alcoholic liver disease ( ald ) represents a spectrum of clinical illness and morphological changes that range from fatty liver , hepatic inflammation , and necrosis ( alcoholic hepatitis ) to progressive fibrosis ( alcoholic cirrhosis ) .
many of the toxic effects of ethanol in the liver have been associated with its metabolism .
ethanol oxidation generates toxic products such as acetaldehyde , and reactive oxygen species result in oxidative stress that initiates apoptosis and cell injury [ 25 ] .
it is activated by endotoxin , cytokines , and oxidative stress . in unstimulated cells ,
nf-b is a heterodimeric complex that is sequestered in the cytoplasm by its interaction with ib family of inhibitors .
when these cells are stimulated , ib is phosphorylated with subsequent release of nf-b resulting in the translocation of nf-b from the cytoplasm to the nucleus where it activates the expression of target genes [ 7 , 8 ] .
activation of nf-b increased expression of proinflammatory cytokines and chemokines that were key factors in ethanol - induced liver injury rats [ 912 ] .
peroxisome proliferators activated receptors gamma ( ppar ) is a family of ligand - activated nuclear transcriptional factor which regulates cell differentiation , apoptosis , lipid metabolism , and inflammation . more recently , decreased expression of ppar has been found in rats with alcoholic liver fibrosis .
these suggested that ppar may play an important role in the development of hepatocellular inflammation , necrosis , and fibrosis in rats with ethanol consumption .
curcumin ( diferuloylmethane ) , an antiinflammatory and antioxidant compound , is isolated from the rhizomes of the plant curcuma longa   linn .
importantly , it has been showed that curcumin suppressed the activation of nf-b in ethanol - induced liver injury in rats .
activation of ppar by curcumin resulted in inhibition of nf-b trans activating activity and increased expression of ppar at both the transcriptional and translational levels in activated hepatic stellate cells ( hscs ) .
however , it is unclear whether curcumin had any effect in early stage of ethanol - induced liver injury . therefore , the present study determined the effect of curcumin on early stage of ethanol - induced liver inflammation and improved pathology in rats .
female sprague - dawley rats , weighing 180220 grams , purchased from the national laboratory animal center , mahidol university , salaya , nakorn pathom , were used .
the rats were kept in a controlled temperature room at 25  1c under standard conditions ( 12-hour day - night rhythm ) .
all rats were received well care in accordance with the ethical committee , faculty of medicine , chulalongkorn university , thailand .
curcumin in powder form ( cayman chemical company , usa ) is dissolved in 50% ethanol that freshly prepared for the experiment .
all rats were fed with the controlled diet which contained 35% of energy from fat , 18% from protein , and 47% from carbohydrate for 4 weeks ad libitum .
group 1 ( control , n = 8) : rats were fed distilled water ( 2.0 ml ) orally via an intragastric tube once per day for 4 weeks .
group 2 ( ethanol , n = 8) : rats were fed 50% ethanol ( 7.5 g / kg bw a day ) orally via an intragastric tube twice a day for 4 weeks .  
group 3 ( ethanol + curi , n = 6 ) : rats were fed curcumin ( 200 mg / kg bw ) dissolved in 50% ethanol ( 7.5 g / kg bw a day ) via intragastric tube twice a day for 4 weeks .
group 4 ( ethanol + curii , n = 7 ) : rats were fed curcumin ( 600 mg / kg bw ) dissolved in 50% ethanol ( 7.5 g / kg bw a day ) by using intragastric tube twice a day for 4 weeks . at the end of the study
three small pieces of livers were collected , frozen in liquid nitrogen , and stored at   80c for mda analysis , sod activity , and ppar protein expression .
the remaining of liver was fixed in 10% formalin solution to determine histopathology , nf-b activation , and hepatic apoptosis .
after the liver samples were fixed in 10% formalin solution at room temperature , they were processed by the standard method .
briefly , tissues were embedded in paraffin , sectioned at 5 m , and stained with hematoxylin - eosin , and then picked up on glass slides for light microscopy .
