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CHAPTER- 6

Hepatoprotective Mechanism of Crinum asiaticum L. and Lycorine In Carbon Tetrachloride Induced Oxidative Stress in Swiss Albino Mice

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CHAPTER- 6

MATRIX METALLOPROTEINASE LACTATE

DEHYDROGENASE EXPRESSION, DNA FRAGMENTATION,

HISTOCHEMICAL ANALYSIS OF MAST CELLS,

HISTOPATHOLOGICAL AND ULTRA STRUCTURAL

OBSERVATION OF LIVER BY LIGHT, SCANNING AND

TRANSMISSION ELECTRON MICROSCOPES

6.1. INTRODUCTION

Matrix metalloproteinases (MMPs) are a family of highly homologous protein-

degrading zinc dependent enzymes endopeptidases.This family currently includes more

than 25 members that can be divided into collagenases (MMP-1, -8, and -13),

gelatinases (MMP-2 and 9), stromelysins (MMP-3 and 10), matrilysins (MMP-7 and

26), and the membrane-type MMPs (MMP-14 to 17 and 24). MMPs are important in

many normal biological processes including embryonic development, angiogenesis,

and wound healing, as well as in pathological processes such as inflammation, cancer,

and tissue destruction. MMPs collectively cleave most, if not all, of the constituents of

the extracellular matrix (ECM) and are involved in the breakdown and remodeling of

many tissues and organs.

Lactate dehydrogenase is a cytosolic enzyme, which is essentially present in all

tissues involved in glycolysis and exists in five different isoforms designated as LDH1

to LDH5. In cardiac muscle, kidney and erythrocytes, LDH1 and LDH2 predominate,

whereas in liver and skeletal muscle, LDH4 and LDH5 predominate. LDH3 accounts

for many other tissues such as lung, brain, endocrine glands and platelets (Palmer,

2001). With any destructive process of these tissues, the enzyme leaks into

extracellular fluids and then into body fluids. Hence, detection of elevated

concentration of this enzyme released into the blood stream from the damaged tissues

has become a definitive diagnostic and prognostic criterion for various diseases and

disorders and a study of its isoenzyme has found importance in the location of tissue

damage.


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Mast cells are derived from pluripotent haematopoietic stem cells in the bone

marrow that leave and circulate as immature cells only to mature once they reach their

destination (Ishizaka et al., 1993). This is distinctly different from basophils that

mature in the bone marrow before being released into circulation (Yong, 1997). Once

released, mast cells undergo a maturation process that involves numerous factors

including the specific cytokine, stem cell factor (SCF) (Ishizaka et al.,1993, Galli et al.,

1993). The SCF receptor, c-Kit, is abundantly expressed in mature mast cells and plays

a critical role in the maturation, development and secretory action of mast cells (Galli

et al., 1993).

A mast cell is a resident cell of several types of tissues and contains many

granules rich in histamine and heparin. Although best known for their role in allergy

and anaphylaxis, mast cells play an important protective role as well, being intimately

involved in wound healing and defense against pathogens. Mast cells play a key role in

the inflammatory process. When activated, a mast cell rapidly releases its characteristic

granules and various hormonal mediators into the interstitium. Mast cells can be

stimulated to degranulate by direct injury (e.g. physical or chemical) cross-linking of

Immunoglobulin E receptors, or by activated complement proteins.The microscopic

study of diseased tissue is an important tool in anatomical pathology, since accurate

diagnosis of cancer and other diseases usually requires histopathological examination

of samples. Trained medical doctors, frequently board-certified as pathologists, are the

personnel who perform histopathological examination and provide diagnostic

information based on their observations.

6.2. MATERIALS AND METHODS 6.2.1. Gelatin zymography (Nandini Bhattacharjee et al., 2009).

Sample and substrate gel sample buffer (10% SDS, 4% sucrose, 0.25 M Tris

HCl, PH 6.8, 0.1% bromophenol blue) were mixed in 3:1 ratio. Each sample (20 µg)

was loaded under non-reducing conditions onto electrophoretic mini-gels (SDS-PAGE)

containing 1 mg/mL of type-1 gelatin (Sigma, USA). The gels were run at a running

buffer temperature of 4 °C. After SDS-PAGE , the gels were washed twice in 2.5%

Triton X-100 for 30 min each, rinsed in water and incubated overnight in a substrate

buffer at 37 °C (Tris-HCl 50 mM, CaCl2 5 mM, NaN3 0.02%, pH 8 ). The gels were


65

stained with Coomassie brilliant blue R250 and gelatinolytic activity of matrix

metalloproteinases was detected as clear white bands on a blue background.

