A Study on the possible therapeutic effects of wheyprotein products against hepatic fibrosis in

experimental rats

Thesis

Submitted as a partial fulfillment

For the Master Degree

In Pharmaceutical Sciences

(Pharmacology and Toxicology) (2015)

Submitted to:

Faculty of Pharmacy, Cairo University

By:

NohaFawzy Hassan

Pharmacology and Toxicology department

Faculty of Pharmacy

MTI University

Under supervision of:

Prof.Dr./ Ezz El-din Said El Denshary

Pharmacology and Toxicology department

Faculty of Pharmacy

Cairo University

Abstract

Inflammation and hepatic stellate cell (HSC) activation are the most crucial steps inthe formation of hepatic fibrosis. Hepatocytes damaged by viral or bacterial infection,alcohol or toxic chemicals initiate an inflammatory response that activates collagenproduction by HSCs. Through this mechanism chronic administration ofthioacetamide for 8 weeks at dose (100 mg/kg body weight) effectively induced liverinjury and dysfunction and caused hepatic fibrosis. In this study, the effect of Beta-lactglobulin (β-LG), Lacprodan R alpha-10(LAC) and L-methionine were evaluated inTAA-induced hepatic fibrosis in rats. Method: seventy two Sprague Dawley rats weredivided into nine groups. Group 1 received saline only; all groups were injected withTAA two times / week, concurrently, Group3-4 received daily β-LG (100mg/kg and200 mg/kg given orally), respectively. Group 5 received a combination of β-LG (50mg/kg body weight) and l-methionine (40 mg/kg body weight).Groups 6,7 and 8werereceived the same as groups 3,4 and 5 except we use LAC instead of β-LG ,whilegroup 9 was received L-methionine (40 mg/kg body weight) only .Results:TAAinduced extensive liver damage that is evidenced by significant increase in serumlevels of alanine amino transferase (ALT), Aspartate aminotransferase (AST), alkalinephosphatase (ALP) and total bilirubin(TB) .Moreover, TAA induced oxidative stressas indicated by significant increase in the hepatic lipid peroxidation (LPO)markermalondialdehyde (MDA) and nitric oxide content(NO) as well as significant decreasein antioxidant enzymes like hepatic reduced glutathione concentration (GSH) andsuperoxide dismutase (SOD) activities. The administration of whey protein products(WPs) and l- methionine concurrently with TAA succeeded in protecting hepatocytesfrom damage and attenuating hepatic fibrosis could be induced by TAA .That isevidenced by the decline in serum AST, ALT, ALP and TB levels. WPs and L-methionine reduced LPO and NO free radicals while increased GSH concentrationSOD activity in hepatic tissues. Conc.: our study

indicated that whey proteins products useful as pharmacological agent in enhancinghepatic fibrosis suppression and preventing its occurrence.

Keywords: Hepatic fibrosis; Thioacetamide; Lacprodan-alpha-10; beta-lactglobulin;L-methionine; oxidative stress, rats

INTRODUCTIONToxicological properties of thioacetamide (TAA)

CH3-C(S) NH2

Thioacetamide has been used as an organic solvent in the leather,

textile, and paper industries, as an accelerator in the vulcanization of buna rubber

(synthetic polybutadiene), and as a stabilizer of motor fuel.

Thioacetamide is an organosulfur compound. This white crystalline

solidis soluble in water and serves as a source of sulfideions in the synthesis of

organic and inorganic compounds. It is a prototypical thioamide(Ambrose et

al., 1950), among the various toxicity models, thioacetamide (TAA) is frequently

used to produce liver injury in animals and further to evaluate the therapeutic

potential of drugs. TAA administration has been shown to cause fibrosis and

cirrhosis in liver eventually leading to hepatocarcinoma (Tsukamoto et al., 1990).