all fields in each section were examined for grading of steatosis and necroinflammation according to colantoni et al .
steatosis was scored as the percentage of parenchymal cells containing fat ( micro- or macrosteatosis ) :    0 = no parenchymal cells containing fat ,   1 =   < 20% of parenchymal cells containing fat ,   2 = 2039% of parenchymal cells containing fat ,   3 = 4050% of parenchymal cells containing fat ,  
inflammation and necrosis were scored by the number of foci of inflammation and necrosis identified under low - power field of light microscope :     0 = no inflammation and necrosis ,   1 = 1 focus per low - power field of inflammation and necrosis ,   2 = 2 foci per low - power field of inflammation and necrosis ,   3 = 3 or more foci per low - power field of inflammation and necrosis .
mda was assayed by determining the rate of production of thiobarbituric acid - reactive components .
an aliquot of 0.2 ml was mixed with solution containing 20% acetic acid , 0.8% thiobarbituric acid , and 8.1% sodium dodecyl sulfate , heated in water bath at 95c for 60 minutes .
the solution was centrifuged for 10 minutes at 4 000 rpm , and the absorbance of the supernatant fraction was determined at a wavelength of 546 nm .
sod was determined using the method of winterbourn , in which the light - triggered release of superoxide radicals from riboflavin leads to the formation of a blue complex through reaction with nitroblue tetrazolium .
one gram of the liver was homogenized in 0.1 m phosphate buffer ph   7.4 on ice and cleared by centrifugation at 3 000 rpm at 4c for 15 minutes .
the supernatant fraction was incubated in solution containing 0.067 m phosphate buffer ph   7.8 , 0.1 m edta , 1.5 mm nbt and 0.12 mm riboflavin for 10 minutes in an illuminated chamber with an 18 w fluorescent lamp .
absorbance was recorded at 560 nm , and sod activity was expressed as units / mg protein .
apoptosis was measured by the identification of apoptotic nuclei in sections of liver by fragment end labeling of dna ( apoptosis detection kit , chemicon , usa ) . in brief ,
the dna fragments were allowed to bind an antidigoxigenin antibody that was conjugated to a peroxidase .
diaminobenzidine ( dab ) was applied to develop dark brown color and then the slides were counterstained with hematoxylin .
the results were expressed as the number of positive stained cells per high - power field .
the liver sections were deparaffinized with xylene and ethanol for ten minutes . after water washing , sections retrieved the antigen ( nf-b p65 , santa cruz , usa ) with citrate buffer ph   6.0 in microwave for thirteen minutes .
next , 3%   h2o2 and 3% normal horse serum were performed on the slides to block endogenous peroxidase activity for five minutes and blocked nonspecific binding for twenty minutes , respectively .
then , the primary antibody used for nf-b p65 , a polyclonal antibody against the p65 subunit , was applied at a dilution of 1:150 for one hour at room temperature and incubated with the secondary antibody for thirty minutes .
when the development of the color with dab was detected , the slides were counterstained with hematoxylin . under light microscopy , the positive stained cells presented dark brown in nucleus .
the results were expressed as the number of positive stained cells per high - power field . liver sample ( 0.1 g )
was homogenized in 1 ml of lysis buffer for 30 minutes on ice and cleared by centrifugation at 12 000 rpm for 15 minutes at 4c .
a 60 g of protein was applied to 10% sds - page gel , and the fractionated proteins were transferred to polyvinylidene fluoride membrane .
membrane was blocked in tbst containing 5% dry nonfat milk for 1 hour and then incubated with ppar monoclonal antibodies ( 1:400 , santa cruz , usa ) overnight at 4c .
then washed three times and incubated with secondary antibody , goat antimouse igg horseradish peroxidase ( 1:4,000 , cayman , usa ) for 1 hour .
