NO is generated during endothelial NO synthase

(eNOS)-mediated conversion of L-arginine to L-citrulline in

the presence of various substrates and co-factors [10, 11].

NO produced by the eNOS is considered as a fundamental

determinant in maintaining the vascular function [12]. In

October 12, 1998, the Nobel Prize in Medicine or Physi-

ology was awarded to Robert Furchgott, Louis Ignarro, and

FeridMurad for their great discoveries concerning NO as a

key signaling molecule in regulating cardiovascular phys-

iology [13]. In the normal vascular physiology, NO-med-

iated signaling has a potential to inhibit vascular

inflammation, thrombosis, and cellular proliferation [13,

14]. VED occurs as a result of eNOS downregulation and

inactivation, eNOS uncoupling, high oxidative stress,

diminished production and bioavailability of NO, and

imbalance in the relative contribution of endothelium-

derived relaxing and contracting factors [11, 15–17].

The phosphatidylinositol 3-kinase (PI3K) pathway plays

a key role in maintaining vascular function by activating

the serine/threonine protein kinase (protein kinase B/Akt),

which subsequently enhances eNOS phosphorylation/acti-

vation and NO production [12, 18]. However, impairment

in PI3K/Akt pathway has been demonstrated in the aorta of

diabetic mouse [19, 20]. Moreover, enhanced oxidative

stress might additionally play a role in the pathogenesis of

diabetes mellitus-induced vascular complications [21, 22].

Catechins are polyphenolic compounds obtained mainly

from green tea leaves of the plant Camellia sinensis [23].

Catechins have vascular protective effects through multiple

actions that include anti-oxidant, anti-inflammatory, anti-

thrombogenic, anti-proliferative, and anti-hypertensive

potentials [24, 25]. Catechin affords anti-oxidant effect by

scavenging free radicals and inducing anti-oxidant

enzymes [26–28]. Interestingly, the constituent of green tea

improved endothelium-dependent vasodilation in vitro by

activating eNOS through endothelial PI3K/Akt pathways

[29]. Therefore, the present study has been designed to

investigate the effect of catechin hydrate in diabetes mel-

litus-induced experimental vascular endothelial abnormal-

ities (VEA) and to investigate the possible role of PI3K and

eNOS signaling in this context.

Materials and Methods

The experimental protocol used in the present study was

approved by the ‘Institutional Animal Ethics Committee’

in accordance with the guidelines given by the ‘Committee

for the Purpose of Control and Supervision of Experiments

on Animals’ (CPCSEA, Chennai, India). Wistar albino

female rats weighing about 250–350 g were used in the

present study. The animals were acclimatized in the

‘Institutional animal house’ and maintained on rat chow

(Ashirwad Industries, Mohali, Punjab, India) and tap water.

Rats were allowed for ad libitum access to food and water.

They were exposed to normal day and night cycles.

Experimental Protocol

Six groups were employed in the present study and each

group comprised six rats. Catechin hydrate treatment in the

dose of 50 mg/kg/day per os was employed in the present

study. Based on a previous report [27], the dose of catechin

hydrate was selected. Catechin hydrate was dissolved in

warm distilled water. Normal and diabetic rats were housed

in polypropylene cages with stainless steel top grill for

1 month before starting experiments. Wortmannin was

dissolved in 0.01 % DMSO, while L-NAME was dissolved

in distilled water.

Group I (normal control): Rats were maintained on

standard food and water, and no treatment was given.

Group II (diabetic control): Rats were administered strep-

tozotocin (STZ, 50 mg/kg, i.p., once) dissolved in citrate

buffer of pH 4.5 and were allowed for 8 weeks to develop

experimental VEA. Group III (catechin per se): Normal

rats were administered catechin hydrate (50 mg/kg/day,

p.o.) for 3 weeks. Group IV (catechin treated): The dia-

betic rats after 5 weeks of STZ administration were treated

with catechin hydrate (50 mg/kg/day, p.o.) for 3 weeks.