Thioacetamide is a very effective, reliable, and satisfactory model in

producing liver cirrhosis in laboratory rodents. Various investigators have used

different methods of TAA administration in experimental animals for producing

fibrosis and cirrhosis, such as intraperitoneal or subcutaneous administration,

mixing the toxin with the diet or in drinking water. Use of TAA in hepatic fibrosis

animal models has many advantages, including highly specific hepatotoxicity

(Barker andSmuckler,1974), similar progression of human hepatic fibrosis

development and damage regions of the liver to those observed in human hepatic

fibrosis induced by chronic liver injury.

The experimental hepatotoxicity of TAA in rodents was studied in

response to its detection in orange juice following its use as a fungicide in orange

groves (Fitzhugh and Nelson, 1948).Single doses of TAA in the range of 1–2

mmol/kg cause classical centilobular necrosis accompanied by rise in serum

transaminases and bilirubin. Chronic administration of TAA in the diet causes liver

cancer in male rats including hepatocellular carcinoma, cholangiocarcinoma and

papillary adenocarcinoma (Yeh et al., 2004). More recently, the in vivo use of

TAA in rodents as a model hepatotoxin produced highly selective liver damage

including cirrhosis, fibrosisas well as hepatic necrosis/apoptosis (Li et al.,

2002).The initiation of the hepatotoxic effect of TAA requires metabolic activation

which finally leads to oxidative stress (Wang et al., 2000;Ramaiah et al.,

2001;Chilakapati et al., 2007).

Reactive oxygen species and their role in induction of diseases

The causes of the poisonous properties of oxygen were obscure prior to

the publication of Gershman’s free radical theory of oxygen toxicity, which states

that the toxicity of oxygen is due to partially reduced forms of oxygen (Gerschman

et al., 1954). In the same year, observations of a weak electron paramagnetic

resonance (EPR) signal attributable to the presence of free radicals in a variety of

lyophilized biological materials were reported by (Commoner et al., 1954). The

world of free radicals in biological systems was soon explored by Denham Harman

who proposed the concept of free radicals playing a role in the ageing process

(Harman, 1956). This work gradually triggered intense research into the field of

free radicals in biological systems.

A second epoch of the research of free radicals in biological systems was

explored when McCord and Fridovich discovered the enzyme superoxide

dismutase (SOD) and thus provided convincing evidence about the importance of

free radicals in living systems (McCord and Fridovich, 1969).

A third era of free radicals in biological systems when Mittal and Murad

provided evidence that the hydroxyl radical, OH, stimulates activation of guanylate

cyclase and formation of the “second messenger” cyclic guanosine monophosphate

(cGMP) (Mittal and Murad, 1977 ). Since then, a large body of evidence has been

accumulated that living systems have not only adapted to a coexistence with free

radicals but have developed various mechanisms for the advantageous use of free

radicals in various physiological functions.

Oxygen free radicals or, more generally, reactive oxygen species (ROS),

as well as reactive nitrogen species (RNS), are products of normal cellular

metabolism. ROS and RNS are well recognized for playing a dual role as both

deleterious and beneficial species, since they can be either harmful or beneficial to

living systems. Beneficial effects of ROS occur at low/moderate concentrations

and involve physiological roles in cellular responses to noxia, as for example in

defense against infectious agents and in the function of a number of cellular

signalling systems. One further beneficial example of ROS at low/moderate

concentrations is the induction of a mitogenic response(Valko et al., 2006).

The harmful effect of free radicals causing potential biological damage is

termed oxidative stress and nitrosative stress. This occurs in biological systems

when there is an overproduction ofROS/RNS on one side and a deficiency of

enzymatic and non-enzymatic antioxidants on the other. The excess ROS can

damage cellular lipids, proteins or DNA inhibiting their normal function. Because

of this, oxidative stress has been implicated in a number of human diseases as well

as in the ageing process (Kovacic and Jacintho, 2001;Ridnour et al., 2005). The

delicate balance between beneficial and harmful effects of free radicals is a very

important aspect of living organisms and is achieved by mechanisms called “redox

regulation”.