all data were presented as means and standard deviation ( sd ) . for comparison among all groups of animals , one way analysis of variance ( one - way anova ) and
the histologic appearance of the liver in the control group was normal ( figure 1(a ) ) . in the ethanol - treated group , the histologic features showed mild to moderate steatosis and mild necroinflammation ( figure 1(b ) ) .
rats treated with ethanol and curcumin 400 mg / kg bw a day improved the liver histopathology that showed only mild steatosis but not necroinflammation ( figure 1(c ) ) . the high dose of curcumin treatment ( 1,200 mg / kg bw a day ) also improved the liver histopathology that showed mild steatosis and mild necroinflammation ( figure 1(d ) ) . the summary of steatosis and necroinflammation score were shown in table 1 .
the level of hepatic mda , a marker of lipid peroxidation , increased significantly in ethanol - treated group as compared with control group ( 3.42  1.36 versus 1.44  0.24 nmol / mg protein , p < .05 ) .
curcumin treatment ( 400 or 1,200 mg / kg bw a day ) decreased the elevation of hepatic mda level significantly when compared with ethanol - treated group ( 1.43  0.14 versus 3.42  1.36 and 1.43  0.29 versus 3.42  1.36 nmol / mg protein , resp . ;
our results showed that the level of hepatic sod activity of the control group was 1081.36  145.01 units / mg protein , while that of the ethanol - treated group was 1135.86  209.48 units / mg protein . in rats treated with ethanol and curcumin ( 400 or 1,200 mg / kg
bw a day ) , the levels of hepatic sod activity were 966.28  139.44 and 967.84  116.66 units / mg protein , respectively .
the number of apoptotic nuclei in the liver of control group was very low ( 0.38  0.28 cells / high - power field ) .
in contrast , the numbers of apoptotic cells were observed frequently in centrilobular area in ethanol - treated group when compared with control group ( 2.43  2.68 versus 0.38  0.28 cells / high - power field , p < .05 ) ( figures 4   and 5 ) . there was a trend of decreased apoptosis in low dose of curcumin treatment , but the difference did not reach a statistical significance ( figure 4 ) .
the number of positive stained cells in the liver of ethanol - treated group was significantly higher than control group ( 1.08  0.52 versus 0.04  0.04 cells / high - power field , p < .05 ) .
in contrast , curcumin treatment ( 400 or 1,200 mg / kg bw a day ) decreased the number of positive stained cells significantly when compared with ethanol - treated group ( 0.15  0.02 versus 1.08  0.52 and 0.17  0.09 versus 1.08  0.52 cells / high - power field , resp . ;
p < .05 ) ( figures 6   and 7 ) . in order to examine the change of ppar protein expression in early stage of ethanol - induced liver injury
the ppar protein expression in control group was 0.57  0.19 , and ethanol group was 0.68  0.16 . rats treated with ethanol and curcumin ( 400 or 1,200 mg / kg bw a day ) had 0.44  0.03 and 0.54  0.23 , respectively .
these data did not show a significant change in ppar protein expression in the liver in all groups ( figure 8) .
ethanol oxidation generates toxic metabolites , free radicals ; and induces a state of oxidative stress which contributes to the pathogenesis of ald .
importantly , oxidation of ethanol through the cytochrome p450 2e1 ( cyp 2e1 ) generates superoxide anion radical and hydrogen peroxide [ 2 , 21 , 22 ] .
these free radicals are capable of damaging many cellular components such as dna , protein , and lipid .
one of the characteristic features of oxidative stress is enhancement of lipid peroxidation . a number of studies
have been demonstrated that ethanol intake increased the formation of lipid peroxidation product , such as mda [ 2426 ] .
we found that an increase in hepatic mda level as well as pathological changes were observed in ethanol - treated group .   to counteract this oxidative stress ,
cells have a variety of antioxidant enzymes , including sod , catalase , and glutathione peroxidase .
the effects of chronic ethanol exposure on activity of sod are controversial , with reports of decrease or no changes [ 25 , 28 ] .
these studies may reflect variations in experimental design , diet , and duration of ethanol feeding .
