Journal of Asian Natural Products Research. Vol. 8. No. 3. April-May 2006, 201-208

Effects of crocetin on the matrix metalloproteinases in cardiachypertrophy induced by norepinephrine in rats

X.-C. SHEN. Y.-LU and Z.-Y. QIAN*

Department of Pharmacology, China Pharmaceutical University, Nanjing 210009 China

(Received 2S February 2004: revised 20 April 2004: in final form 2^ April 2(X)4)

The cffecl.s of crocetin on the cardiac hypt-rtrnphy induced hy lony-lerm ireatnieni with norepinephrine(NE) in rats have beer investigated. The activities of matrix nictalloproteinases (MMP-2 iind MMP-M)have been assayed by gelatin SDS-PAGE /ymography. The expressions of MMP-2 and MMP-9 weredetected by RT-PCR, ATPa.se activity and hydroxyproline contents were [ncasured with a commercial kil.The results show ihai crocctin blocked Ihe dcvelupnienl of left ventricular hypertrophy induced by NE.decreased Ihc level of collagen in myiKardium, enhanced both The Na' -K ' ATPase activity in cardiactissue and the Ca""'-Mg~^ ATPase activity in niilochondria and inhibited significantly the activity ofMMP-2 and the expressions of MMP-2 and MMP-9. These results suggesi ihai crocetin may preventcardiac hypertrophy indticed by NE in rats.

Keywords: Crocetin: Norepinephrine: Cardiac hypertrophy; Matrix metalloproieinase

1. Introduction

Crocelin {figure I), a carotenoid, is a primary active ingredienl in extraction from Crocussativus L (Saffron) [I ]. Saffron, a traditional Chinese medicine, has long been used to treatcoronary heart disease and hyperlipemia and can promote blood circulation by removingblood slasis. Previous studies indicate that crocelin could inhibit protein kinase A (PKA) andprotein kinase C (PKC) [2]. In our studies it exhibited a strong anti-oxidative effect [3], theability of anti-myocardial ischemia induced by deligating coronary artery in rats and dogs.It also significantly inhibited the cardiac hypertrophy induced by constriction of theabdominal aorta [4] and decreased the activities of MMP-2 and MMP-9 [5|.

Chronic heart failure (CHF) is a cardiovascular disorder with multiple-etiology, bigh-prevalence and poor-prognosis. The levels of catecholamines circulating in the heart areassociated with both heart failure 161 and its prognosis [7]. The enhanced activity of thesympathetic nervous system results in increased release of norepinephrine (NE) fromthe sympathetic nerve endings in the myocardium. Recent studies have shown that NEstimulates the production of intracelluiar reactive oxygen species (ROS) in cultured neonatal

•Corresponding author. Tel.: -1-86-025-83271322. Eax: -f 86-025-832713.\'i. E-mail: sxc9860@sina.com

.limrnal ofA.iian Naiurvl Piiic/itcis ResearchISSN 1028-6021) print/ISSN 1477-221.1 online ® 2006 Taylor & Francis

titl p://w w w. 1 u ndf. e o. uk/j ou mal sDOI: I0,IO80/10286(l2O4l2.i:il2B6452

202 X. • C. Shen eial

Figure I. Chemical structure of crocetin.

ral cardiomyocytes [8], The abnormal ROS can play an important role in signal iransductionof the neonatal rat cardiomyocyte hypertrophy [91.

Matrix metalloproteinases (MMPs) belong to a family of protenolytic enzymes that basbeen implicated as playing a key role in extracellular matrix (EXM) degradation in .severaldisorders 11()|. Experimental animal hearts (11.12] and buman hearts 113] wiib advanced heartfailure or myocardial remodeling have also shown increases in activity of MMPs or messengerribonucleic acid (mRNA) levels. MMP-2 (gelatinase A) and MMP-9 {gelatinase B) areparticularly relevant in myoeardial remodeling 1141. Mohamad reported that the patients ofmyocardial remodeling had a significant increase in zymographic activity of MMP-2 andMMP-9 [151. Rouet-Benzineb suggests that MMP-2 and MMP-9 may be involved indisorgani/alion of tbe contractile apparatus in dilated cardiomyopathy |I6]. Concomitantly,activity and mRNA of MMP-2 had been increased in cardiac bypertrophy induced bylong-tenn NE treatment in rats [ 171. The inhibition of MMPs activities can attenuale tbe degreeof cardiac byperirophy [18,19]. In tbe present studies, we investigated the effects of crocetin oncardiac hyperlropy, activities and mRNA expressions of MMP-2 and MMP-9 in NE-inducedcardiac bypertrophy in rats.