The process of “redox regulation” protects living organisms from various oxidative

stresses and maintains “redox homeostasis” by controlling the redox status in vivo

(Droge, 2002).

Biological consequences of oxidative stress

At high concentrations, ROS can be important mediators of damage to

cell structures, nucleic acids, lipids and proteins (Valko et al., 2006). The hydroxyl

radical is known to react with all components of the DNA molecule, damaging

both the purine and pyrimidine bases and also the deoxyribose backbone

(Halliwelland Gutteridge,1999).

The most extensively studied DNA lesion is the formation of 8-

hydroxyguanine (8-OH-G). Permanent modification of genetic material resulting

from these “oxidative damage” incidents represents the first step involved in

mutagenesis, carcinogenesis and ageing. It is known that metal-induced generation

of ROS results in an attack not only on DNA but also on other cellular components

involving polyunsaturated fatty acid residues of phospholipids, which are

extremely sensitive to oxidation (Siems et al., 1995). Once formed, peroxyl

radicals (ROO) can be rearranged via a cyclisation reaction to endoperoxides

(precursors of malondialdehyde) with the final product of the peroxidation process

being malondialdehyde (MDA) (Mao et al.,1999).

Reactive oxygen species and mechanisms of maintenance of “redox

homeostasis”

The intracellular “redox homeostasis” or “redox buffering” capacity is

substantiated primarily by GSH and thioredoxin (TRX). The glutathione

(2GSH/GSSG couple) represents the major cellular redox buffer and therefore is a

representative indicator for the redox environment of the cell (Droge, 2002).

Under enhanced oxidative stress conditions, GSSG content increases, this

in turn increases the content of protein mixed disulphides. A significant number of

proteins involved in signalling that have critical thiols, such as receptors, protein

kinases and some transcription factors can be altered in their function by formation

of mixed disulphides. In this regard, GSSG appears to act as a non-specific

signalling molecule. The high ratios of reduced to oxidised GSH and TRX are

maintained by the activity of GSH reductase and TRX reductase, respectively.

Both of these “redox buffering” thiol systems counteract intracellular oxidative

stress; in addition to antioxidant functioning in the cell, GSH and TRX are

involved in cell signalling process (Droge, 2002).

Under pathological conditions, however, abnormally large concentrations

of ROS/RNS may lead to permanent changes in signal transduction and gene

expression, typical for disease states. The process of redox signalling is adopted by

various organisms including bacteria to induce protective responses against

oxidative stress and to restore the original state of “redox homeostasis” after

temporary exposure to ROS/RNS.

Moreover, the cell cycle is characterized by fluctuations in the redox

environment of a cell, mediated, in particular by intracellular changes in

concentration of glutathione (Schaferand Buettner, 2001;Kernand Kehrer, 2005).

GSH has been shown to play a role in the rescue of cells from apoptosis; depletion

of GSH, which renders the cellular environment more oxidizing, was concomitant

with the onset of apoptosis. A further shift towards a more oxidizing environment

in the cell leads to apoptosis and necrosis. While apoptosis is induced by moderate

oxidizing stimuli, necrosis is induced by an intense oxidizing effect (Evens, 2004).

Reactive oxygen species, human disease and ageing: pathophysiological

implications of altered redox regulation

Oxidative stress has been implicated in various pathological conditions

involving cardiovascular disease, cancer, neurological disorders, diabetes,

ischemia/reperfusion, other diseases and ageing (Valko et al., 2007). Convincing

evidence for the association of oxidative/ nitrosative stress and acute and chronic

diseases lies on validated biomarkers of oxidative stress. Such biomarkers have to

be objectively measured and evaluated on healthy and ill subjects for long periods

(Dalle-Donne et al., 2006).

Reactive oxygen species -induced hepatotoxicity

Reactive oxygen species (ROS) are important cytotoxic and signaling

mediators in the pathophysiology of inflammatory liver diseases. Generated

intracellularly or in the extracellular space by resident and infiltrating phagocytes,

ROS are able to modulate apoptotic and necrotic cell death pathways. In addition,

reactive oxygen can affect the pathophysiology indirectly by enhancing the

formation of pro-inflammatory mediators and by activating antiproteases. Despite

the many detrimental effects, ROS are essential for host defense functions of

phagocytes and may enhance the formation of mediators regulating liver blood

flow and regeneration (Jaeschke, 2000).

Antioxidants and counteraction of reactive oxygen species

The effect of reactive oxygen and nitrogen species is balanced by the

antioxidant action of non-enzymatic antioxidants, as well as by antioxidant

enzymes. Such antioxidant defenses are extremely important as they represent the

direct removal of free radicals (prooxidants), thus providing maximal protection

for biological sites.

Some antioxidants act in a hydrophilic environment, others in a

hydrophobic environment, and some act in both environments of the cell. For

example, vitamin C reacts with superoxide in the aqueous phase while vitamin E

does so in the lipophilic phase. In contrast, -lipoic acid is both water and fat

soluble and therefore can operate both in cellular membranes and in cytosol.

Certain antioxidants are able to regenerate other antioxidants and thus restore their

original function. This process is called an “antioxidant network” (Valko et al.,

2006).

Role of antioxidants

Antioxidants are inter-related and may prevent oxidant damage in several ways:

1. Scavenging of ROS.

2. Decreasing the conversion of less reactive ROS to more reactive ROS.

3. Facilitating repair of damage caused by ROS.

4. Providing an environment favorable for activity of other antioxidants

(Clarkson and Thompson, 2000).

L-methionine and its therapeutic uses

Methionine is an essential amino acid that is required in the diet of

humans and livestock. Plant proteins are frequently deficient in methionine and

consequently an exclusively vegetable diet may fail to meet nutritional

requirements. Methionine deficiency has been linked to development of various

diseases and physiological conditions including toxemia, childhood rheumatic

fever, muscle paralysis, hair loss, depression, schizophrenia, Parkinson’s liver

deterioration and impaired growth (Rose, 1938). Deficiencies can be overcome by

supplementing the diet with methionine therefore methionine is of significant

interest (Parcell, 2002).

In April 2000, the Complementary MedicinesEvaluation Committee

(CMEC) recommended that L-methionine is suitable for use as an ingredient in

therapeutics and does not require any substance-specific restrictions on its use.

Methionine is extensively used in the poultry and feedstock industry (Funfstuck et

al., 1997; Campbell, 2001).

Besides its role in protein synthesis, the essential amino acid L-

methionine exerts tissue protective (antioxidant) actions against free radical-

induced injuryas it contains sulfur and natural antioxidant glutathione.

It may lead to a reduction of oxidative degradation (peroxidation) of lipids

and thus may offer protection against cell membrane damage. L-methionine may

also help reduce liver damage caused by lead poisoning or treatment with

medicines (Erdmannet al., 2005).

L-Methionine may also inhibit multiplication of breast and prostate cancer

cells (without affecting healthy cells). Methionine deficiency has been associated

with heart and blood vessel disorders and with cancer (Erdmannet al., 2005).The

body also needs plenty of methionine to produce two other sulfur-containing amino

acids, cysteine and taurine, which help the body eliminate toxins and build strong

healthy tissues. One of the important functions of methionine is its ability to be a

supplier of sulfur and other compounds required by the body for normal

metabolism and growth. Sulfur is a key element and vital to life. Without an

adequate intake of sulfur, the body will not be able to make and utilize a number of

antioxidant nutrients. Methionine is also a methyl donor for a wide variety of

chemical and metabolic reactions inside our body (Sánchez-Góngora et al., 1997;

Parlesak et al., 1998). Methionine acts a precursor amino acid for glutathione

which protects the cells from oxidative damage and plays vital role in

detoxification (Reed, 1990).