Group V (L-NAME incubated aortic ring of catechin

treated): The diabetic rats were treated with catechin

hydrate (50 mg/kg/day, p.o., 3 weeks) as mentioned in

42 Cardiovasc Toxicol (2014) 14:41–51

123

Group IV. The in vitro effect of L-NAME (100 lM) (the

aortic ring was incubated with L-NAME for 30 min) on

endothelium-dependent and endothelium-independent

relaxation in the phenylephrine-precontracted isolated

aortic ring preparation was evaluated. Group VI (Wort-

mannin incubated aortic ring of catechin treated): The

diabetic rats were treated with catechin hydrate (50 mg/kg/

day, p.o., 3 weeks) as mentioned in Group IV. The in vitro

effect of Wortmannin (100 nM) (the aortic ring was incu-

bated with Wortmannin for 30 min) on endothelium-

dependent and endothelium-independent relaxation in the

phenylephrine-precontracted isolated aortic ring prepara-

tion was evaluated.

Induction of Experimental VEA

The experimental diabetes mellitus was induced by

administering STZ (50 mg/kg, i.p., once) dissolved in cit-

rate buffer of pH 4.5, and STZ-diabetic rats were allowed

for 8 weeks to develop VEA [30, 31].

Assessment of Experimental Diabetes Mellitus

The blood sugar level was monitored once after 72 h of

STZ administration, and serum glucose concentration was

estimated by glucose oxidase–peroxidase (GOD–POD)

method using the commercially available kit (Crest bio-

systems, Goa, India). Rats showing blood glucose level of

greater than 200 mg/dL were selected and termed as dia-

betic rats. The initial and final glucose concentrations

(8 weeks after the STZ administration) were estimated.

The working reagent (1000 lL) was added to 10 lL dis-

tilled water, 10 lL glucose standard solution (100 mg/dL),

and 10 lL serum sample with thorough mixing to prepare

blank (B), standard (S), and test (T), respectively. The

mixture was incubated at 37 �C for 10 min. The absor-

bance of Sand Twas was read against B within 60 min at

505 nm spectrophotometrically (LABINDIA UV 3000,

UV–Visible Spectrophotometer, India). This method is

based on the principle that glucose is oxidized to gluconic

acid and hydrogen peroxide catalyzed by glucose oxidase.

Hydrogen peroxide thus formed reacts with 4-hydroxy

benzoic acid (4-HBA) and 4-aminoantipyrine (4-AAP) in

presence of peroxidase to form a red colored Quinoneimine

dye complex whose absorbance was read. Intensity of the

color formed is directly proportional to the amount of

glucose present in the sample.

The total serum glucose level was calculated using the

following formula:

Glucose concentration mg=dLð Þ ¼ Absorbance of T

Absorbance of S� 100

Assessment of VEA

The development of vascular endothelial structural and

functional abnormalities was assessed by determining ace-

tylcholine (Ach)-induced endothelium-dependent relaxa-

tion using isolated aortic ring preparation, estimating aortic

and serum nitrite/nitrate concentration, and employing

scanning electron microscopic study and hematoxylin–eosin

staining of thoracic aorta.

Isolated Rat Aortic Ring Preparation

The rat was killed and the thoracic aorta was incised. The

aorta was cut into a ring of around 5 mm in length and

mounted in an organ bath containing Krebs-Henseleit

solution (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5 mM;

MgSO4.7H2O 1.2 mM; NaHCO3 25 mM; KH2PO4

1.2 mM; C6H12O6 11.1 mM) of pH 7.4, bubbled with oxy-

gen (95 % O2 and 5 % CO2) and maintained at 37 �C. The

preparation was allowed to equilibrate for 90 min under

1.5 g tension. The isometric reading was recorded using a

force transducer (Ft-2518) connected to Physiography

(INCO, Ambala, Haryana, India). Initially, the aortic ring

preparation was primed with 80 mM KCl to assess its

functional integrity and to improve its contractile response.