2. Results and discussitm

2.1 Ventricular mass

Table 1 shows tbe NE-indueed cardiac bypertrophy evaluated by left ventricular index andbeart index, whicb refer to tbe ratios of left ventricular weight or heart weight and bodyweight. They were increased in bypertrophic heart induced by NE compared witb a normalgroup. After treatment witb crocetin ibe indexes were decreased significantly {p < 0.05,p<O.OI).

2.2 Collagen content and ATPase activity

Tbe hydroxyproline assay sbowed that the level of hydroxyproline was significantly higherin the model group than that of normal group (/.xO.Ol), and it was mucb lower in

Tubte 1. Cardiac weight index after treatment with vehicle (control), captopril. and crocetin in rats.

) /( HW/BW

NormalMode!CapHiprilCrocclin ICrticoiin II

603025

2.88 ± 0.263.26 ± 0.23"2.95 ±0.19"2.97 ± 0.24"3.03 ±0.14'

2.07 ±0.152.40 ±0.16"2.16 ± 0.16"2.18 ±0.19^2.24 ±0.1 r"

BW: weight; LVW: left vciilricular weigtii: HW: liearl weighi. > < U.OI v.\. normal, ^p < 0.0! vs. moik-!, >

Effects of crocetin on the matri.x metatlopwteina.ses 203

Table 2. Effects of crocetin on the level of hydroxyproline and ATPase aclivity on cardiac hypertrophyinduced hy NEf..

Na\K*-ATPase Ca'^Mg'* ATPasel/imol Pi Img prot)~ h ' j

Group

NormalModelCaptoprilCrocellri 1Crocelin 11

Dose (mg kg ')

_-605025

Cardiac tissue

0.994 + 0.710.232 ± 0.19'0.839 ± 0.50"0.927 ± 0.33"0.623 ± 0.32*=

Mitochondrhiiu

1.04:0.137:0.327 :0.265 :0.650 :

t0.61tO.IO"tO.3I''t 0.21"t 0.54'-'

Hydni.xyproline I fig img pmt) /

0.373 ±0.190.783 ± 0.04"0.631 ±0.09^0.636 + 0.04^0.628 ± 0.09"

-/I < 0.{)l vs. normal. > < n.OI v.s. mcMlel, > < 0.05.

crocetin-treated groups than that of model group. The ATPase activity of extracts fromcardiac tissue in mode! group was much lower than that in a sham operation group(p < 0.01). After exposure of rats to crocetin and captopril, ATPase significant activity wasevaluated {p < 0.05) (Table 2).

2.3 MMPs activity analysis

Figure 2 indicates that the activities of MMP-2 and MMP-9 were elevated significantly in themodel group (/) < 0.01). and treatment with crocetin, remarkably, decreased MMP-2 activitysignificantly but not that of MMP-9.

2.4 Levels of MMP-2 and MMP-9 mRNA in cardiac hypertrophy

NE induced up-regulation of MMP-2 and MMP-9 mRNA expression (figure 3). Crocetin andcaptopril down-regulated the mRNA expressions of both MMP-2 and MMP-9.

Many reports have shown that stimulation of myocardiai p- and a adrenergic receptorscan result in the development of cardiac hypertrophy in rats [20,21 ]. ATPases of cardiac cellsare one of the most important enzymes in maintaining the fluxes of vital cations byhydrolysis of the terminal high-energy phosphate of ATP [22]. Intracellular sodiumconcentration is overloaded following the decrease of Na"^,K^-ATPase. This overloadingactivates sodium-calcium exchanger of cardiac myocyte membrane, and calcium ions entercardiac myocyte. Decreased activity of Ca~ .̂Mg~"^-ATPase mainly results in disequilibriumof calcium and magnesium both infra- and exo-cell, and calcium aggregates in the cell.Decreased activity of ATPase leads to intracellular calcium overloading, which activatesmany signal conducting systems and kinase activation. In the end. it can cause cardiachypertrophy. Decreased cardiac mechanical performance is reported to be highly related todecreased ATPiise activity in several models of cardiac hypertrophy. Our results demonstratethat crocetin can increase the cardiac ATPase activity, and then improve the cardiaccontractility.

In the process of the pathology of cardiac hypertrophy, the changes of phenotype ofcardiac myocyte. including the augmentation of the cellular volume, the alteration of the typeof contraction proteins, the proliferation of the myocardium interstitial cell, the hyperplasiaof fibrous tissue, all result in piling upof the collagen and cellulose, and ultimately induce thederangement of myocardium constitution [23[. Our experimental results indicate that themyocardium hydroxyproline increased in cardiac hypertrophy, and that crocetin decreased it.

204 Shen et iil.

MMP-2

MMP-9

2 3 4Effect of Crocetin

B 6

o-= 4

o5 1

0

MMP-2MMP-9

model cap croce 50 croc 25

Effect of Crocetin

Figure 2. Gelatin SDS-PAGE zymognim analysis nl' MMP-2 ;i[id MMP-9 activity in NE-induced rat cardiachypertrophy left vcnlricLilur tis.sue, Ld'I ventricular [issue extracts were analyzed Ibr MMP-2 and MMP-9 activity indifferenl groups tisiiij; SDS-PAGE ?yinograms containing gelatin. (A) Representative gelalin-containing ?ymogrumillustrfiiing MMP-2 und MMP-^ activity iti left venlHcukir (issue extracts collected from ditfereiil groups. Each laneWii.s loaded with S -̂g protein extract from the I,V |tanc: 1, normal: 2. miHlel; .\ caplopril:4. crocetin (25 mg kg" '):

g kg )]. (B) Effect olcnK-etiii un aetivify ot MMP-2 and MMP-') iti NE-induced (.•ardiac hyperlrophyl

."S, cnjcetin (51) mg kg )].tissue. Ratios of IOD]MM|..n/IODn,,ti,mi MMI--: and IODMMj...,/IOD,n,n,,,,] MM -J: " = 4. mean ± i>.*p

hyperlrophy0.05 vs. model.

Subsequently, crocetin suppressed the increased collagen of interstitial cell and amelioratedmyocardium reconsiiiution.

Matrix metalloproteinases (MMRs) are members of a family of zinc-dependent enzymesthat degrade molecules of the extracellular matrix [20.231. They have been implicated incontributing to myocardial remodeling in animal models and human with congestive heartfailure and cardiomyopathy [24]. MMPs are suggested to play an important role in tissueremodeling under both normal and pathological conditions [24|. MMPs activity is indicatedas having a p<isitive coiTelation with cardiac hypertrophy. Treatment with MMP inhibitorsmay prevent myocardial remodeling. The present studies revealed that crocetin preventedcardiac hypertrophy by attenuating activities of MMP-2 and MMP-9 and expressions ofMMP-2 and MMP-9.

In summary, erocetin may depress librillar collagen production, expressions of MMP-2 andMMP-9. and activities of MMP-2 iuid MMP-9 in NE-induced cardiac hypertrophy in rats.

Effects ofcwci'thi on llw nuilrix metalloproleinases 205

MMP-2

MMP-9

OAPDH

1 2 3 4 5

Effect of Crocetin

• MMP-2

D MMP-9

» 0.45

0.4

0.35

0.3

I 0.26oS "'̂ra

0.15

0,1

0.05

0

Normal Model Captopnl Croce 50mg/kg Croce 25mg/k9

Effect of Crocetin

Figure 3. ElVecl of crocetin on mRNA expressions of MMP-2 and MMP-9 by RT-PCR. E.xpression of MMP-2. MMP-9and GAPDH mRNA were dclermined by RT-K'R. PC'R prt)duels were run and separated by 1 .T'/c agamse gel, (A).Represcnlativc mRNA expressions for MMP-2 and MMP-9 determined by the RT-PCR method in ihe lefl ventriculartissues from Nbi-induced cardiac hypertrophy. |lane; 1. normal; 2. m(xlel: 3, eaptopril; 4, cnxrciin l5[)mgkg"');5. crotetin (25mgkg "'l. (B). EITect of crocetin on MMP-2 and MMP-9 expressions in cardiac hj-pcilrophy inducedby NE in rats. Ralios of AMMi>.>/A<,;Ai.i>n and AMMpVA<iAPDn were used to express the levels of MMP-2 and MMP-9mRNA in cardiae tissue, respectively, n — 4. mean ± s. */J < 0.05 vs. model, #p < 0.05 vs. normal.

The detailed mechanisms of crocetin in the prevention and treatment of cardiac hypertrophyremain to be investicated further.

3. Experimental

3.1 Drugs and reagents

Crocetin (C2()H24O4. >90% purity a.s determined by HPLC) was isolated and purified in ourlaboratory. NE was purchased from the Shanghai Hefen Pharmaceutical Factory. ATPase.hydroxyproline assay kits were purchased from Nanjing Jiancheiig Bioengineering Institute.AMV reverse transcriptase was purchased from Gibco. Tacf polymerase was purchased fromSangon (Shanghai. China). Other reagents were all of AR grade.

3.2 Animals

Female Sprague-Dawley (SD) rats (180-220 g) were provided by the Experimental AnimalCenter of China Pharmaceutical University and raised under constant temperature and

206 X.-C. Shenela\.

humidity conditions; they were given free access to a standard chow and tap water before andduring the experimental period.

3.3 Cardiac hypertrophy

Norepinephrine ( l .5mg kg" ' d~ ' ) was injected intraperitoneally in ascorbic acid saline

twice daily lor 15 d 120]. The animals were divided into live groups: (I) control

(normal saline), (2) NE, (3) NE + crocelin (50mg k g " ' - d " ' X 15 d), (4) NE-I-crocetin

(25mg k g " ' d ' ' x l 5 d). (5) NE + captopril (60mg k g ^ ' - d " ' x l 5 d). Crocetin and

captopril were treated i.g. After the rats were sacrificed, their hearts were removed, and the

free wall of the right ventricle was separated from the intra-vcntricular septum, which

remained as a part of the left ventricle (LV). The left ventricles were weighed after freezing in

liquid nitrogen and stored at - 70°C. The left ventricular index and heart index were

calculated by the ratios between left ventricukir weight or heaii weight and body weight.

3.4 Homogenization of LV tissue

LV tissues were sliced into small pieces and thoroughly washed with potassium phosphatebuffer (50 mmol L~') to avoid contamination from hlood. Homogenization of the LV tissuewas performed following the method described by Lee et al. [251. The homogenates were usedto determine ATPase activity, hydioxyproline contents. Protein concentration was determinedhy the Coomassle brilliant blue method, using bovine serum albumin as a standard.

3.5 SDS-PAGE zymography

Extracellular proteins of approximately 25 mg frozen tissue were extracted with a 20-foldvolume ot exuaction hufler [iOmM Tris-HCI pH 7.5. 150niM NaCl, 20mM CaCU, 1 M-MZnSO4. 0.01% (v/v) Triton X-lOO, 1.5 mM NaN> 0.5% PMSF] overnight at 4°C afterhomogenation. These extracts contained approximately l.5mgml ' pn)tein. Myocardialmatrix metalloproteinase activity was measured as previou.sly described [26-281. Briefly,gelatin [0.1% (w/v)] was added to a Laemmli acrylainide polymerization mixture. In a 1:2preparation the extracts were mixed with substrate gel sample buffer [ 10% (w/v) SDS, 4% (w/v)sucmse, 0.25 mM Tris-HCI pH 6.8 and 0.1% (w/v) hromphenol hlue|. Approximately 8 jxg ofmixture was loaded immediately without boiling. The gels were mn at 15 mA per gel throughstacking phase (4%) and at 20 mA per gel for the separating phase (10%). maintaining a runningbulTer temperature 4°C. After SDS-PAGE, the gels were washed twice with 2.5% Triton X- 1(X)solution lor 30 min each, with water and then incubated overnight at 37°C in a 0.05 mol L~'Tris-HCI hulTer. pH 7.5, containing 5mmol L~' dxCU. The gels were then stained for60-90 min in 0.25% Coomassie hrilliant blue R250 in acetic acid-methanol-water (1:4.5:4.5by volume) and siihsequently destained with 10% methano! and 7% acetic acid. Enzyme activityof MMP-2 and MMP-9 can be visualized in the gelatin-containing zymograms as cleai- handsagainst a blue background. The relative clearing of each sample was quantitated by determiningthe inverse optical density, using Labwork 4.0 image acquisition and analysis software.

3.6 Reverse transcription-FCR

Total RNA was isolated according to the manufacturer's instructions. MMP-2 and MMP-9were measured by reverse transcription polymerase chain reaction (RT-PCR). For MMP-2.

Effects of crocetin on the matrix metatloproteinases 207

the sense primer was 5'-CCC AGA AAA GAT TGA CGC-3'. and the antisense primer was5'-CGA CAG CAT CCA GGT TAT-3'. The PCR product size was 314 bp, and the primer wasdesignated by Primer 5.0 according lo MMP-2 cDNA (Genebank NM-031054). For MMP-9,the sense primer was 5'-CGT GGC CTA CGT GAC CTATG-3'. and the antisense primer was5'-GGATAG CTC GGT GGT GTC CT-3'; the PCR product size was 592 bp [29]. As an innercontrol, rat cDNA for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA wasproduced by RT-PCR according to Ref. [30]. Some 5 (xg of total RNA was reverse transcribedinto cDNA and then amplified using RT-PCR procedures. The amplification profile involveddenaturation at 94°C for 5 min and at 94°C for 30 s, annealing at 58°C for 30 s {MMP-2 andGAPDH), 68°C for 30 s (MMP-9). and extension at 72°C for 30 s. followed by extension72°C tor 10 min: the number of necessary cycles was 30. The PCR products were separatedby electrophoresis on a 1.7% agarose gel—the gel was stained in ethidium bromide after theeleclrophoresis was over. Identity was confirmed using Labwork 4.0 image acquisition andanalysis software. The values are expressed as ratios of MMP-2/GAPDH, MMP-9/GAPDH.

3.7 Statistical analysis

All data were expressed as x ± s and analyzed by the /-test.

Acknowledgements

The authors are grateful to Professor Jing-jing Liu for his technical help and to Ph.D HaoHong for valuable comments on the manuscript.

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