6.2.2. Separation of Serum LDH isoenzyme by electrophoresis (McKenzie and

Henderson, 1983)

Agarose gel (1%) was prepared and applied immediately to the glass slide.

After the agar gel sets properly, liver samples were applied into a well. After the run,

the gels were removed and stained by the following method. The staining solution

contained 1.0 ml of 1.0M lithium lactate, 1.0 ml of 0.1M sodium chloride, 1.0 ml of

5.0mM magnesium chloride, 2.5 ml of 0.1% (w/v) nitro blue tetrazolium (NBT), 0.25

ml of 0.1% phenazine methosulphate, 2.5 ml of 0.5M phosphate buffer, pH 7.5 and 10

mg of NAD in a total volume of 10 ml. The gels were incubated with the staining

solution at 37◦C in the dark for a suitable period. The separated LDH isoenzymes

appeared as purple bands. The gels were washed with 7.5% acetic acid, preserved in

5% acetic acid.

6.2.3. DNA fragmentation analysis (Wu et al., 2002)

100 mg of tissue from control and experimental groups of rats were weighed

and homogenized with 1 ml saline-EDTA reagent to get 10% tissue homogenate. 300

µl of the homogenate from all the groups were mixed with 300 µl of Tris saturated

phenol and 300 µl of chloroform-isoamyl alcohol mixture. To this content, 25 µl of

SDS was added. The contents were mixed thoroughly and centrifuged at 11,000 rpm

for 15 min. The resultant aqueous phase was collected; 9 µl of NaCl and 2 volumes of

100% ethanol (twice the volume of aqueous phase) were added. The contents were

mixed and centrifuged at 12,000 rpm for 5 min. The pellet fraction containing DNA

was dissolved in TE buffer. The DNA was detected on a 1.5% agarose gel

electrophoresis, stained with ethidium bromide and visualized by UV light.

6.2.4. Mast cell staining (Ranieri et al., 2002)

5 µm thickness tissue sections were dewaxed in xylene and rehydrated through

decreasing concentrations of ethanol to distilled water. The sections were stained with

toluidine blue for 2 min and washed with distilled water followed by staining with light

green SF for 30 s and washed using distilled water and dehydrated in increasing

concentrations through alcohol series, xylene and mounted using DPX . High power


66

objective field (40X) was chosen for counting total number of mast cells at ten different

fields per slide.

6.2.5. Histological studies (Kleiner et al., 2005)

The classic paraffin sectioning and haematoxylin eosin staining techniques were

used for the histological studies. The various steps involved in the preparation of

tissues for histological studies are as follows:

6.2.5.1. Fixation

In order to avoid tissue by the lysosomal enzymes and to preserve its physical

land chemical structure, a bit of tissue from each organ was cut and fixed in bouin’s

fluid immediately after removal from the animal body. Bouins fluid, which is the

commonly used fixative, was prepared by mixing the following chemicals.The tissues

were fixed in bouin’s fluid for about 24 hurs. The tissues were then taken and washed

in tap water for a day to remove excess of picric acid.

6.2.5.2. Dehydration

The term dehydration means the removal of water from the tissues by alcohol of

varying grades. For dehydration ethanol was used. The tissues were kept in the

following solutions for an hour each

30% alcohol.

50% alcohol

70% alcohol

100% alcohol

Inadequately dehydrated tissues cannot be satisfactorily infiltered with paraffin.

At the same time over dehydration results in making the tissues brittle, which would be

difficult for sectioning. So the tissues were carefully dehydrated.

