is one of the most popular tonic herbs in asia . in a traditional herbal prescription ,
eu is used either as a single herb or in combination with one or two of the other herbs .
eu is also a popular folk drink and is used as a functional food reinforcing the muscles and lungs , lowering blood pressure , preventing miscarriages , improving liver and kidney tone , and increasing longevity .
eu , prepared from leaves or bark , is commonly used as liver and kidney tonic , thus , improving detoxification and circulation by the liver   and kidney [ 4 , 5 ] , respectively .
eu leaves have been used for treatment of hepatic lipid accumulation [ 6 , 7 ] and hepatic damage .
cortex extracts ( euce ) contain the same components as eu leaves , which have been the focus of medical research .
studies have shown that eu leaf extracts have potent protective effects in various lipid peroxidation models and reduce oxidative damage of biomolecules [ 3 , 811 ] .
hepatic accumulation of triglyceride ( steatosis ) is a major complication associated with obesity , insulin resistance , and alcoholic and nonalcoholic fatty liver disease .
this is because of increasing lipogenesis and decreasing -oxidation followed by lipid peroxidation and mitochondrial dysfunction . if left untreated
carbon tetrachloride ( ccl4 ) is a colorless liquid that was commonly used as an anesthetic in the 19th and early 20th century .
however , ccl4 was banded after establishment of its hepatotoxicity in the first 25 years of the 20th century .
ccl4-induced liver damage in rats is the best characterized animal model of xenobiotic - induced free radical - mediated liver diseases .
depending on the dose and duration , the effects of ccl4 on hepatocytes are manifested histologically as hepatic steatosis , fibrosis , hepatocellular death , or carcinogenicity .
triglyceride secretion depends on the function of endoplasmic reticulum ( er ) which assembles and secrets apolipoproteins in the liver .
if er function is damaged , secretion of apolipoproteins such as apolipoprotein b ( apob ) is inhibited , leading to hepatic lipid accumulation .
after oral administration , ccl4 concentrates in the liver , resulting in rapid accumulation of triglycerides in the liver .
recently , ccl4 was shown to induce reactive oxygen species ( ros ) through activation of cytochrome p450 , leading to er stress - mediated dysfunction .
specifically , p450 2e1 interacts with nadph - dependent cytochrome p450 reductase ( npr ) .
electron uncoupling between npr and p450 2e1 is a major source of ros on the er membrane .
ros attack polyunsaturated fatty acid portions of membrane lipids to propagate a chain reaction , leading to lipid peroxidation and disruption protein synthesis , which results in the accumulation of proteins in the er lumen and induction of er stress .
it has been reported that severe and prolonged er stress causes the accumulation of free radicals and disruption of protein secretion , leading to alteration of pathological conditions .
regulation of er stress has been suggested as one of the therapeutic / preventive approaches for the treatment of pathological conditions / diseases with er stress .
proteins accumulated during er stress are degraded by lysosomal enzymes through the endoplasmic reticulum - associated degradation ( erad ) pathway .
the lysosome - induced erad pathway has been suggested as one of the regulatory mechanisms of er stress because the lysosomal activation can relieve intra - er unfolded protein folding requirement .
er stress regulation can be one of the potential mechanisms for ros - associated hepatic steatosis .
the secretion of apob is also altered in the presence of er stress [ 21 , 22 ] .
ccl4-induced steatosis is related to er stress and its related dysfunctions such as the alteration of apolipoproteins and ros accumulation .
accordingly , the aim of this study is to investigate the effect of euce in ccl4-induced hepatic steatosis and er stress .
this study suggests that the preventive / therapeutic effect of the cortex extracts is due to the regulation of er stress through lysosomal activation .
carbon tetrachloride ( ccl4 ) and oil red o were purchased from sigma - aldrich company ( mo , usa ) . dried cortex of eucommia ulmoides oliv .
the cortex was ground into a powder , mixed with extraction solvent ( 25% ethanol ) at the ratio of 1  :  12 , and then incubated in a 70c water bath for 2 hours .
following incubation , the extraction solution was filtered , evaporated , and then dried to a powder by freeze drying at 55c under low pressure .  
forty - eight sprague dawley ( sd ) male rats weighing 240250  g ( 8-week - old ) were purchased from samtako inc .