The cumulative dose responses of Ach (10-8, 10-7, 10-6,

10-5, and 10-4 M) or sodium nitroprusside (SNP; 10-8,

10-7, 10-6, 10-5, and 10-4 M) were recorded in phenyl-

ephrine (3 9 10-6 M)-precontracted aortic ring preparation

with intact and denuded endothelium, respectively [32, 33].

Estimation of Aortic and Serum Nitrite/Nitrate

Concentration

A part of isolated aortic tissue was homogenized in 5 mL

of phosphate-buffered saline of pH 7.4 and centrifuged at

10,000g for 20 min. The supernatant was used for esti-

mating the aortic concentration of nitrite/nitrate and protein

content. The carbonate buffer (400 lL, pH 9.0) (equal

volume of 500 mM sodium bicarbonate and 50 mM

sodium carbonate were mixed to obtain 50 mM carbonate

buffer) was added to 100 lL supernatant from homoge-

nized aortic sample or 100 lL of serum sample in separate

tubes, followed by addition of small amount (*0.15 g) of

copper–cadmium alloy (copper and cadmium in the ratio of

1:10). The tubes were incubated at room temperature for

1 h with thorough shaking for reducing nitrate to nitrite.

The reaction was stopped by adding 100 lL of 0.35 M

NaOH. Following this, 400 lL of 120 mM zinc sulfate

solution was added in order to deproteinize the samples.

The samples were allowed to stand for 10 min and then

centrifuged (REMI Cooling Centrifuge, India) at 4,000g for

Cardiovasc Toxicol (2014) 14:41–51 43

123

10 min. Greiss reagent (mixture of 250 lL of 1.0 % sul-

fanilamide prepared in 3 N HCl and 250 lL of 0.1 %

N-naphthyl ethylenediamine prepared in water) was added

to aliquots (500 lL) of clear supernatant, and aortic and

serum nitrite/nitrate concentrations were measured spec-

trophotometrically (LABINDIA 3000, India) at 545 nm

[34]. The standard curve of sodium nitrite (0.1–3 nM) was

plotted to calculate the concentration of aortic nitrite/

nitrate (lM/mg of protein) and serum nitrite/nitrate (lM/

L). The protein concentration in the homogenized aortic

preparation was estimated using the commercially avail-

able kit (AGAPPE Diagnostics Ltd., Kerela, India).

Scanning Electron Microscopic Study

The scanning electron microscopic study was performed in

order to examine the integrity of vascular endothelium [35].

This study was carried out in the Division of Entomology,

Indian Agricultural Research Institute, New Delhi, India. The

longitudinal strips of thoracic aorta (3–4 mm) were fixed in

2.5 % glutaraldehyde and 2 % para formaldehyde (PF) in

0.1 M phosphate buffer (pH 7.4) for 6–12 h at 4 �C, and

subsequently dehydrated in a series of acetone solution (50 %

for 20 min, 70 % for 20 min, 80 % for 20 min, 90 % for

20 min, and 100 % for 50 min), followed by isoamylacetate

(100 %) and acetone (100 %) solution in the ratio of 1:1 for

20 min, and subsequently by isoamylacetate (100 %) alone

for 20 min. Aortic segments were further dried using hex-

amethyldisilazane (Sigma-Aldrich Ltd., St. Louis, MO,

USA). The segments were then mounted on aluminum stubs

and coated with palladium (30 nm) (JFC-1100) and viewed

using Zeiss EVOMA-10 scanning electron microscope in

order to examine the integrity of aortic vascular endothelium.

Histological Assessment of the Integrity of Aortic Vascular

Endothelial Layer

The histological assessment of the integrity of aortic vas-

cular endothelial layer was performed with the help of

Mangalam Pathological Laboratory, Haryana, India. A part

of excised aorta was immediately immersed in 10 % neu-

tral-buffered formalin, dehydrated in graded concentrations

of ethanol, immersed in xylene, and embedded in paraffin.