6.2.5.3. Clearing

Dealcoholization or replacement of alcohol from the tissues with a clearing agent

is called as clearing. Xylene was used as the clearing agent for one or two hours, two

or three times. Since, the clearing agent is miscible with both dehydration and

embedding agents, it permits paraffin to infilterate the tissues. So, the clearing was

carried out as the next step after dehydration to permit tissue spaces to be filled with


67

paraffin. The tissues were kept in the clearing agent till they become transparent and

impregnated with xylene.

6.2.5.4. Impregnation

In this process the clearing agent xylene was placed by paraffin wax. The

tissues were taken out of xylene and were kept in molten paraffin embedding bath,

which consists of metal pots filled with molten wax maintained at about 50o

C. The

tissues were given three changes in the molten wax at half an hour intervals.

6.2.5.5. Embedding

The paraffin wax used for embedding should be fresh and heated upto the

optimum melting point at about 56o

C- 58 o

C. A clear glass plate was smeared with

glycerine. L-shaped mould was placed on it to from a rectangular cavity. The molten

paraffin wax was poured and air bubbles were removed by using a hot needle. The

tissue was placed in the paraffin and oriented with the surface to be sectioned. Then

the tissue was pressed gently towards the glass plate to make settle uniformly with a

metal pressing rod and allowed the wax to settle and solidity room temperature. The

paraffin block was kept in cold water for cooling.

6.2.5.6. Section cutting

Section cutting was done with a rotatory microtome. The excess of paraffin

around the tissue was removed by trimming, leaving ½ cm around the tissue. Then the

block was attached to the gently heated holder. Additional support was given by some

extra wax, which was applied along the sides of the block. Before sectioning, all set

screws holding the object holder and knife were hand tightened to avoid vibration. To

produce uniform sections, the microtome knife was adjusted to the proper angle in the

knife holder with only the cutting edge coming in contact with the paraffin block. The

tissue was cut in 7 µ thickness.

6.2.5.7. Flattening and mounting of sections

This was carried out in tissue flotation warm water bath. The sections were

spread on a warm water bath after they were detached from the knife with the help of

hair brush. Dust free clean slides were coated with egg albumin (not for

histochemistry) over the whole surface. Required sections were spread on clean slide

and kept at room temperature.


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6.2.5.8. Staining

The sections were stained as follows; deparaffinization with xylene two times

each for five minutes

Dehydration through descending grades of ethyl alcohol

100% alcohol (absolute) - 2 minute

90% alcohol - 1minute

50% alcohol - 1 minute

Staining with Ehrlich’s haemaoxylin for 15-20 minutes. Throughly washed in tap

water for 10minutes. Rinsined with distilled water.stained with eosin. Dehydration

again with ascending grades of alcohol.



100%alcohol - 1minute

Clearing with xylene two times, each for about 3 minutes interval.

6.2.5.9. Mounting

On the stained slide, DPX mountant was applied uniformly and microglass

cover slides were spread. The slides were observed in Nikon microscope and micro-

photographs were taken.

6.2.6. Ultrastructural studies by transmission electron and scanning electron

microscopy

Animals were anesthetized as described previously and then perfused with

35–50 ml of 4% paraformaldehyde and either 0.5% glutaraldehyde. Then the colon

tissue from control and experimental groups of rats were fixed with 3% glutaraldehyde

in 0.1 M phosphate buffer, pH 7.4, for 18 h. Post fixation was done using 2% osmium

tetroxide in 10mM sodium phosphate buffer (pH 7.4) and left over night. Then sections

were dehydrated using series of ethanol solutions. The tissue was embedded in a

mixture of (1:1) 1, 2-epoxy propane and Epon (Epikote resin). The tissue was then

hardened using dodecyl Succinic Anhydride (DDSA) and Methyl Nadic anhydride

(MNA). A diamine catalyst N-benzyl-N diethylamine was used for hardening. The

specimen was kept in a block holder and placed in hot air oven at 60ºC for 48 h.

Ultrathin sections were cut, stained with uranyl acetate and lead nitrate, and collected


69

on mesh grids coated with a thin Formvar film, and viewed in a Philips EM201C

transmission electron microscope. (Cogger et al., 2001). Intact liver sample was

examined using Philips Scaning Electron Microscopy. The images were taken

(15000 magnification) in each animal tissues according to methods described

previously ( Couteur et al., 2001 and McLean et al., 2003).