( osan , korea ) and housed in an air - conditioned room at 22  2c with a 12  h light / dark cycle .
animals were fed with rodent chow and tap water ad libitum . to study the protective effect against the ccl4-induced acute liver lipid accumulation ,
all rats were randomly divided into six groups of eight rats each : ( a ) control group , ( b ) ccl4 group , ( c ) ccl4 + euce 0.25  g / kg , ( d ) ccl4 + euce 0.5  g / kg , ( e ) ccl4 + euce 1  g / kg , and ( f ) euce 1  g / kg .
rats of groups b , c , d , and e were intraperitoneally injected with 1  mg / kg ccl4 mixed in olive oil , and rats of groups a and f were intraperitoneally injected with the same volume of pure olive oil .
rats in groups c , d , e , and f were treated with euce 8 times ( twice / day for four days ) before the injection of ccl4 ( except for rats in group f ) .
four hours after ccl4 injection , each rat was anesthetized , blood was drawn , and liver tissues were removed .
livers were excised from the animals and assayed for gsh levels , mda formation , and pathological histology , according to the procedures described below .
this experiment was approved by the institutional animal care and use committee of chonbuk national university , jeonju , korea .
then , liver samples were embedded in oct compound and cut into 10  m sections for oil red o staining .
fixed samples were then stained with 0.3% oil red o in 60% isopropanol for 30  min and washed with 60% isopropanol .
sections were counterstained with hematoxylin , washed with running water for 5  min , and mounted with an aqueous solution .
free radical scavenging activity of the euce was measured using the 1 , 1-diphenyl-1-picrylhydrazyl ( dpph ) radical scavenging assay .
euce solution ( 0.3  ml ) at a range of concentrations was mixed with 0.2  mm dpph in methanol ( 2.7  ml ) .
the mixture was shaken vigorously and allowed to stand for 1  h before the absorbance was measured at 517  nm .
free radical scavenging activity was calculated as the following percentage : [ ( as  ai)/as]100 ( as is absorbance of dpph alone and ai is absorbance of dpph in the presence of various extracts ) . butylated hydroxyl toluene ( bht ) at a concentration identical to the experimental samples was used as a reference .
serum levels of triglyceride ( tg ) , total cholesterol ( tc ) , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , liver glutathione ( gsh ) , and malondialdehyde ( mda ) were determined using a commercial analysis kit obtained from the asan institute of biotechnology ( seoul , korea ) and jiancheng institute of biotechnology ( nanjing , china ) .
liver samples ( 115  mg ) mixed with 500  l d - pbs were homogenized and centrifuged at 3500  g for 5 minutes . the supernatants were removed and centrifuged briefly after the addition of 400  l chloroform - methanol ( 1  :  2 ) .
then , 250  l chloroform and 250  l water were added , and the samples were centrifuged at 3000  rpm for 5 minutes . the lower phase was transferred to a new tube , and residual chloroform was evaporated by heating at 55c .
after chloroform evaporation , 25  l of ripa buffer was added and the samples were resuspended by heating at 90c for 3 minutes .
proteins were separated under nonreducing conditions , transferred to nitrocellulose membranes , and incubated for 2  h at room temperature in blocking buffer ( 20  mm tris , ph 7.5 , 137  mm  nacl , 0.1% tween 20 , and 5% nonfat dry milk ) .
blots were washed three times and incubated overnight at 4c in the same buffer containing 0.5% dry milk and primary antibody ( 1  :  1000 dilution ) .
the blots were then washed and incubated with mouse horseradish peroxidase - conjugated secondary antibody ( 1  :  4000 ) in 1.0% skim milk for 1 hour at room temperature .
then , the intensities of band were measured and quantified as described by luke miller ( http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/ ) .