A transverse section of 5 lM was stained with hematox-

ylin–eosin [36, 37]. The aortic section was examined using

Motic Microscope BA310 (Motic, USA) at 40X to assess

the integrity of endothelial layer.

Assessment of Oxidative Stress

The oxidative stress was assessed by estimating aortic

superoxide anion generation and serum thiobarbituric acid

reactive substances (TBARS).

Estimation of Aortic Superoxide Anion

Aorta was incised into transverse rings of 5–6 mm in length

and placed in 5 mL Krebs-Henseleit solution buffer con-

taining nitroblutetrazolium (NBT, 100 lM/L) and incubated

at 37 �C for 90 min. The NBT reduction was terminated by

adding 5 mL of 0.5 N HCl. The rings were then minced and

homogenized in a mixture of 0.1 N NaOH and 0.1 % sodium

dodecyl sulfate in water containing 40 mg/L of diethylene-

triaminepentaacetic acid (DTPA). The mixture was then

centrifuged (REMI Cooling Centrifuge, India) at

20,000g for 20 min, and the resultant pellets were re-sus-

pended in 1.5 mL pyridine and kept at 80 �C for 90 min to

extract formazan. The mixture was centrifuged at

10,000g for 10 min, and the absorbance of developed for-

mazan was determined spectrophotometrically (LABINDIA

3000, India) at 540 nm [38, 39]. The amount of reduced

NBT (picoM/min/mg) was calculated using the following

formula = A�V/(T�Wt�e�l), where A is absorbance, V is the

volume of solution (1.5 mL), T is the time for aortic rings

incubated with NBT (90 min), Wt is the blotted wet weight

of aortic rings, e is an extinction coefficient (0.72 L/mmol/

mm), and l is the length of light path (10 mm).

Estimation of Serum TBARS

One milliliter trichloroaceticacid (20 %) was added to

100 lL serum and 1.0 mL of 1 % TBARS reagent (mix-

ture of equal volume of 1 % thiobarbituric acid aqueous

solution in 1 M NaOH (50 mg/mL) and glacial acetic

acid), which were mixed and incubated at 100 �C for

30 min. The samples, after cooling on ice, were centrifuged

(REMI Cooling Centrifuge, India) at 1,000g for 20 min.

Serum concentration of TBARS was measured spectro-

photometrically at 532 nm [40]. The standard curve of

1,1,3,3-tetramethoxypropane (0.1–1 nM) was plotted in

order to calculate the concentration of serum TBARS.

Drugs and Chemicals

Streptozotocin was purchased from Himedia Pvt. Ltd., Mum-

bai, India. Catechin hydrate was obtained from Sigma-Aldrich

Ltd., St. Louis, MO, USA. It was 98 % (HPLC) pure com-

pound. Wortmannin, L-phenylephrine, 1,1,3,3-tetramethoxy-

propane and L-NAME were also purchased from Sigma-

Aldrich Ltd., St. Louis, MO, USA. Acetylcholine iodide and

DTPA were purchased from Otto kemi, Mumbai, India. SNP,

glutaraldehyde, and trichloroacetic acid were purchased from

RANKEM, New Delhi, India. NBT was purchased from SD

fine, Mumbai, India. Thiobarbituric acid was purchased from

Otto Chemika-Biochemika, Mumbai, India. All other chemi-

cals used in the present study were of analytical grade.


123

Statistical Analysis

All values were expressed as mean ± SD. Data for isolated

aortic ring preparation were statistically analyzed using

repeated measures of analysis of variance (RM ANOVA),

followed by Student–Newman–Keuls Method. The endo-

thelium-dependent relaxation (Ach, 10-8, 10-7, 10-6,

10-5, and 10-4 M) and endothelium-independent relaxa-

tion (SNP, 10-8, 10-7, 10-6, 10-5, and 10-4 M) in

between all experimental groups were statistically analyzed

using one-way ANOVA, followed by Tukey’s multiple

comparison test. The data for aortic and serum levels of

nitrite/nitrate, aortic superoxide anion generation, serum

TBARS, and glucose were statistically analyzed using one-

way ANOVA, followed by Tukey’s multiple comparison

test. A ‘p’ value of less than 0.05 was considered statisti-

cally significant.

Results

Rats administered STZ (50 mg/kg, i.p., once) produced

hyperglycemia, and those rats exhibited the serum glucose

level of more than 200 mg/dL after 72 h of STZ admin-

istration were selected and termed as diabetic rats, which

were included in the present study. At the end of the

experimental protocol (8 weeks after the administration of

STZ), the fasting blood glucose level was noted to

be markedly increased in diabetic rats (Table 1). Catechin

hydrate treatment (50 mg/kg/day, p.o., 3 weeks) submaxi-

mally and significantly reduced the elevated serum glucose

level in diabetic rats (Table 1).

Catechin hydrate (50 mg/kg/day, p.o., 3 weeks) pro-

duced no statistically significant per se effects on various

parameters assessed in normal rats. Around 10 % reduction

in body weight was noted in STZ (50 mg/kg, i.p., once)-

administered diabetic rats that was not significantly altered

in the treatment group.

Effect of Catechin Hydrate on Endothelium-Dependent

Relaxation

In a phenylephrine (3 9 10-6 M) pre-contracted isolated

rat aortic ring preparation, administration of Ach (10-8,

10-7, 10-6, 10-5 and 10-4 M) produced markedly the

endothelium-dependent relaxation in a dose-dependent

manner. However, Ach-induced endothelium-dependent

relaxation was markedly diminished in the aorta isolated

from diabetic rats. Interestingly, a significant restoration of

Ach-induced endothelium-dependent relaxation was

observed in the aorta isolated from catechin hydrate

(50 mg/kg/day, p.o., 3 weeks)-treated diabetic rats. How-

ever, catechin hydrate-induced restoration of Ach-pro-

voked endothelium-dependant relaxation in the aorta of

diabetic rats was significantly attenuated upon the incuba-

tion of the aortic ring with either L-NAME (100 lM) or

Wortmannin (100 nM) (Fig. 1).

Effect of Catechin Hydrate on Endothelium-

Independent Relaxation

Sodium nitroprusside (10-8, 10-7, 10-6, 10-5, and 10-4 M)

dose-dependently produced endothelium-independent

relaxation in the phenylephrine (3 9 10-6 M) pre-con-

tracted isolated rat aortic ring preparation in all diabetic and

non-diabetic groups employed in the present study with or

without catechin hydrate treatment (Fig. 2).

Effect of Catechin Hydrate on Aortic and Serum

Nitrite/Nitrate Concentration

The aortic concentration of nitrite/nitrate was noted to be

significantly diminished in diabetic rats as compared to

normal rats. Similarly, serum nitrite/nitrate concentration

was decreased in diabetic rats as compared to normal rats.

Catechin hydrate treatment, however, significantly elevated

the diminished levels of aortic and serum nitrite/nitrate in

diabetic rats (Figs. 3, 4).

Effect of Catechin Hydrate on the Integrity of Vascular

Endothelium

A marked disruption in the integrity of vascular endothe-

lium was noted in the aorta of diabetic rats examined in

scanning electron microscopic study (Fig. 5). Likewise, the

histopathological study using hematoxylin–eosin staining

Table 1 Effect of catechin hydrate on serum glucose in STZ-administered rats

Assessments (mg/dL) Normal rats STZ-administered rats Catechin per se Catechin treated

Serum glucose 103.23 ± 8.34 284.59 ± 26.2a 92.53 ± 8.21 175.86 ± 6.58b

All values were represented as mean ± SDa p \ 0.001 versus normal ratsb p \ 0.001 versus STZ-administered rats


123

revealed the disruption of endothelial cell layer of the aorta

isolated from diabetic rats as compared to the normal rat

aorta, which showed uniform endothelial cell layer

(Fig. 6). However, catechin hydrate treatment to diabetic

rats markedly improved the integrity of aortic vascular

endothelium (Figs. 5, 6).

Effect of Catechin Hydrate on Aortic Superoxide Anion

Generation and Serum TBARS

A marked increase in aortic superoxide anion generation

was noted in diabetic rats as compared to normal rats

(Fig. 7). Likewise, serum TBARS concentration was noted

to be markedly increased in diabetic rats as compared to

normal rats (Fig. 8). However, treatment with catechin

hydrate significantly prevented diabetes mellitus-associated

marked increase in aortic superoxide anion generation and

serum TBARS (Figs. 7, 8).

Discussion

The VED is an initial event that roots for the development

of cardiovascular disorders, including atherosclerosis and

hypertension [11, 41–43]. The present study reveals for the

first time the novel therapeutic ability of catechin hydrate

in preventing diabetes mellitus-induced vascular endothe-

lial structural and functional abnormalities. The key finding

of this study is that the protective effect of catechin in

8 7 6 5 40

20

40

60

80

100

Normal ControlDiabetic Control

Catechin Per se

Catechin Treated

L-NAME Incubated Aortic Ring of Catechin TreatedWortmannin Incubated Aortic Ring of Catechin Treated

-Log M(SNP)

% P

reco

ntra

ctio

n

Fig. 2 Effect of catechin hydrate on SNP-induced endothelium-

independent relaxation. SNP dose-dependently produced endothe-

lium-independent relaxation in the phenylephrine (3 9 10-6 M) pre-

contracted isolated rat aortic ring preparation in all diabetic and non-

diabetic groups with or without catechin hydrate treatment. Responses

were represented as percentage of maximum contraction induced by

phenylephrine (3 9 10-6 M). All values were expressed as

mean ± SD

0

5

10

15

20

25

a

b


Catechin Per seCatechin Treated

Aor

tic

Nit

rite

/ Nit

rate

Con

c. (

mol

/ mg

of p

rote

in)

Fig. 3 Effect of catechin hydrate on aortic nitrite/nitrate concentra-

tion (l mol/mg of protein). Catechin hydrate treatment significantly

elevated the diminished level of aortic nitrite/nitrate in diabetic rats.

All values were expressed as mean ± SD. a = p \ 0.001 versus

normal control; b = p \ 0.001 versus diabetic control

Fig. 1 Effect of catechin hydrate on Ach-induced endothelium-

dependent relaxation. In a phenylephrine pre-contracted isolated rat

aortic ring preparation, administration of Ach produced markedly the

endothelium-dependent relaxation in a dose-dependent manner that

was markedly diminished in the aorta isolated from diabetic rats. A

significant restoration of Ach-induced endothelium-dependent relax-

ation was observed in the aorta isolated from catechin hydrate-treated

diabetic rats. However, catechin hydrate-induced restoration of Ach-

provoked endothelium-dependant relaxation in the aorta of diabetic

rats was significantly attenuated upon the incubation of the aortic ring

with either L-NAME or Wortmannin. Responses were represented as

percentage of maximum contraction induced by phenylephrine

(3 9 10-6 M). All values were expressed as mean ± SD.

a = p \ 0.001 versus normal control; b = p \ 0.001 versus diabetic

control. c, d = p \ 0.001 versus catechin treated


123

preventing diabetes mellitus-induced experimental VED

might be mediated through the activation of PI3K and

eNOS signaling system.

A reduction in Ach-induced endothelium-dependent

vasorelaxation is considered as an index of VED [32, 44,

45]. In the present study, a marked reduction in Ach-

induced endothelium-dependent relaxation was noted in

the aorta isolated from diabetic rats, indicating the

development of VED. Further, the scanning electron

microscopic study revealed a marked impairment in the

integrity of vascular endothelium in the diabetic rat aorta.

Moreover, a marked damage in the vascular endothelial

layer was noted in the diabetic rat aorta as indicated in

histological study of hematoxylin–eosin staining. These

results suggest the development of vascular endothelial

structural and functional abnormalities in the diabetic rat

aorta.

The occurrence of oxidative stress is one of major events

for the induction of VEA [46, 47]. The development of

oxidative stress is pertained with the elevation of aortic

superoxide anion generation and serum TBARS [39, 48–

50]. In the present study, a significant increase in aortic

superoxide anion generation, as assessed in terms of esti-

mating reduced NBT, was noted in diabetic rats. In addi-

tion, serum TBARS concentration was noted to be

significantly increased in diabetic rats as compared to

normal rats. These results highlight the development of

marked vascular oxidative stress in diabetic rats. It could

therefore be possible that induction of significant vascular

oxidative stress in rats afflicted with diabetes mellitus

might have played a pivotal role in damaging vascular

endothelial layer and subsequently impairing the vascular

endothelial functional integrity.

It is worth mentioning that vascular oxidative stress is

involved in reducing the bioavailability of NO in the

Fig. 5 Effect of catechin

hydrate on the integrity of

vascular endothelium. The

scanning electron microscopic

study was performed to examine

the integrity of vascular

endothelium of the rat thoracic

aorta. This study revealed a

marked disruption in the

integrity of vascular

endothelium in the thoracic

aorta of diabetic rats. Catechin

hydrate treatment to diabetic

rats markedly improved the

integrity of aortic vascular

endothelium. a Normal control;

b diabetic control; c catechin

per se; d catechin treated

0

5

10

15

20

a

b



Seru

m N

itri

te /

Nit

rate

( m

ol/L

)µ

Fig. 4 Effect of catechin hydrate on serum nitrite/nitrate concentra-

tion (l mol/L). Catechin hydrate treatment significantly elevated the

diminished level of serum nitrite/nitrate in diabetic rats. All values

were expressed as mean ± SD. a = p \ 0.001 versus normal control;

b = p \ 0.001 versus diabetic control


123

vascular bed [38] and subsequently inducing the dysfunc-

tion of the endothelium [51–53]. In the present study, the

developed high degree of vascular oxidative stress might

therefore have played a role in diminishing the

bioavailability of NO in the aortic vascular bed of diabetic

rats. This pathological event might have caused VED in

diabetic rats. This contention is strongly supported by the

fact of results obtained in the present study that a signifi-

cant decrease in aortic and serum nitrite/nitrate

A B

C D

Bar 100µmFig. 6 Effect of catechin

hydrate on the integrity of

vascular endothelial layer. The

histological examination was

performed using Motic

Microscope BA310 (Motic,

USA) at 40X (scale

bar = 100 lM).

Histopathological study using

hematoxylin–eosin staining

revealed the disruption of

endothelial cell layer of the

aorta isolated from diabetic rats

as compared to the normal rat

aorta, which showed uniform

endothelial cell layer. Catechin

hydrate treatment to diabetic

rats markedly improved the

integrity of aortic vascular

endothelial layer. a Normal

control; b diabetic control;

c catechin per se; d catechin

treated

0

10

20

30

40a

b

Normal Control

Diabetic Control


Red

uced

NB

T (

pic

o m

ol/m

in/m

g )

Fig. 7 Effect of catechin hydrate on aortic superoxide anion

generation as assessed in terms of estimating reduced NBT

(picomol/min/mg). Treatment with catechin hydrate significantly

prevented diabetes mellitus-associated marked increase in aortic

superoxide anion generation. All values were expressed as

mean ± SD. a = p \ 0.001 versus normal control; b = p \ 0.001

versus diabetic control

0

2

4

6

8

10

b

a


Catechin Per se Catechin Treated

Ser

um T

BA

RS

( m

ol/L

)µ

Fig. 8 Effect of catechin hydrate on serum concentration of TBARS

(lmol/L). Treatment with catechin hydrate significantly prevented

diabetes mellitus-associated marked increase in serum TBARS. All

values were expressed as mean ± SD. a = p \ 0.001 versus normal

control; b = p \ 0.001 versus diabetic control


123

concentration accompanying with high oxidative stress was

noted in diabetic rats.

Catechin is a polyphenolic compound obtained mainly

from green tea leaves of the plant C. sinensis [23]. In the

present study, 3-week treatment with catechin hydrate

markedly improved Ach-induced aortic endothelium-

dependent relaxation in diabetic rats. However, in vitro

incubation of L-NAME markedly prevented catechin

hydrate-induced improvement in Ach-provoked endothe-

lium-dependent relaxation in the isolated aorta of diabetic

rats. Likewise, in vitro incubation of Wortmannin mark-

edly prevented catechin-induced improvement in Ach-

provoked endothelium-dependent relaxation in the isolated

aorta of diabetic rats. L-NAME is an inhibitor of NOS [54,

55], while Wortmannin is a selective inhibitor of PI3K [56,

57]. It is a known fact that activation of PI3K could acti-

vate eNOS and generate NO to mediate vascular endo-

thelial functional regulation [12, 18]. Thus, it could be

suggested that catechin has a potential to prevent diabetic

VED that might be mediated through the activation of

endothelial PI3K and subsequent activation of eNOS and

generation of nitric oxide. This contention is strongly

supported by the results obtained in the present study that

catechin-induced restoration of Ach-provoked endothe-

lium-dependent relaxation in the aorta of diabetic rats was

significantly attenuated upon the incubation of the aortic

ring with either L-NAME or Wortmannin.

Catechin has a potent anti-oxidant action [25, 26, 58]. In

the present study, aortic endothelial layer damage and

impairment of endothelial integrity in diabetic rats were

markedly prevented by catechin hydrate treatment. In

addition, catechin hydrate treatment significantly elevated

the aortic and serum concentration nitrite/nitrate in diabetic

rats. These beneficial vasculo-protective actions of catechin

hydrate might be associated with its significant vascular

anti-oxidant action. This contention is well-supported by

the results obtained in the present study that the treatment

with catechin hydrate significantly prevented diabetes

mellitus-associated marked increase in aortic superoxide

anion generation and serum TBARS. Thus, it might be

suggested that the vascular anti-oxidant potential of cate-

chin hydrate could have contributed to preventing endo-

thelial layer damage and impairment of endothelial

integrity in diabetic rats. Catechin hydrate-mediated vas-

cular anti-oxidant action and reduction in lipid peroxida-

tion could also have played a role in improving the

bioavailability of NO in diabetic rats, and this might have

caused vascular functional improvement as noted in the

present study in catechin hydrate-treated diabetic rats.

In conclusion, our study suggests that catechin hydrate

has a therapeutic potential in preventing diabetic VED that

might be mediated through the activation of endothelial

PI3K and subsequent activation of eNOS and generation of

NO. In addition, significant reduction in glucose level,

vascular oxidative stress, and lipid peroxidation might have

additionally contributed to catechin hydrate-associated

prevention of diabetes mellitus-induced vascular endothe-

lial structural and functional abnormalities.

Acknowledgments We express our gratefulness to Dr. Rajendar

Singh Sra, MD, Chairman, Shri Om Parkash, Secretary, and Mr.

Sanjeev Kalra, Administrator, Rajendra Institute of Technology and

Sciences, Sirsa, Haryana, India, for their support.

Conflict of interest The authors declare that there is no conflict of

interest.

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Documents

Catechin Averts Experimental Diabetes Mellitus-Induced Vascular Endothelial Structural and Functional Abnormalities