6.3. RESULTS

The expression of matrix metalloproteinase in control and experimental group

of mice were evaluated. A substantial increase in MMP was observed following

treatment with CCl4 compared with control group of mice. No significant changes

observed in C. asiaticum and lycorine alone administered group of mice for period of

8weeks compared to that of control group of mice. However, the expression of matrix

metalloproteinase gradually decreased by the C. asiaticum and lycorine treatment as

compared to the CCl4 induced group of mice. Similarly, silymarin treatment to

CCl4 induced mice showed significant reduction in matrix metalloproteinase expression

(Fig. 6.1). So these results confirmed that C. asiaticum and lycorine have ability to

reduce the expression of matrix metalloproteinase during free radical produced

oxidative damage in liver tissues.

Fig. 6.1. Effect of C. asiaticum and lycorine on matrix metalloproteinase expression

1. Control 2. C. asiaticum alone 3. Lycorine alone 4. CCl4 alone

5. CCl4 + C. asiaticum 6. CCl4 + lycorine 7. CCl4 + silymarin


70

LDH plays a central role in the intermediary metabolism of all cells in

mammalian organs; The present study LDH expression were analysed in control and

experimental animal. The CCl4 induced group of mice exhibited over expression of

lactate dehydrogenase as compared to control group of mice. But, no significant

alterations were observed in C. asiaticum and lycorine alone treated group of mice.

Hence, it confirmed that the CCl4 can damage the liver tissues. However,

administration of C. asiaticum and lycorine to CCl4 induced mice gradually decreased

the expression of lactate dehydrogenase compared with CCl4 induced group of mice.

Likewise Silymarin treatment to CCl4 induced group of mice shows the reduced

expression of lactate dehydrogenase compared with CCl4 induced group of mice (Fig.

6.2).

Fig. 6.3 shows the effect of C. asiaticum and lycorine on liver DNA

1. Control

2. C.asiaticum alone

3. Lycorine alone

4. CCl4 alone

5. CCl4 + C.asiaticum

6. CCl4 + lycorine

7. CCl4 + silymarin

1. Control

2. C. asiaticum alone

3. Lycorine alone

4. CCl4 alone

5. CCl4 + C. asiaticum

6. CCl4 + lycorine

7. CCl4 + silymarin

Fig. 6.2. Effect of C. asiaticum and lycorine on lactate dehydrogenase


71

More DNA fragmentation was observed in the CCl4 induced group of mice

compared with control group of mice. So, it confirmed that the CCl4 produced

oxidative stress can damage the nucleus, nucleolus and finally DNA molecules. But, no

significant variation is observed in C. asiaticum and lycorine alone treated group of

mice compared with control group of mice. However, after administration of C.

asiaticum and lycorine to CCl4 induced group of mice shows the less DNA

fragmentation as compared to that of CCl4 induced group of mice. Likewise, silymarin

treatment to CCl4 induced group of mice shows the minimum DNA fragmentation (Fig.

6.3). This results intimate DNA protective nature of C. asiaticum and lycorine as

compared to silymarin treated group of mice.

Fig. 6.4 represents toluidine blue stained liver mast cell count found in control

and experimental group of mice. When the mice administered with 1 ml of CCl4 twice a

week for 8 weeks shows the more number of mast cells liver tissues. It refers to

inflammation made by CCl4. However, no significant deviation observed in the C.

asiaticum and lycorine alone administered group of mice. Nevertheless, the number of

mast cell count decreased by the C. asiaticum and lycorine treatment compared with

those groups induced by CCl4. In addition to silymarin treatment to CCl4 induced group

of mice which shows the decreased mast cell count compared with CCl4 induced group

of mice.

Fig. 6.5 A–G represents the photomicrographs of hematoxylin– eosin staining

of hepatic tissues section of control and experimental groups of mice. Fig. A shows the

hepatic tissue of control mice exhibiting a concentric arrangement of the hepatocytes

with sinusoidal cards around the central vein and portal tracts. The portal tracts show

portal triad with portal vein, hepatic artery and bile duct. Likewise, the sections of

hepatic tissues of control group of mice treated with C. asiaticum (L) and lycorine

alone also revealed an equivalent architecture (Fig. B and C). Fig. D portrays the

section of hepatic tissues of CCl4 induced group of mice exhibiting distortion in the

arrangement of hepatocytes around the central vein, periportal fatty infiltration with

focal necrosis of hepatocytes, congestion of sinusoids around central vein regions,

granular degeneration, microvesicular vacuolization, focal necrosis, hyperemia in the

sinusoids and portal tract inflammation.