, microsomal proteins were incubated in assay buffer ( 1  mm ascorbic acid , 2  mm magnesium chloride , 1  mm nadph , and 50  mm phosphate buffer , ph 6.8 ) at 37c in a shaking water bath . following incubation ,
samples were concentrated to 50  l , and then the supernatants were transferred to a 96-well plate .
prior to reading , 2  m naoh was added to each sample or standard .
absorbance was measured at  = 517  nm on a 96-well plate reader .  
isolation of lysosomes was performed using a method based on differential and density - gradient centrifugation techniques .
after isolation of lysosomes , the 100  l assay mixtures consisted of the following : -galactosidase assay , 0.5  mm 4-methylumbelliferyl- ( mu- ) -galactoside in 100  mm citrate - phosphate buffer ( ph 4.35 ) with 0.4  m  nacl ; -glucuronidase assay , 1  mm 4--glucuronide in 100  mm acetate buffer ( ph 4.0 ) ; -mannosidase assay , and 4  mm -mannopyranoside in 100  mm acetate - phosphate buffer ( ph 4.0 ) .
assay mixtures were incubated at 37c for 30 minutes with an excitation wave length from 360  nm to 410  nm . all data are expressed as mean  sd . all comparisons were done by one - way analysis of variance ( anova ) with dunn 's test for post hoc analysis .
liver tissues were collected to assess the effect of euce on liver pathological changes . as shown in figure 1(a ) , h&e staining of liver sections demonstrated normal liver architecture in ccl4-treated rats .
oil red o staining showed the development of acute lipid accumulation in rats 4 hours after injection of ccl4 , whereas no histological abnormalities were observed in normal control or euce - treated rats ( figure 1(b ) ) .
administration of euce prevented fatty deposition in hepatocytes . as demonstrated by histological results , h&e staining indicated no morphology changes in the ccl4-treated rats compared with the control group , while oil red o staining showed that ccl4-induced lipid accumulation was blocked by pretreatment with euce .
specifically , a significantly reduced in lipid accumulation was observed with administration of euce at a dose of 1  g / kg . to analyze the possible role of euce in lipid metabolism , which plays a major role in fatty liver formation , triglyceride ( tg ) and total cholesterol ( tc ) in liver and serum were investigated . as shown in figure 2 , tg and tc levels
were significantly increased by acute ccl4 injection , and this was blunted by euce pretreatment .
the effect of euce on dpph free radical scavenging activity was tested , and the results are presented in figure 3(a ) . as shown in figure 3(a ) , ic50 values of euce were about 310  g / ml . euce exhibited a curve of antioxidant activity .
gsh levels were significantly depleted by ccl4 administration ; however , depletion of gsh induced by ccl4 was significantly reversed in a dose - dependent manner by pretreatment with euce ( figure 3(b ) ) . ccl4-induced
compared with the control group , the ccl4-treated group showed significantly increased mda content ( figure 3(c ) )
. however , euce treatment at dose of 0.5 and 1.0  g / kg significantly decreased mda content .
these results indicate that euce has the potential to reduce lipid peroxidation induced by ccl4 .
furthermore , alt and ast , hepatic enzymes that are released into the blood stream by liver damage , were not increased ( figures 3(d ) and 3(e ) ) .
apoa1 is responsible for carrying hdl , and apob is responsible for carrying ldl and triglyceride .
as shown in figure 4 , the expression of apob was increased in the liver after 4  hr of exposure to ccl4 , but apoa1 was no affected . increased expression of apob in the liver
was suppressed by pretreatment with euce in a dose - dependent manner , particularly at a dose of 1  g / kg .
transient accumulation of triglyceride and cholesterol in liver induced by ccl4 occurred via decreased plasma apob production and vldl secretion .
these results show that pretreatment with euce may improve apob secretion compared with ccl4-treated group .
a previous study reported on the identification of proteins that play an important role in the survival of liver cells after induction of er stress by ccl4 . as shown in figures 4(a ) and 4(b ) in ccl4-treated rats , the expression of er stress proteins grp78 , chop , ire-1 , and spliced xbp-1 was increased , and eif-2 was phosphorylated in liver tissue .
pretreatment with euce reduced the expression of er stress proteins in a dose - dependent manner .
p450 2e1 , the major isozyme involved in the bioactivation of ccl4 and responsible for er stress - induced ros , was also increased in ccl4-treated rats .
consistently , pretreatment of euce significantly reduced the p450 2e1 activity ( figure 4(c ) ) .
enhanced activity of lysosomal enzymes has been suggested to have a regulatory role on er stress .