decreased sod activity in ethanol fed rats was associated with enhancement of lipid peroxidation and severe pathology of liver .
therefore , the sod activity in liver did not change in early ethanol - induced liver injury .
oxidative stress can also initiate or amplify inflammation through upregulation of several genes involved in the inflammatory response .
one such gene is nf-b , whose activation results in the upregulation of proinflammatory cytokines .
activation of nf-b and upregulation of cytokine production occurred in ethanol - induced liver injury and are associated with lipid peroxidation [ 9 , 10 ] .
evidence was presented that curcumin prevented ethanol - induced liver injury in rats by inhibiting the expression of nf-b - dependent genes .
although , a high dose of curcumin treatment ( 1,200 mg / kg bw ) was not better than low dose ( 400 mg / kg bw ) , the present study showed that curcumin improved ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation .
ethanol - induced liver injury has been linked to oxidative stress caused by the production of reactive oxygen intermediates that cause mitochondrial dysfunction , leading to a release of proapoptotic factors such as cytochrome c that can activate caspases and initiate the apoptotic cascade in hepatocytes .
jin and coworker observed the pathological changes and investigated the correlation of hepatocyte apoptosis with cyp2e1 expression and oxygen free radical in rats with ald . using the tunel assay , we detected a difference in apoptosis between the control and ethanol - treated group that was similar to human alcoholic hepatitis and experimental rat model of ald [ 3335 ] .
cells in centrilobular area are low o2 and nutrient supply thus the distribution of apoptotic cells is observed frequently in centrilobular area . in this study curcumin treatment
did not detect a difference in hepatocyte apoptosis ; however , this was a trend of decreased apoptosis in low does of curcumin treatment .  
this stage showed severe liver injury and hsc activation . in normal liver , hscs undergo a process known as activation , which upregulate cytokines and growth factor .
for instance , platelet - derived growth factor is capable of inhibiting ppar expression via mitogen - activated protein kinase - mediated phosphorylation of ppar .
also , tnf- , inflammatory cytokine , is known to inhibit ppar expression in adipocytes and an early phase of hsc activation in liver fibrosis [ 38 , 39 ] , thus alcoholic liver fibrosis rats could decrease ppar expression .
our model showed only mild steatosis , necroinflammation , and no hsc activation ; therefore , no change of ppar protein expression was found in ethanol - treated group .
further studies should be determined roles of ppar in different stages of ald . in conclusion
, our study demonstrated that curcumin , a representative phenolic antioxidant and antiinflammmation , could improve histopathology of liver in early stage of ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation .
for hepatocyte apoptosis , curcumin treatment might have a trend of decreased apoptotic cells in ethanol - fed rats .
 to study the mechanism of curcumin - attenuated inflammation and liver 
 pathology in early stage of alcoholic liver disease , female sprague - dawley rats were 
 divided into four groups and treated with ethanol or curcumin via an intragastric tube 
 for 4 weeks . a control group treated with distilled water , and an ethanol group was 
 treated with ethanol ( 7.5 g / kg bw ) . 
 treatment groups were fed with ethanol 
 supplemented with curcumin ( 400 or 1 200 mg / kg bw ) . 
 the liver histopathology in 
 ethanol group revealed mild - to - moderate steatosis and mild necroinflammation . 
 hepatic 
 mda , hepatocyte apoptosis , and nf-b activation increased significantly in ethanol - treated group when compared with control . 
 curcumin treatments resulted in improving 
 of liver pathology , decreasing the elevation of hepatic mda , and inhibition of nf-b 
 activation . 
 the 400 mg / kg bw of curcumin treatment revealed only a trend of decreased 
 hepatocyte apoptosis . 
 however , the results of sod activity , ppar protein expression 
 showed no difference among the groups . in conclusion , 
 curcumin improved liver 
 histopathology in early stage of ethanol - induced liver injury by reduction of oxidative 
 stress and inhibition of nf-b activation . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion