Effect of curcumin on cetrain lysosomal hydrolases in isoproterenol-induced myocardial infarction in rats

Biochemical Pharmacology, Vol. 51, pp. 47-51, Copyright 0 1995 Elsevier Science Inc.

ISSN 0006-2952/96/$15.00 + 0.00 SSDI 0006.2952(95)02118-3

1996.

ELSEVIER

Effect of Curcumin on Cetrain Lysosomal Hydrolases in Isoproterenol-Induced Myocardial Infarction in Rats Chandrasekar Nirmab and Rengarajulu Puwanakrishnan* DEPARTMENTOF BIOTECHNOLOGY,CENTRAL LEATHERRESEARCHINSTITUTE, MADRAS-600020,INDlA ABSTRACT. ‘The effect of curcumin on lysosomal hydrolases in serum and heart was studied by determining the activities of P-glucuronidase, P-N-acetylglucosaminidase, cathepsin B, cathepsin D, and acid phosphatase. Rats treated with isoproterenol (30 mg/lOO g body weight) showed a significant increase in serum lysosomal hydrolase activities, which were found to decrease after curcumin treatment. Isoproterenol administration to rats resulted in decreased stability of the membranes, which was reflected by the lowered activity of cathepsin D in mitochondrial, lysosomal, and microsomal fractions. Curcumin treatment returned the activity levels almost to normal, showing that curcumin restored the normal function of the membrane. Histopathological studies of the infarcted rat heart also showed a decreased degree of necrosis after curcumin treatment. BIOCHEMPHARMA-

COL 51;1:47-51, 1996. KEY WORDS.

Considerable ations

attention

that might

damage. somes, have

has been focused

accompany

Formation

ischemic

of autophagic

and spread been

isoproterenol;

of lysosomal

observed

crosis is mediated

hydrolases,

some

of the

hydrolases

lysosomal creases tabolism

from other

vacuoles

mentally

[3]. A d ecrease

of different induced

ISOt-induced

myocardial myocardial

hydrolase

damage

and infarcted

activities

in particulate

this

heart

disruption

of cellular could

leading

tissue

the cell

cumin

constitthat

by ischemia to

stability

in-

to altered

me-

constituents,

[4], and collagen

(mouse

ne-

viz. gly-

[5], in experi-

the

membranes

infarction

results

in increased

that may be responsible [15].D ecreased

fractions

of ischemi,:

tant

constituent

antiinflammatory

of Curcumtl

the

bone

been

target

proposed

that

particularly

viability

changes

treatment. nase,

cytes rather

stabilization

[17],

cell mem-

may prolong

The purpose

the

of this study

the action of curcumin on the lysosomal in heart and serum and on the histopathtaking

place

in the heart

of the major cardiac

D, which

than

membranes,

Cur-

action

of myocardial

muscle.

[ 15, 161

of curcumin.

a membrane-stabilizing

cardiac

Distribution

cathepsin

and non-target

mechanisms

the action

the lysosomal

of ischemic

was to investigate hydrolase activities ological

forestomach)

to explain

to have

and it is possible branes,

(mouse

sites [ 141. Several

marrow)

is known

D

is localized

in interstitial

during

curcumin

lysosomal

predominantly

proteiin myo-

cells [18], also was studied.

infarction.

activity

of ischemic

ly-

for tissue

myocytes,

heart

including

homoge-

lysosomal

the cells from autolytic and het(diferuloyl methane), an imporlonga, possesses

[lo], and anticancer

antioxidant

[ll] actions,

MATERIAL

AND

METHODS

of lysosomal

nates has been a common finding [7, 81. One approach to ameliorate the damage due to myocardial injury is to stabilize membranes, and to protect erolytic damage. Curcumin

lysosome; cathepsin

age at both have

sites in addition

in lysosomal

curcumin;

of lyso-

indicate

are labilized

enzymes,

connective

glycoprotein

sosomal enzymes

that

alter-

[ 1, 21. Irreversible

membrane-bound

the levels of lysosomal

cosaminoglycan,

and

infarction;

myocellular

throughout

degradation

by lysosomal latent

vacuoles, hearts

by abnormal

uents

may originate

on lysosomal

or hypoxic

enzymes

in ischemic

heart; myocardial

[9],

in addition

to antiaggregatory and antlthrombolytic properties [12, 131 and is observed to prevent benzo[a]pyrene-induced DNA dam-

Isoproterenol-HCl,

cosaminide,

p-nitrophenyl-P-D-glucuro-

phosphate, hemoglobin, N-a-benzoyl HCl, p-nitrophenyl-P-D-N-acetyl-glu-

and bovine

serum

albumin

were purchased

arfrom

the Sigma Chemical Co., St. Louis, MO, U.S.A. Curcumin was received as a gift from CFTRI, Mysore, India. All other chemicals

used were of analytical

grade.

Female rats (Wistar, inbred at the CLRI animal facility), weighing approximately 100 g, were housed in solid-bottomed polypropylene cages. The animals received a commercial rat diet (Hindustan

*Corresponding author: Dr. R. Puvanakrishnan, Department of Biotechnology, Central Leather Research Inst Itute, Adyar, Madras . 600 020, India. Tel. 91-44-4911386; Fax 00 91-44-4911589. t Abbreviation: ISO, isoproterenol. Received 17 October 1994; accepted 2 August 1995.

p-nitrophenol,

nide, p-nitrophenyl ginine p-nitroanilide

Lever,

Bombay)

and water ad lib. The animals

were divided into four groups as follows: (1) normal control group; (2) ISO-administered group (30 mg/lOO g body weight, subcutaneously, twice at an interval of 24 hr) as described by Wexler and Kittinger [19]; (3) curcumin-treated control group; and (4) ISO-administered group treated with curcumin.

C. Nirmalaet al.

48

TABLE

1. Effect of curcumin

P+Glucuronidase*

Group

Control Isoproterenol Curcumin C urcumin + isoproterenol Values

on lysosomal hydrolase activities in serum in experimentally

are means

8.63 f 0.69 12.60 + 0.75” 8.76 f 0.88 9.06 f 0.65

in p’mol p-nitrophenol/hr/lOO

t Expressed

in ~mol

p-nitroanllineihr/l00

1: Expressed

m pmol

tyrosine/hr/lOO

I Expressed

m nmol

p-nitrophenol/mln/mg

compared

Curcumin

17.23 f 22.03 f 17.46 f 18.42 f

Cathepsin

0.96 1.56” 0.86 1.02

Cathepsin

Bt

10.32 f 0.96 14.88 f 1.20” 10.52 + 1.02 11.56 f 1.23

10.46 f 15.99 * 10.95 f 11.79f

Acid phosphatase#

DS

1.02 1.13” 1.69 1.17

5.06 f 0.36 9.25 f 0.60” 4.89 + 0.56 5.22 f 0.87

f SD of SIX determmeians.

* Expressed

” P < 0.001.

P-N-Acetylglucosaminidase*

induced myocardial infarction

with

mg protem. mg protem. mg protein. protem.

control.

(200 mg/kg body weight)

suspended

in 1% gum

mination

of enzymatic

activities.

P-Glucuronidase

activity

was

acacia in water was given to each animal orally for 2 days prior to IS0 administrations, and the same dose was continued dur-

determined according to the method described by Kawai and Anno [20] and P-N-acetylglucosaminidase by the method of

ing IS0

Moore and Morris [21]. Cathepsin D activity was assayed the method of Sapolsky et al. [22] using 1.5% hemoglobin

by in

0.1 M acetate

of

administration.

After the experimental of ISO), the rats were blood

was collected

period (24 hr after the second killed by cervical decapitation,

to obtain

dose and

tyrosine

serum.

as described Separation

of subcell&r

troanilide

fractions

assayed The heart

tissue samples,

obtained

24 hr after the second

of ISO, were cut open and placed the blood.

Then

M sucrose,

blotted,

were prepared sucrose mine

the heart weighed,

at 4”. A portion

was subjected fractions

separated

and cell debris

5,000

g for 10 min;

portion

The enzyme

proteins, mitochondria

g for 10 min;

at 120,000 g for 30 min, and supernatant of en-tic

Measurement

nuat mi-

cytosol.

actiwities

Myocardial subfractions were treated with Triton X-100 (final concentration 0.2%, v/v) in ice for 15 min prior to the deter-

TABLE 2. Effect of curcumin

Group

Control Isoproterenol C urcumin Curcumin + isoproterenol Values

are means

20.32 f 32.00 f 21.26 f 21.29 f

0.72 1.24” 0.95 1.08

k SD of SIX determmations.

* Expressed

in gmol

p-nitrophenol/hr/lOO

t Expressed

m pmol

p_nitroank4hr/lOO

$ Expressed

m p’mol

tyrosine/hr/lOO

§ Expressed

in nmol

p-nitrophenol/min/mg

“P

< 0.001,

compared

wth

control.

41 P < 0.01,

compared

wrh

conrrol.

phosphate

mg protein. mg proten. mg proten protan.

of Barrett

and Heath

as substrate.

extracts

method

of Lowry

albumin

as the reference

arginine

Acid phosphatase Activities

as total activity/hr/lOO

enzyme

Tissues

and

in serum

p-ni-

activity

was

[24] using p-niof these

mg protein.

et al. [25] using

taken

enzymes Protein

was estimated

crystalline

in

by the

bovine

serum

standard.

P-N-Acetylglucosaminidase*

44.13 + 4.18 56.68 + 5.0191 42.68 f 4.23 44.30 * 4.94

for histological

4) were removed

as quickly

in 10% buffered complete,

formalin

were

embedded

were cut at 5 pm,

and stained

The sections

were examined

tomicrographs

were taken.

The t-test.

results

examination as possible

neutral

tissues

on heart lysosomal hydrolase activites in experimentally

P-Glucuronidase*

[23] using N-a-benzoyl

by the method

trophenyl

the amount B was determined

Histological studies

and the different

structural

at 15,000

extracts

in 0.25 M

by Barret

of cathepsin

HCl as the substrate.

were expressed all the

pH 3.0, and estimating

The activity

was used to deter-

at 600 g for 10 min; lysosomes

0.25

of the homogenate

centrifugation, as follows:

dose

to remove

in ice-cold

the tissue samples

Another

to differential

cleus,

crosomes

and minced.

of this preparation

activity.

were

saline

tissues were rinsed

by homogenizing

the total

in isotonic

buffer,

liberated.

were

1, 2, 3 and and placed

solution.

After

in paraffin, with

under

statistically

(groups at autopsy

fixation serial

hematoxylin

was

sections

and eosin.

light microscope,

and pho-

evaluated

Student’s

using

induced myocardial infarction

Cathepsin

27.32 f 36.98 + 25.92 f 28.78 f

Bt

1.87 2.90” 2.02 2.52

Cathepsin

24.68 32.62 23.24 26.23

f f f f

D$

1.67 2.32” 1.64 1.25

Acid phosphatase3

22.72 f 30.55 + 21.98+ 23.89 f

1.42 1.05” 1.56 1.86

49

Lysosomal Hydrolases in Myocardial Infarction TABLE 3. Effect of curcumin on the subcellular distribution of heart cathepsin D in experimentally cardiil

induced myo-

infarction

Cathepsin D (pm01 tyrosine liberated/hr/lOO mg protein) Nuclear fraction

Group Control Isoproterenol Curcumin Curcumin + isoproterenol

12.05 14.45 11.95 12.3;’

Values arc means + SD of

f f * f

of cytoaol

(free)

to lysxomal

t B: ratio

of cytosol

(free)

to tail

0.001,

compared

1.63 1.04 1.75 1.19

13.29 8.44 13.94 14.25

f + f f

Lysosomal fraction

1.67 1.023 1.35 1.42

26.41 16.04 25.77 26.91

1.36 0.94$ 1.46 1.69

7.39 3.22 7.69 6.13

f f f +

Cytosolic fraction

0.66 0.3% 0.71 0.83

16.18 26.78 16.07 18.49

wth

(hound)

in Table 1. Serum lysosomal hydrolases (P-glucuronidase, acetylglucosaminidase,

cathepsin

B, cathepsin

were found to be elevated significantly

ISO-induced

myocardial

A significant

infarction.

at near normal

The

P-N-

D, and acid

phosphatase)

both curcumin

following

levels of these en-

levels in rats that received

and ISO. increase

in the activity

of P-glucuronidase,

cathepsin B, cathepsin D, P-N-acetylglucosaminidase

0.655 0.816 0.691 0.705

and acid

in the ratio of cytosol to

bound lysosomal and cytosol to total activities,

which were

found to be near normal values. Figure 1 represents histological

the normal architecture

examination.

of heart upon

A massive necrosis of the heart mus-

cle fibers with disruption of muscle bundles was observed in ISO-treated

groups (Fig. 2). The

curcumin-treated

control

group revealed normal cardiac muscle fiber architecture 3). In the ISO-administered

(Fig.

group treated with curcumin,

a

decreased degree of necrosis was observed (Fig. 4).

in the heart was noticed in rats treated with IS0

(Table 2) curcumin treatment caused a decrease in the enzyme

DISCUSSION

activity to near control levels. Table 3 shows the activities

It was reported previously that the localization

of cathepsin

D in the subcel-

lular fractions of the heart. The data presented clearly indicate that significant

changes

were not observed

in cathepsin

D

activity in the nuclear fraction of the heart in rats treated with IS0

0.613 1.669 0.624 0.687

control.

of lysosomal hydrolases in serum are presented

zymes remained

1.23 2.48$ 1.97 1.75

Bt

activity

cumin showed a marked decrease

The activities

+ + + f

A*

activity.

RESULTS

phosphatase

+ f f f

Microsomal fraction

SIX samples.