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Fig. 6.4. Effect of C. asiaticum and lycorine on Mast cell expression

Mast cells of (A) control, (B) C. asiaticum alone, (C) Lycorine alone, (D) CCl4

induced group (E) C. asiaticum (L) + CCl4, (F) lycorine + CCl4 and

(G) Silymarin + CCl4 treated mice hepatic tissue. Arrows indicate the mast cells of

control and experimental group of mice

Fig. E demonstrates the section of hepatic tissues of CCl4 induced group of mice treated

with C. asiaticum presenting the normal hepatocytes arrangement around the central

vein with abridged necrosis, declined fat accumulation and mild sinusoidal dilatation.

Fig. F shows the liver histological sections shows the well preserved hepatocytes,

uniform cytoplasm, well-known nucleus and central veins in lycorine treated group of

mice. Similarly, the hepatic tissues of CCl4 induced group of mice treated with

silymarin shows similar pattern of hepatocytes arrangement (Fig. G) and are

comparable with control group of mice.

A B

C D

E F

G


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Fig. 6.5. Histopathological alteration in control and experimental mice

(A) Control histology liver showed intact hepatocytes [H], prominent nucleus [N],

sinusoidal space [S] and central vein [CV]. (B and C) C. asiaticum and lycorine

alone similar architecture was showed when compared with control group of mice.

(D) Oral administration of CCl4 induced damage deformation in central vein,

degeneration and loss of cell boundaries. Well developed hepatocytes with

prominent nucleus and maintained sinusoidal space after C. asiaticum (L) (E)

lycorine (F) and Silymarin (G) administered

A B

C D

E F

G


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Fig. 6.6 A – G represents the Scanning electron photomicrographs of liver

tissues of control and experimental groups of mice. Hepatocytes damage, membrane

deformation was found in CCl4 induced mice liver. This histopathological examination

showed that the hepatocytes regeneration by the C. asiaticum and lycorine treatment

during the oxidative damage induced by CCl4. Similarly the scanning electron

micrograph of hepatocytes of CCl4 administered group of mice treated with silymarin

showed the similar pattern of hepatocytes protection and are comparable with control

group of mice.

Fig. 6.6. A – F. represents the Scanning electron photomicrographs of liver tissues of

control and experimental groups of mice. (A) control, (B) C. asiaticum alone, (C) Lycorine alone (D) CCl4 induced group

(E) C. asiaticum + CCl4) (F) lycorine + CCl4 and (G) Silymarin + CCl4

A B

DC

E F

G

Hepatoprotective Mechanism of

Fig. 6.7. Transmission ele

mice

Transmission elect

alone (D) CCl4 ind

Silymarin + CCl

magnification. End

mitochondria. [DM

A

C

E

of Crinum asiaticum L. and Lycorine In Carbon Tetrachloride Induced Oxidative Stress in Swiss Alb

ion electron microscopes investigation of control and

electron micrographs of (A) control, (B) C. asiaticum alon

induced group (E) C. asiaticum + CCl4) (F) lycorine +

CCl4 treated mice hepatic tissue sections were show

. Endoplasmic reticulum [ER], nucleus [N], mitochondria [M

. [DM], Residual[R], Golgi apparatus [G]

G

s Albino Mice

75

ol and experimental

alone, (C) Lycorine

rine + CCl4 and (G)

showed at 15,000×

ria [M] Disintegrated

B

F

D


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The ultrastructural changes occurred in hepatocytes of control and experimental

groups of mice are shown in Fig. 6.7 A – G. Fig. A represents the electron micrograph

of hepatocytes of control group of mice showing the normal cellular organelles, Golgi

complex, mitochondria, nucleus with intact nuclear membrane, rough endoplasmic

reticulum. Similar architecture observed in control group of mice treated with C.

