R.

Rukkumani et al

Research Article

Curcumin Influences Hepatic Expression Patterns of Matrix Metalloproteinases in Liver Toxicity

Rajagopalan Rukkurnani, Kode Aruna, Penumathsa Suresh Varma, and Venugopal Padmanabhan Menon* Department of Biochemistry, Faculty of Sciencc. Annamalai University, Annamalainagar - 608 002, Tamil Nadu, India.

. . .. - ........ --._ ....... _._ ... _--. -----

Abstract. Hepatic fibrosis is a result of an imbalance between enhanced matrix synthesis and diminished

breakdown of connective tissue proteins, the net result of which is increased deposition of Extra Cellular Matrix. In this concept Matrix Metalloproteinases play an important role because their activity is largely responsible for extra cellular matrix breakdown. In the present study we have tested the influence of curcumin, the' active principle of turmoric. on matrix metalloproteinase expression during alcohol and thermally oxidised sunflower oil induced liver toxicity. Male albino Wistar rats were used for the study. The matrix metalloproteinase expressions worn found to be increased significantly in alcohol as well as thermally oxidised sunflower oil groups and on treatment wIlIl curcumm there was a significant decrease. In alcohol + thermally oxidised sunflower oil group, we found a significant decrease in matrix metalloproteinase activities.· Administration of curcumin significantly improved their activities. From the results obtained, we could conclude that curcumin influences the hepatic matrix metalloproteinases and effectively protects liver against alcohol and i\ PUFA induced toxicity. il

I

Introduction

liver fibrosis and cirrhosis result from the majority of chronic liver insults and represent a common ,lnd difficult clinical challonge of worldwide importance. At present. the only curative treatment for end stage cirrhosis is transplantation. Hence there is a considerable imperative to develop antifibrotic strategies that are applicable to liver fibrosis. Development of liver fibrosis entails major alterations in both the quantity and quality of extracellular matrix (ECM) (1,2). The remodeling of ECM both in normal and pathological conditions is controlled by a group of enzymes called matrix metalloproteinases (MMPs) (3).

MMPs are a family of structurally and functionally related zinc endopeptidases which are capable of in vitro and in VIVO degradation of all kinds of EeM protein components such as interstitial basement mcrnbr. me collaqr-n. proteoqlycans, fibronectin. larninin ; mel thus a rr~ implicated in connective tissue remodolling procosso-. associated with various pathological conditions (<1) Available data suggest that hepatic fibroproliferation IS associated with alterations in hepatic MMP exprossrons. Expression of MMP increases with advancing fibrosis and allows an assessment of the amount of connective tissue deposited (5). Due to their crucial role in regulation of the ECM, MMPs are known as potential biochemical markers for assessing liver toxicity (6).

The Italian Journal of BH)chernlslry Vol. 53 (2) 2004

Alcoholic liver disease is a major medical complication of alcohol abuse and a common liver disease in western countries. Increasing evidence demonstrates that prolonged alcohol intake results in fibrosis and ultimately cirrhosis (7). In take of high fat diet along with alcohol primes the toxic effects of alcohol (8). In our previous studies, we developed the fibrotic animal model by inducing alcohol and thermally oxidised sunflower oil ( L\PUFA) (9, 10).

The present study was undertaken in order i) to identify the hepatic expression patterns of MMPs during alcohol and I\PUFA induced toxicity and ii) to study the influence of curcumin over MMP expressions during alcohol and L\PUFA induced toxicity.

Curcumin is a natural phenolic curcuminoid present in turmeric (Curcuma tonga) (11), a spice used in Indian food. Curcumin, the active principle of turmeric has been extensively investigated for its hepatoprotective potential. In experimental animals, curcumin has been shown to prevent lipid peroxidation (12, 13). We had already reported the protective role of curcumin -ever alcohol and PUFA induced toxicity (10). Since little or no work has been done to evaluate its effects on MMP expression patterns, in the present study, we planned to test its protective role by monitoring its influence over MMPs, the repair enzymes, during alcohol and I\PUFA induced hepatotoxiclty.

61

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Research Article

Materlals and methods

Animals

.Male Albino rats, Wistar strain of body weight ranging 140-160 g bred in Central Animal House, Rajah Muthiah Medical College, Tamil Nadu, India, fed on pellet diet (Agro Corporation Private Limited, Bangalore, India) were used for the study and water was given ad libitum. The standard pellet diet comprised 21% protein, 5% lipids, 4% crude fibre, 8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2% vitamin and 55% nitrogen free extract (carbohydrates). It provides metabolisable energy of 3600 K Cal.

The animals were housed in plastic cages under controlled conditions of 12h light / 12 h dark cycle, 50% humidity and at 30:t 2 "C. The animals used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Iridian Council of Medical Research, Hyderabad, India and approved by the Animal Ethical Committee,

Annamalai University. '

Materials used

Ethanol

Absolute othanol (AR) was obtamed from Hayman tnnited. England,

Thermally oxidised

suntlowev oil (L\PUFA) Sunflower oil was subjected to heating at 180 C for 30 minutes, twice,

Curcumin

Curcumin was obtained from Central drug house private

limited, Mumbai, India,

Experimental design

The animals were divided into 8 groups of 6 rats each.

Group 1 (Control)

Control rats

Group 2 (Alcohol)

Rats given 20% ethanol (7.9 g/kg body weight) (14) orally, using an intragastric tube,

Group 3 (L\pUFA)

Rats given high fat diet (15% thermally oxidised sunflower oil) (9) mixed with the diet.

Group 4

(Alcohol + L\PUFA)

Rats given 20% ethanol + 15% thermally oxidised sunflower oil.

Group 5 ,

(Alcohol + Curcurhin)

Rats given curcumin (80 mg/kg body weight) dissolved in 20% ethanol.

Group 6 ,

( L\PUFA + Curcutnin)

Rats given 15% thermally oxidisecl sunflower oil + curcumin (80 mg/kg body weight) dissolved in distilled water.

62

"~'

H Hukkuru.nu f~t ,II

Group l

(Alcohol +\PUA + CIJICliITlin) flats D'V('" curcurnin (80 mg/kg body weiqnt) dissolved in 20"0 ethanol t 15"" thermally oxidisod ,.lHlflower oil.

Group fl (Curcumin)

Flats qrven curcurnin (80 rng/kg body wetgllt) dissolved in distilled water orally using an mtraqastr«: tube.

Rats were maintained on isocalorific diet using glucose solution. (Total calories per clay 508 K Cal/kg body weight). At the end of the exporimental period of 45 days, the rats wore killo(j by cervical decapitation and liver was collected, cleared off blood and immediately transferred to ICO cold cont.iinors containinq O.9(~o NaCI for assossmq tile acnvrnos of MMPs.

Biochemical investigation

Total MMP activity W(tS as!'!'!.'.cd by multiwell zymography (15) and uidivrdunl MMP expression was analysed by qelatin Iyrnography tIll), MMp-2 anr:

MMP-'), tho major tissue remodohnq qetatiuase.i. worn estimated by succinylation rnothoo (17). Donsitometric analysis was carried out llsing Gel/chenu Doc, Bio-rad using software quantity one 4.4.1,

Results & Discussion:

Fig 1 shows the multiwell zymogram of the liver samples and fig 1 a gives the densitometric reading of the same. From the figures we could infer that the total activities of MMPs were increased significantly in alcohol (II) and i\PUFA (IV) groups when compared to norrnal (I) and significantly decreased in alcohol + ,1PUFA group (V). In alcohol + curcumin (VI) and in !~PUFA + curcumin(VII), there was a significant decrease in MMP activities and in alcohol + .\PUFA t curcumin group (VIII) there was a significant increase. when compared to corresponding untreated groups ( II, IV & V respectively). Curcumin control group (IX) showed no change in MMP activities when compared

to normal (I). ,

Fig 2 and 3 give the gelatin zymogram of liver samples and 2a and 3a gives the densitometric reading of the same. Four different kinds of MMPs; 130 KD, 92 KD, 72 KD and 45 KD were found to be expressed during alcohol and L\PUFA ingestion and subsequent curcumin treatment. The activities of all four MMPs were found to be increased in alcohol and l\PUFA groups (Fig 2 - ii & iii) and decreased in alcohol + ,\PUFA group (Fig 2 -iv) when compared to normal (Fig 2 -i). Curcumin treatment to alcohol (Fig 3 -i) and ,\PUFA (Fig 3 -ii) significantly decreased the MMP

The Italian JOUfIl,t1 of Biocnenusuv Vol. 53 (2) 2004

R. Rukkurnani et ill.

\1

; IX

'vl :\1 ... ·.1.,!;. ( 111":1:1:1111 \"11 .\1'1":\.( .uctuuiu: \'II! Alcohol ' • .:lPT T . .\. ·--.:t.1I..:HIIllIl.

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Figure 1

Multllvell Zvtnoqrarn of Liver Samples

Research Article

92 KO 72KD

45KD·

Norm.1

Il. Alcohol

III .\ PUfA

tv Alcohol + ,\ PUFA

Figure 2

Zymogram of liver samples

IHI

-------~

-- Ii

I

I

.~60 rn

i :: l. &_ _'_'__'_[--.J=-w

130 KO t2 KO 12 KO i .a KO

• Normal CAlcohol CHeated PUFA CAlcohol + ~!eated PUI'A ,

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Figure 2a

Densitometry of Liver Zymogram

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Group,

activities and curcumin treatment to alcohol + /~PUFA significantly improved the MMP activities (Fig 3 -iii) when compared to corresponding untreated groups. Curcumin control group (Fig 3 -iv.) showed no significant alteration in the patterns when compared to normal (Fig 2 -i).

Fig 4 shows the changes in the activities of MMP 2 (72 KD) and MMP 9 (92 KD). The activities of MMP 2 and MMP 9 were significantly increased in alcohol

_ and ;\PUFA groups and decreased in alcohol + ~PUFA group when compared to normal. On treatment with curcumin there was a significant decrease in MMP activities in alcohol and .t\PUFA groups and significant increase in alcohol + ,~PUFA !}roup when compared to correspondinq untreated group.

Research Article

Liver fibrosis represents the common pathological outcome for the majority of chronic liver insults. Hepatic stellate cells (HSC) which surround the liver sinusoids are considered to playa pivotal role in the development of liver fibrosis. In the nor.nalliver, these cells are compact and non fibrogenic (quiescent HSC). Chronic liver injury induces a prQgramme of changes in HSC morphology and synthetic capabilities. HSC proliferate and transform to enlarged myofibroblast cells. These activated HSC are considered to be the major source of collagenous and non-collagenous matrix proteins which accumulate in fibrotic liver (18). MMPs are a family of Zn proteases that play an important role in the process of tissue remodelling and repairing: in physiological and pathological states by degrading: ECM proteins and collagen in particular. Studies sugg!est that MMPs may participate in pathological response involved in liver fibrosis (19). Among MMP S' MMp·2 & MMP- 9 are mainly responsible for the deqradat'on of type IV collagen, a major component of the basement membrane, 'whose deposition becomes prominent during fibrosis. Hence in our stu-

130 )0(0

1'} KD

'_:.il

45 KO

AlcOhol + Curcurmn

j, PUFA • CUfCUI'(\Ir

Figure 3

Zymogram of liver samples

70 -r----------. 60

~50 ~ 40

c

= 30

.. '

" .

0: 20

10 o

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D.!cohllt CLrctmn a:tk,II""'.'11' ,\.1:11(\1',,11

Fottll, titalt-Of'lIfA.. (; •• rlllrm m:;h'1 dl"\

L.. ._ .

Figure3a

Densitometry of Liver Zymogram

64

K Hukkumam et al

dy. we further quantified the MMp-2 and MMP-9 in particular.

In our study, we observed <tn Increase in MMP activities in alcohol and i\PUFA treated groups. This may be mainly due to the increased expression of MMPs to break down and repair the excessive matrix protein that is deposited during alcohol and \PUFA intake. Ingestion of ethanol inducos various processes including changes in NAD+ INADH (20), production of acetaldehyde protein adducts (21), induction of CYP2EI (22), formation .ot 1-hydroxyethyl free radicals (22) and endotoxin derived activation of Kupffer cells which inturn produce tumour necrosis tactor« (23). These changes cause severe damage to the liver. Moreover acetaldehyde the main product of ethanol metabolism affects hepatic collagen synthesis, both invivo and in vitro, leading to increased collagen accumulation, resulting in fibrosis (24)

In [\PUFA treated rats, the increased intake of PUFA increases the degree of unsaturation of the biomembrane and makes them more susceptible to lipid peroxidation (25). Reports have shown that wide utilization of fats which are highly susceptible to oxidation during cooking and frying may alter physiological effects of their PUFA content and generate lipid peroxides that cause membrane damage and increase lipid infiltration predisposing fatty liver and fibrosis (26), Thus these changes during alcohol and .\PUFA ingestion damage the liver and perturb the homeostasis of ECM. Furthermore cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase pathways are also activated by PUFA, which may increase liver damage by enhanced production of free radicals resulting in fibrosis (27).

Two possible explanations can be given for the increased activities of MMPs during alcohol and PUFA ingestion. First. MMP expression may be indu-

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Cliafl!lcs in the Activities of MMp-2 .ind MMI'.9lfl i.ivet

R Hukkumani et ;11

ced by increased amount of matrix proteins with compensatory or homeostatic mechanism against excessive deposition of matrix components. Second the increased transforming growth factor (TGF) -111, which is known to be increased in chronically diseased liver. may uprequlate MMPs at transcriptional and post transcriptional levels (28).

Previous reports suggest that in acute phase of liver injury Cine! in progressive liver fibrosis, there is increased expression of MMPs (29). Evidences indicate that the MMP expression is uprequlated in the early phase of exporunental liver injury (30). Reports have also shown that MMP-2 and 7 reveal stronger • expressions in liver dUring early liver injury (31), Hence the increased MMP expression in alcohol and L\PUFA groups in OIJr study suggests that it is due to the counter balancinq action of MMPs to degrade the excessive matrix components deposited due to alcohol and \PUFA toxicrtv.

Administration of curcumin significantly decreased the activities of MMPs in alcohol and ~PUFA treated groups. This may be mainly due to the hepatoprotectivn role of curcurrun Normally oxidative stress plays an important role in the development of alcohol and L\PUFA induced liver injury (32). Curcumin due to its effective antioxidant property might have inhibited the liver damage. Curcumin, is a well-known antioxidant and known to protect liver against toxicity (33). Curcumin acts either by preventive mode, where it prevents free radical tormation or by intervenient mode, whereby preformed free radicals are quenched by curcumin. The antioxidant mechanism cf curcumin may include one or more of the following interactions. Scavenging or neutralizing of free radicals (34), interacting with oxidative cascade and preventing its outcome (3~j), oxygen quenching and making it less available for oxidative reaction (34), inhibition of OXIdative enzymes likn cytochrome P450 (34), chelating .tnd disarming oxidative properties of metal ions such <:1S iron (:36).

Thus curcunun due to its effective antioxidant property might have Inhibited liver damaqe and thus decreased the doposnion of ECM proteins. Hence we observed decreased MMP activities in alcohol + curcumin and .\PUFA \ curcumin groups.

In our study, the expressions of MMPs were de'creased in alcohol + .\PUFA group in contrast to alcohol and .\PUFA treated groups. Reports have shown that intake of PUFA along with alcohol increases the toxicity to the liver (9). Ingestion of i\PUFA along with alcohol increases the induction of CYP2EI, which results in increased generation of ROC; and enhanced liver injury (8). The decreased activities of

The Italian Journal 01 (I1f,chernlstry Vol. ~)3 (2) 2004

Research Article

~.~~ ... ~~- .. -.-~.---

MMPs in alcohol + i\PUFA group suggest end stage liver fibrosis. Studies have demonstrated that there is a decrease in MMP activities in the later progressive stage of liver fibrosis. Possible explanations for this include decreased procollagen gene expression and biosynthesis, decreased activation of proMMPs or specific inhibition of native MMPs (37).

Arthur has reported that following a single toxic liver injury, MMPs are increased during early phase of liver injury, is maximal during inflammation and is diminished at the end fibrotic stages (38). Hence the increased MMP expression in alcohol and ~PUFA mqestion in our study correlates with inflammation or progressive liver injury anp decreased MMP expres-

. sion in alcohol + l\PUFA indicates advanced fibrosis.

Administration of curcumin to Alcohol + ~PUFA il rats significantly increased the MMP activities. This I may be due to the increase in the defense mechanism against fibrosis by curcumin. Curcumin being an effective antioxidant might have decreased LPO and prevented liver damage progressing towards end fibrotic stage.

Thus we conclude that curcumin effectively decreases the liver toxicity by influencing the activities of MMPs and thus protects structural integrity of the liver.

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Corresponding Author

Dr. Venugopal P. Menon, Professor and Head, Department of Biochemistry, Annamalai University. . Annamalainagar - 608 002, Tamil Nadu. India.

Phone: 0091 -4144-238343 Fax: 0091-4144-238343 E-mail: cmrana@sify.com

The Italian Journal of Biochomistry Vol. 53 (2) 2004