* A: ratio

$ P s

Mitochondrial fraction

or with curcumin and ISO. However, there was a signif-

icant decrease in the activity of cathepsin D in mitochondrial, lysosomal, and microsomal fractions of the heart in rats treated with ISO. Cathepsin

D activity in the cytosolic fraction of the

of acid hydro-

lases in cardiac myocytes is in the lysosome and that the release of these enzymes from the lysosome to the cytosol leads to myocardial cellular injury and death in the ischemic state of the heart [2, 7, 261. This is in agreement showing that in ISO-administered

with our findings

rats the activities of serum

lysosomal acid hydrolases increased, lysosomal bound acid hydrolases decreased,

and cytosol

enzyme activities

increased.

rats, but this increase in activity was observed to be reduced

Ravens and Gudbjamason [27] observed that the release of hydrolytic enzymes from lysosomes after coronary occlusion

markedly in rats treated with curcumin and ISO. ISO-administered groups showed an increased ratio of cytosol to bound

cellular destruction.

heart was found to be elevated

lysosomal activities

significantly

in ISO-treated

and cytosol to total activity for cathepsin

D in the heart. Treatment

of ISO-administered

rats with cur-

FIG. 1. Heart tissues of control rats under light microscope showing normal architecture (200x ).

may be a causative factor for the development

of myocardial

Using the immunofluorescence

method,

Decker et al. [2] have also shown the release of cathepsin from lysosome to cytosol 15-30

D

min after coronary occlusion.

FIG. 2. Rat heart showing massive necrosis of muscle fibers 1 day after ISO-induced myocardial infarction (200~ ).

50

C. Nirmala et al

/

,-,

,.“.1

FIG. 3. Heart tissues of curcuminetreated control normal muscle fiber architecture (200x).

Furthermore, sosomes function other

these

cytosolic

acid hydrolases

and from the sarcoplasmic and

disruption

organelle

In a previous hydrolases thepsin

of mitochondria,

study carried

composed

mainly

reticulum,

inside sarcopiasmic

was found

to decrease

enzymes somal

and

microsomal

that the distribution

membranes. of enzyme

fractions,

of

which

on the lyso-

It is interesting

to note

between

the cytosol

reported

in our studies

indicates

decreased

our studies demonstrated

lysosomal

stability

of lysosomal

in ISO-treated

that curcumin

enzymes

to

rats. In might in-

as well as decrease

the

activity of the total lysosomal acid hydrolases, thereby enhancing the stability of the lysosomes. Histological studies carried out on the heart

samples

support

our findings,

creases

the degree

It is possible curcumin branes,

since

the release indirectly

this property

[3 11. It has been reported lysosomal

groups

that curcumin

(Figs.

l-4)

also

pretreatment

de-

of necrosis.

that

may help

of different

showing

of endogenous in stabilizing

of glucocorticoids

corticoids lysosomal

by

mem-

is well known

that small doses of ibuprofen

emit

Thus,

pretreatment

integrity

hearts.

of hydrolytic

enzymes

in rats, exercises

with

curcumin

and delay signs of necrosis

This protective

since curcumin, is non-toxic

infarction

action

could

preserve

in severely

is all the more

in

a protecisch-

interesting

at a dose range of 0.05 to 2 g/kg body weight,

and non-mutagenic

[36, 371 in rats.

The authors rhank Dr. K. V. Rughaoun, Director, CLRi, {or hrs permission to publish this work. The financial assistance by CSIR, New Deihl, to C. Nirmakc is gratefully acknowledged.

treat-

release

activity

addition,

role.

in the liberation

myocardial

fraction

to total activities

and cytosol

the release

the

effect of curcumin

lysosomal

hibit

inhibit

and microsomal

could be due to the stabilizing

was

injury and

rats. Curcumin

rats could

from the lysosomal

reticulum

myocardial

D in the microsomal

in ISO-treated

of EGO-administered

is

and that activa-

of ischemic

cathepsin

and cawhich

tive

diminution

lysosomal

of acid

fraction,

important

ment

and

[28, 291.

acid phosphatase,

of sarcoplasmic

In our study,

ISO-induced

earlier

tion of these acid hydroiases death.

quent

the dys-

sarcolemma,

in the microsomal

for the evolution

from ly-

induce

out in dogs [30], activities

such as P-glucuronidase, D were observed

released

reticulum

in the same way as reported

FIG. 4. Heart tissues of ISO-administered rats treated with curcumin, showing a decreased degree of necrosis (200x ).

rats showing

suppress

enzyme

release by stabilizing the lysosomal membrane [32]. Curcumin is found to be more potent than ibuprofen as a stabilizer of lysosomal membrane and as an uncoupler of oxidative phosphorylation [17]. Kalra and Prasad [33] have suggested that oxygen free radicals generated during ischemia, in addition to the direct myocardial damaging effect, may also be responsible for the cardiac damage through the release of lysosomal enzymes. The beneficial effect of curcumin may also be mediated by the scavenging of oxygen free radicals [34, 351 with the resultant preservation of cellular viability serving secondarily to preserve lysosomes as well. The results of this study illustrate that curcumin, due ro its stabilizing action on the lysosomal membrane and the conse-

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