asiaticum and lycorine alone (Fig. B and C). The electron micrograph of hepatocytes

of CCl4 induced group of mice (Fig.D) showed that low organelle regeneration,

swelling in the cisternae of rough endoplasmic reticulum and mitochondrial cristae,

fusion or disappearance of mitochondrial crests, degranulation of rough endoplasmic

reticulum, with damaged nuclear membrane, lipid accumulation, decreased glycogen

content. The electron micrograph (Fig. E and F ) apparently shows the hepatocytes

protective nature of C. asiaticum and lycorine in CCl4 administered group by virtue of

an apparent appearance of nuclear membrane and chromatin, either absent or

significant reduction in the swelling in the cisternae of the rough endoplasmic reticulum

and mitochondrial cristae, dilation in the perinuclear space, reduction of smooth

endoplasmic reticulum, accumulation of glycogen and parallel rough endoplasmic

reticulum cisternae with ribosome. Likewise, the electron micrograph of hepatocytes of

CCl4 administered group of mice treated with silymarin showed similar pattern of

hepatocytes protection (Fig.G) and are comparable with control group of mice.

6.4. DISCUSSION

Interaction of cells with the ECM is critical for the normal development and

function of organisms. The turnover and remodeling of the ECM must be highly

regulated to prevent abnormal development and generation of a pathological condition.

Many studies support the role of MMPs in ECM remodeling and its function

(Westermarck and Kahari, 1999). In our study we observed an increase in the MMP

activities in liver during CCl4 administration. Two possible explanations can be

attributed for the increased activities of MMPs during CCl4 treatment. MMPs are

secreted by a wide range of cells including neutrophils and macrophages

(Zeng and Guillem, 1995). This is carried out by increasing the production of cytokines

and other inflammatory mediators. Reports have shown that cytokines and

inflammatory mediators induce the promoter regions of MMP genes (Russell et al.,

2002). Increased MMP causes increased degradation of ECM. During CCl4 treatment,


77

the active metabolites activate free radical (CCl.-

3) production which causes damage to

the liver tissue. This triggers the deposition of collagen and ECM. The increased

concentration of collagen and ROS activates MMP, which degrades collagen and ECM,

thereby affecting the liver architecture. Treatment with C. asiaticum and lycorine

significantly decreased the activities of MMPs when compared to CCl4 treated mice. C.

asiaticum and lycorine by its antioxidant property it scavenges the free radical

production moreover scavenging of ROS prevents or diminishes the activation of

MMP.

LDH is an enzyme present in all human cells catalyzing the pH dependent

interconversion of lactate into pyruvate. Characteristically, human LDH can be

separated into five different isoenzymes (LDH1 through LDH5), based on their

electrophoretic mobility (Kory and Susan 1993). In the present study, CCl4 was used to

induce liver damage in rats. It significantly elevated hepatic enzyme activity of total

LDH. However, LDH activity was reversed by the C. asiaticum and lycorine treatment

when compared with CCl4 induced group of mice. Laila Faddah et al., (2007) reported

that the CCl4 induced group of rats showed significant increase of total LDH. However,

after treating with DDB (Diphenyl Dimethyl Dicarboxylate) with vitamin C and E

reduced the activity of LDH.

DNA fragmentation is a vital characteristic in apoptosis. With respect to the

mechanism, it has been known that there are plenty of species of endogenous DNases

existing as a status of zymogen in the cytoplasm. DNase molecules can recognize a

specific sequence between adjacent chromosomes. When the zymogen of a given

DNase is activated, DNA molecules within cells will be chopped up into various

fragments with different lengths, thus leading to DNA fragments with 180-200 bp and

their integral times, where a typical DNA laddering can be seen (Granville et al., 1998,

Nagata, 2000, Nicholson et al., 1997) in agarose-gel after electrophoresis, which has

been regarded as a vital marker in apoptosis that is distinctively different from necrosis.

The present study, in CCl4 induced group of mice expressed more DNA fragmentation

when compared with control group of mice. However, mice administered with C.

asiaticum and lycorine shows less DNA fragmentation. These results indicate that the

C. asiaticum and lycorine can protect the DNA from oxidative damage produced from

CCl4.


78

Hepatic mast cells consistently increase in number with progression of various

liver diseases. Studies of cirrhosis, fibrosis, hepatitis and other cholangiopathies

demonstrate that, histologically, hepatic mast cell counts generally increase as diseases

progress (Koda et al., 2000, Matsunaga and Terada, 2000, Stoyanova, 2004)

implicating a significant role for mast cells in hepatic disorders. During systemic mast

cell activation syndrome patients with elevated cholesterol levels also display increased

liver transaminases and bilirubin levels suggesting that mast cell activation plays a part

in liver abnormalities of unknown etiology (Alfter et al., 2009). These studies, and

others, warrant an evaluation of mast cells during liver disease and progression. Our

present study shows the number of mast cells in the CCl4 treated groups were increased

gradually, where as C. asiaticum extract and lycorine administered group of mice

showed significant decrease in number of the mast cell. Our results also concurrence

with Da Hee Jeong et al., (2005).

Centrilobular necrosis, ballooning of hepatocytes, infiltration of lymphocytes

and steatosis of liver cells were characteristic alterations occurred due to CCl4

intoxication (Shukla et al., 2005, Bhadauria et al., 2007). Histopathological assessment

of different liver segments of the control and experimental animals by light microscope

and scanning microscope has been examined. CCl4 treatment caused disorganization of

hepatocytes and deformation of surface membranes. Moreover, administration of the C.

asiaticum and lycorine after CCl4 intoxication reduced such alteration and maintains

the organ quite similar to that of control group of mice. The results of the

histopathological studies supported and well interrelated with data obtained from

biochemical analysis.

Transmission electron microscopic studies also potentiate the histological

observations showing the degeneration of the hepatocytes ultrastructure. The markedly

depressed glycogen granules, disturbed mitochondria and disintegration of hepatocytes

rough endoplasmic reticulum found during CCl4 induction state possibly correlate with

the decreased level of hepatic protein synthesis. This degeneration in CCl4 induced

mice also leads to change in intracellular calcium level as rough endoplasmic reticulum

is renowned to sequester calcium. Moreover, the pathology circumstance of

hepatocytes mice contained swollen mitochondria (Ernster and Schatz, 1981) which are


79

ultimately responsible for the enhancement of mitochondrial membrane permeability

leading to the alteration in the mitochondrial matrix enzymes activity. These alterations

might affect the redox state of the mitochondrial thiol groups by modifying the

mitochondrial NAD reduction level (Balázs and Halmos, 1985) which ultimately

disturbs the intracellular free calcium level and its function, it leads to change the

permeability of the mitochondrial membrane as results membranes damages,

cytoskeleton disassembly, chromatin condensation and decreasing of ATP

(Nelson Fausto, 2006). The most significant ultrastructural recovery with

C. asiaticum (L) and lycorine treatment occurred in mitochondria and sER.

Mitochondria is the energy source of the cell and specifically demonstrated in

hepatocytes with two membranes, one of which limits the organelle and the other is

inside the organelle and is thrown into folds that project inward in a tubular nature

called cristae mitochondria (Ernster and Schatz, 1981).The molecules in the electron

transport chain which play the central role in ATP synthesis are found in the cristae. It

is also well known that mitochondria is both a major source of endogenous production

of ROS (Balaban et al., 2005). However, C. asiaticum (L) and lycorine treatment to

CCl4 induced group of mice confirmed the regeneration of rough endoplasmic

reticulum, normalization of the mitochondrial size and increases in hepatic glycogen

granules confirming its protective activity during oxidative stress generated by CCl4.

In conclusion, our results signify that C. asiaticum extract and lycorine was able

to reverse the matrix metalloproteinase, Lactate dehydrogenase. DNA fragmentation

and mast cell expression of liver damage induced by CCl4 and also it maintained the

cellular integrity of liver during CCl4 induction. The C. asiaticum extract and lycorine

indeed retarded the liver injury by blocking the oxidative stress. Hence, this

investigation should be considered an innovative assessment for the hepatoprotective

nature of C. asiaticum and lycorine in mice injured by CCl4.Therefore, the C.

asiaticum extract and lycorine may be useful as a stress reducing agent against

chemical-induced chronic liver diseases.

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