Milk whey proteins

Source, composition and uses

In recent years, milk constituents have become recognized as functional

foods, suggesting their use has a direct and measurable effect on health outcomes.

Whey, a by-product of cheese and curd manufacturing, was once considered a

waste product. The discovery of whey as a functional food with nutritional

applications elevated whey to a co-product in the manufacturing of cheese (Gill et

al.,2000).

Milk contains two primary sources of protein, the caseins and whey.

Whey is separated from the curd during the cheese-making process. It contains

proteins, lactose, vitamins, minerals and traces of fat. Whey protein, which

represents 20% of the total protein content of milk, is sold as a nutritional

supplement, and is particularly popular in the sport of bodybuilding. Whey

contains only five major proteins, namely β-lactoglobulin, -lactalbumin,

glycomacropeptide (depending on the method of whey manufacture), proteose

peptone 3, immunoglobulins and serum albumin, which together make up 85% of

whey protein. In addition, whey derived from buttermilk versus cheese contains the

lipid sphingomyelin (Walzem et al., 2002).

Today, whey is a popular dietary protein supplement purported to provide

antimicrobial activity, immune modulation, improved muscle strength and body

composition and to prevent cardiovascular disease and osteoporosis. Advances in

processing technology, including ultrafiltration, microfiltration, reverse osmosis

and ion-exchange, have resulted in development of several different finished whey

products. Whey protein concentrates (WPC, ranging from 80-95 percent protein);

reduced lactose whey, whey protein isolate, demineralized whey and hydrolyzed

whey are now available commercially. Each whey product varies in the amount of

protein, carbohydrates, immunoglobulins, lactose, minerals and fat in the finished

product.

These variables are important factors in the selection of whey fractions for specific

nutritional applications. (Table 2) describes the variouscomponents of whey

protein(Marshall, 2004).

Table 2: Primary components of whey protein

Whey Component % ofWhey Protein

Benefits

Beta-Lactoglobuin 50-55%

Excellent source of essential and branched chain

amino acids -Binds fat soluble, vitamins,increasing

bioavailability

Alpha-Lactalbum 20-25%

Primary protein found in human breast milk ,excellent

sourceofessential and branched chain amino acids

High in the essential aminoacid tryptophan, which

helps regulate sleep, mood, stress

lmmunoglobulins 10-15%

IgA.IgO,IgE.IgG,IgM-primarily IgG Primary protein

found in colostrums ImmunB enhancing benefits to all

ages, particularlyinfants

Lactofenin 1-2%

An S oxidant found in breast milk, tears, saliva,

antiviral,antibacterial, antifungal. Potentiate growth of

beneficial bacteria.

Lactoperoxidase 0.5% Inhibits growth of bacteria

Bovine Serum

Albumin5-10%

Lage-sized protein with good profiie of essential

aminoacid ,fat-binding properties

Glycomacropeptide 10-15%

Does not contain aminoacid phenylalanine. So is often

used ininfant formulas for infants

withphenylketonuria. It inhibits formationof dental

plaque and cavities

1. Lacprodan alpha-10®

Lacprodan alpha 10® (LAC) (food supplement) is a new product from whey

protein with high concentration of alpha lactalbumin reaching 43%.

Table 3: Lacprodan alpha 10® composition

Protein 79-87 %

α-lactalbumin of protein content 43 %

Lactose 10 %

Fat 2 %

Ash max 5 %

Moisture max 5.5 %

Alpha-lactalbumin (α-lactalbumin) is rich in essential and conditionally

essential amino acids and is the dominant whey protein in human milk, comprising

25-35 % of the milks protein content. Due to low content of α-lactalbumin in

cow’s milk, standard infant formulas and human milk vary significantly in their

content of α-lactalbumin (Heine et al., 1991). Alpha -lactalbumin is a key protein

in producing infant formula, which is closer to human milk (Brück et al., 2006).

Several clinical studies have looked into the functionality of α-

lactalbumin and identified important parameters for infant nutrition, development

and health linked to this protein (Madhu et al., 2008).

Lacprodan alpha 10® formula considered as:

• Ideal for infant formulas with low protein content.

• Infant formula with an increased content of α-lactalbumin induces a growth curve

that is more similar to breast-fed infants(Lien et al., 2004).

• Alpha-lactalbumin enriched infant formula improves infant sleep pattern due to

its high content of the amino acid Tryptophan.

• Reduce feeding related gastrointestinal events such as constipation, reflux and

vomiting.

Alpha-lactalbumin from cow’s has been fractionated and concentrated

frommilk to be suitable for infant nutrition marketed as Lacprodan® alpha-

10.These products are ideal for modifying the protein content in infant formula to

more closely replicate human milk.Lacprodan alpha-10® enhances the nutritional

value of infant milk formula and Arla foods (Denmark) markets the product to

producers of infant milk formula.

At Arla foods ingredients, key protein in bovine milk, alpha-lactalbumin,

has been isolated and carefully refined it to produce Lacprodan alpha-10®.

Used in infant formula, it offers manufacturers an important opportunity

to make their products an even better second-best to breast milk enabling formula

fed infants to obtain a nutritional and physiological status closer to that of breast-

fed.

Alpha lactalbumin has a nutritional quality documented as being higher

than typical milk proteins. Not only does it have a high content of essential amino

acids, conditionally essential amino acids, too, are present in higher than normal

quantities. The protein can also bind micronutrients and so clinical research

suggests, may benefit the immune and neurological systems and promote gut

maturation and healthy gut microflora (Saint-Sauveura et al., 2008). One

pronounced difference between human milk and infant formula based on bovine

milk is the content of alpha-lactalbumin, which is considerably lower in bovine

milk. The easiest way to obtain an approximate amino acid composition similar

to that of human milk is to use a cow’s milk protein with a high content of alpha-

lactalbumin, which contains high levels of tryptophan and cysteine and a

small amount of methionine. Bovine alpha-lactalbumin has a very good

nutritional value compared to other proteins, as can be seen from its high protein

efficiency ratio of 4.0 compared to 3.6 for whey and 2.9 for casein. Its biological

value is also higher (Haug et al., 2007).

In clinical tests, in vitro digestion of Lacprodan® alpha-10 showed

higher apparent digestibility than standard whey protein concentrate. And the

apparent digestibility of a formula enriched with alpha-lactalbumin was even

higher than that of human milk. One of the most important differences is the fact

that the whey proteins in breast milk mainly consist of alpha-lactalbumin

constitutes about 30% of the total protein in breast milk whereas the percentage in

traditional infant formula is only 10% (Forsum and Lonnerdal, 1980).

Studies with rat pups showed higher net protein utilization with

Lacprodan alpha-10® than with standard Whey protein concentrate, indicating

that this protein serves as efficient nutrition for growth and development. The

high content of tryptophan in alpha-lactalbumin is another important factor in

neurological development. Not only does it add to the overall amino acid profile of

infant formula, tryptophan is the precursor for the brain neurotransmitter serotonin

which the brain metabolizes into melatonin (Ahmed et al., 2011). Seratonin and

melatonin are responsible for many neurobehavioral characteristics such as

appetite, satiation, mood, pain perception and sleep-wake rhythm. Increased

serotonin levels may also improve the ability to deal with stress and prevent

depression (Ahmed et al., 2011).

Studies have linked Lacprodan® alpha-10 to the biological processes

involved in gut maturation.Studies shown alpha-lactalbumin reduces apoptosis, a

morphological and biochemically distinctive form of programmed cell death,

without effecting cell division and differentiation.

Biologically active peptides derived from alpha-lactalbumin have also been shown

in vitro to influence gut cell maturation and gut motility (Boudry et al., 2010).

Laprodan® alpha-10 has a significant content of sialic acid, a micronutrient

which serves as a substrate for the gut microflora and plays an important role in

the development of the intestinal cells and neurological system (Lindberg et al.,

1998).

2. Beta lacto globulin

Βeta-Lactoglobulin (β-LG) is the major whey protein found in the milk of

most mammalian species, representing 60 % of the total whey proteins. β-LG is a

globular protein consisting of 162 amino acid residues with a molecular weight

(MW) of 18.3 kDa(Liuet al., 2007).BLG has been reported to have various

biological effects (Xiao et al., 2006;LeMauxet al., 2012). In terms of cancer, β-

lactoglobulin reduced dimethylhydrazine-induced colonic aberrant crypt foci

formation in rats (McIntosh etal., 1998).

AIM OF THE WORKThe purpose of the current study is to assess the possible effectiveness of wheyprotein products such as LAC andβ-LG and their combination with L-methioninein suppression and prevention of thioacetamide- induced hepatic fibrosis model inexpermintal rats.

There is a growing interest towards the utilization of new whey protein

products to be used functionally in place of traditional therapies.

To fulfill this aim, liver fibrosis was induced by intraperitoneal (i.p.) injections of

thioacetamide (TAA) (100 mg/kg) twice a week for 8 weeks, concurrently with

LAC, β-LGadministered orally in two dose levels (100 and 200 mg/kg) and their

combination with L-methionine.

Since TAA caused generation of reactive oxygen species (ROS),

biomarkers of oxidative stress such as reduced hepatic glutathione (GSH) content,

hepatic lipid peroxidation (LPO) estimated as malondialdehyde (MDA) level,

hepatic nitric oxide (NO) level, hepatic superoxide dismutase (SOD) activity were

assessed to reveal TAA-induced hepatic fibrosis and evaluate the antioxidant

effects of whey proteins products (WPs) and their combinations withL-methionine.

Moreover, liver function tests, such as alanine aminotransferase (ALT),

aspartate aminotransferase (AST), alkaline phosphatase(ALP) activities and total

bilirubin level in serum, were carried out to assess hepatocytes damage induced by

TAA and possible therapeutic effect of WPs and their combinations.

Eventually, the extent of TAA-induced hepatic fibrosis was evaluated by

assessing morphological changes in liver sections stained with Hematoxylin &

Eosin to verify histological details and Masson’s trichrome staining to demonstrate

the collagen fibers, using standard techniques.

Summary & ConclusionsSummary

Fibrosis resulting from activation of stellate cells is considered to be

provoked by oxidative stress and cytokines. Indeed induction of free radical

generation, mitochondrial dysfunction and depletion of antioxidants are effective

in the progression of fibrosis and cirrhosis in several experimental models.

Hepatotoxicant agent such as thioacetamide (TAA)is a widely used agent to

develop liver fibrosis in rats that is similar to human fibrosis. TAA is metabolically

activated to thioacetamide sulfoxide and further to thioacetamide-S, S-dioxide. In

fact, toxic effects of TAA are attributed to those reactive metabolites. TAA-

induced liver fibrosis is observed to be associated with lipid peroxidation and

depletion of antioxidants. Accordinglyreduction of oxidative stress appears to be

helpful for theregression of fibrosis. Thus use of radical scavengersand

antioxidants to prevent occurrence of fibrosis have been suggested beneficial.

The purpose of the current study was to assess the possible effectiveness

of whey protein products such as Lacprodan® alpha-10 (LAC) andBeta-

lactglobulin (β-LG) and their combination with L-methionine in suppression and

prevention of TAA- induced hepatic fibrosis model in rats. There is a growing

interest towards the utilization of new whey protein products to be used

functionally in place of traditional therapies.

To fulfill this aim,administration of LAC, β-LG at two dose levels (100

and 200 mg/kg, oral) and their combination with L-methionine concurrently

withTAA-inducedliver fibrosis (100 mg/kg, i.p.) twice a week for 8 weeks.

Since TAA caused generation of reactive oxygen species (ROS) and nitric

oxide (NO), biomarkers of oxidative stress such as reduced hepatic glutathione

(GSH) content, hepatic lipid peroxidation (LPO) estimated as malondialdehyde

(MDA) level, hepatic nitric oxide (NO) level, hepatic superoxide dismutase (SOD)

activity were assessed to reveal TAA-induced hepatic fibrosis and evaluate the

antioxidant effects of whey protein products and their combinations.

Moreover, liver function tests, such as alanine aminotransferase (ALT),

aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities and total

bilirubin level in serum, were carried out to assess hepatocytes damage induced by

TAA and possible therapeutic effect of WPs and their combinations.

Eventually, the extent of TAA-induced hepatic fibrosis was evaluated by

assessing morphological changes in liver sections stained with Hematoxylin &

Eosin to verify histological details and Masson’s trichrome staining to demonstrate

the collagen fibers, using standard techniques.

The results of the present study could be summarized as follows:

Concurrent administration ofTAA(100 mg/kg) and LAC, β-

LGadministered orally (100 mg/kg) and their combination in dose(50 mg/kg) with

L-methionine(40 mg/kg), is capable of combating TAA-induced hepatic fibrosis by

resisting the depletion of reduced GSH which retained very close to normal level .

Further, the level of MDA showed significant reduction value compared to TAA-

intoxicated group. Furthermore, NO level in hepatic tissue retained in its normal

levels in those treated groups. Hence, this may explain the relation between high

levels of NO and lipid peroxidation. Hepatic activity of antioxidant enzyme, SOD,

significantly increased in all treated groups.

Those groups have the ability to protect the hepatocytes from TAA damage

effect,this observation was supported first by liver function test asestimation of

ALT which retained in its normal level in LAC and the combination groups while

in β-LG ,it appeared higher than normal but still lower than TAA-treated group.

However in AST test they all retained its level very close to the normal

levelcompared to TAA-treated group.

Although in ALP, only LAC (100 mg/kg) could preserve the normal level. In TB,

each of LAC, β-LG (100 mg/kg) and LAC (50 mg/kg) with L-methionine (40

mg/kg) retained the normal value. As supported by the histopathological

examinations. Highlighting that group LAC (50 mg/kg) with L-methionine (40

mg/kg) and group β-LG (50 mg/kg) with L-methionine (40 mg/kg) was the best in

almost all results. This confirmed our hypothesis that combination therapy may

have greater benefits in protecting from liver fibrosis than monotherapy, asthey

potentiate the antioxidant effects of each other. The group which showed very

close in its results to them was LAC (100 mg/kg).

Concurrent administration of TAA(100 mg/kg) and LAC or β-

LGadministered orally (200 mg/kg) exhibited a mild to moderate effectiveness

especially LAC (200 mg/kg) in suppression of hepatic fibrosis induced by TAA as

evidenced by the mild improvement in liver function tests ALT, AST, TB, GSH

and MDA when compared to LAC and β-LGadministered orally (100 mg/kg).

Conclusion

On the light of the current results, it could be concluded that:

1. Whey protein products (Lacprodan alpha-10® and Beta lacto globulin)

are effective pharmacological agents against liver fibrosis induced by

TAA as evidenced by the histopathological examination, significant

reduction in liver biomarker enzymes (ALT, AST and ALP), TB, MDA,

NO and the significant elevation in the antioxidant enzymes (GSH and

SOD).

2. The combination between whey protein products and L-methionine

could reverse fibrosis induced by TAA and potentiates their antioxidant

effects through gathering between the free radical scavenging properties

and the antioxidant promoting activity.