pretreatment with euce significantly increased the activity of lysosomal enzymes compared with the control group , particularly at a dose of 1  g / kg ( figure 5 ) , thus , indicating the potential role of euce in enhancing lysosomal enzyme activity .
this study showed that rats pretreated with euce were protected against ccl4-induced hepatic lipid accumulation , as confirmed by histological observation and decreased levels of triglyceride and total cholesterol compared with the control group .
euce regulated er stress response by decreasing p450 2e1 activity and ros accumulation . ccl4-induced er stress response , enhancing p450 2e1 activity . in our previous study
, we showed that er stress and its related p450 2e1 activity play an important role in ccl4-induced steatosis in rats . in the ccl4-induced hepatic steatosis ,
possible role of ros accumulation has been suggested , er stress and its consequent increase of p450 2e1 , leading to ros accumulation .
a converse mechanism , p450 2e1 activation and ros accumulation induced by ccl4 , leading to er stress , is not able to be ruled out . although the cause or consequence of ros production in relation to er stress has not been clearly established , the pieces of evidence of ccl4-induced ros production have been already accumulated .
recently , ccl4 was shown to induce hepatotoxicity by enhancing the formation of free radicals through their metabolism , leading to lipid peroxidation of cellular and organelle membranes as a primary pathogenic step . in this study ,
p450 2e1 activity was significantly increased 4 hours after exposure to ccl4 compared with the control group , while the euce - pretreated group showed decreased p450 2e1 activity compared with the ccl4-treated group ( figure 4(c ) ) .
ccl4 is widely used for experimental induction of liver steatosis / cirrhosis in relatively acute settings .
cytochrome p450 2e1 , a member of the cytochrome p450 mixed - function oxidase system , can catalyze ccl4 to form trichloromethyl free radicals which interact with molecular oxygen to form trichloromethyl peroxy radicals [ 16 , 3537 ] .
these free radicals play an important role in the pathogenesis of liver steatosis by binding to proteins or lipids , which then initiates lipid peroxidation .
it has also been reported that p450 2e1 is the primary enzyme responsible for low - dose carbon tetrachloride metabolism in human liver microsomes .
the hepatotoxic effects of ccl4 are dependent on the cosubstrate nadph because conversion of ccl4 to ccl3oo occurs in conjunction with the nadph - cytochrome p450 electron transport chain in the liver endoplasmic reticulum [ 13 , 40 ] .
cytochrome p450 transfers an electron from nadph to ccl4 , causing ccl4 to be reduced to ccl3oo and cl .
a previous study showed that the expression of p450 2e1 and its interaction with npr both increase after ccl4 treatment ; however , both were also shown to decrease after 12 hours .  
this study also showed that euce pretreatment increased gsh level that had been lowered by ccl4 .
gsh helps prevent damage of important cellular components caused by reactive oxygen species such as free radicals and peroxides .
moreover , gsh plays a preventive / therapeutic role in ccl4-induced hepatic toxicity via the p450 2e1 pathway .
gsh is an antioxidant that contributes to the detoxification of ccl4 , which induces hepatic lipid accumulation through its free radical derivatives .
as reported , increased production of ros induced by ccl4 plays a role in liver steatosis / cirrhosis through two distinct pathways .
one pathway involving p450 2e1 leads to the formation of toxic peroxyl and alkoxyl radicals that initiate lipid peroxidation .
the second pathway involves a detoxification reaction that lowers gsh levels [ 41 , 42 ] .
therefore , the dpph assay was used in this study to evaluate the effect of euce on free radical scavenging activity .
in addition , oxidative stress , which is considered to play an important role in the development of hepatic steatosis / cirrhosis , is associated with lipid peroxidation and lower levels of gsh .
in addition , the ccl4-treated group had significantly increased levels of mda , whereas the euce - pretreated group had significantly decreased levels of mda in liver .
mda is a metabolite of the free radical - mediated lipid peroxidation cascade and therefore is used as a marker of lipid peroxidation .
thus , the biochemical mechanism underlying the development of ccl4 steatosis / cirrhosis may involve mda .
in ccl4-treated rats , significantly increased levels of mda have been shown . in this study , the expressions of er stress proteins and hepatic apob were both increased in ccl4-treated rats , whereas they were decreased in the euce groups ( figures 4(a ) and 4(b ) ) .
this result suggests that euce might modify apob synthesis and therefore could impact on liver and plasma triglyceride content .
apolipoproteins , lipid binding proteins that form lipoproteins to transport lipids through the lymphatic and circulatory systems , are regulated by normal function of the er . with er stress , protein folding and secretion can be significantly affected .
as reported , apob , a member of the apolipoprotein family , is reduced in ccl4-treated rats .
ccl4 decreases secretion of very low density lipoproteins and rapidly increases triglycerides in rat livers .
consistently , in ccl4-treated groups , triglycerides rapidly accumulate in liver , contributing to the failure of secretory mechanisms . it has been reported that decreased apob secretion is responsible for hepatic lipid accumulation [ 50 , 51 ] .
the results of lysosomal enzymes activity shown in figure 5 indicate that euce increased the activity of lysosome enzymes by improving er function . through the protein degradation machinery activation ,
grp78 , also known as binding immunoglobulin protein ( bip ) , is of a particular importance as a regulator of the er stress response .
grp78 normally binds to three main transmembrane proteins : the protein kinase rna- ( pkr- ) like er protein kinase ( perk ) , activating transcription factor 6 ( atf-6 ) , and inositol - requiring enzyme 1 ( ire-1 ) .
in addition , grp78 also serves as the master regulator of the er stress response by binding and inactivating stress sensors at the luminal surface of the er . initiation of er stress response occurs when the quantity of misfolded or unfolded proteins in the er exceeds the capacity of chaperone proteins that trigger the activation of upr pathways . to reduce the accumulation of proteins in the er , perk phosphorylates eukaryotic initiation factor 2 ( eif-2 ) to attenuate translation of proteins .
ire-1 is related to genes involved in the transport of unfolded proteins out of the er and in their degradation by er - associated degradation ( erad ) pathway . as reported , misfolded and unstable proteins in the er are degraded by the erad pathway .
lysosomes mediate degradation of the majority of intracellular proteins , and lysosomal activity is involved in the erad ii pathway . through the stably maintained lysosomal activity , it may be suggested that euce regulates er stress and its subsequent reduced bioactivation of ccl4 to ccl3oo by p450 2e1 , which can activate er stress in response . in conclusion , the results of this study indicate that pretreatment with euce effectively decreases hepatic lipid accumulation induced by ccl4 .
euce increases lysosomal enzyme activity , relieving the protein folding requirement leading to the attenuation of er stress .
the regulatory effect of er stress is suggested to improve apob secretion as well as to regulate the biotransformation of ccl4 and its resultant inhibition of ros accumulation .
future research is necessary to unravel the mechanism of underlying the ability of euce to increase lysosomal enzyme activity , a suggested er stress regulation mechanism .
 
 eucommia ulmoides oliv . ( eu ) 
 has been used for treatment of liver diseases . 
 the protective effects of eucommia ulmoides oliv . 
 cortex extracts ( euce ) on the carbon tetrachloride- ( ccl4- ) induced hepatic lipid accumulation were examined in this study . 
 rats were orally treated with euce in different doses prior to an intraperitoneal injection of 1  mg / kg ccl4 . 
 acute injection of ccl4 decreased plasma triglyceride but increased hepatic triglyceride and cholesterol as compared to control rats . 
 on the other hand , the pretreatment with euce diminished these effects at a dose - dependent manner . 
 ccl4 treatment decreased glutathione ( gsh ) and increased malondialdehyde ( mda ) accompanied by activated p450 2e1 . 
 the pretreatment with euce significantly improved these deleterious effects of ccl4 . 
 ccl4 treatment increased p450 2e1 activation and apob accumulation . 
 pretreatment with euce reversed these effects . 
 er stress response was significantly increased by ccl4 , which was inhibited by euce . 
 one of the possible er stress regulatory mechanisms , lysosomal activity , was examined . 
 ccl4 reduced lysosomal enzymes that were reversed with the euce . 
 the results indicate that oral pretreatment with euce may protect liver against ccl4-induced hepatic lipid accumulation . 
 er stress and its related ros regulation are suggested as a possible mechanism in the antidyslipidemic effect of euce . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion