nonalcoholic steatohepatitis ( nash ) is a progressive form of non - alcoholic fatty liver disease ( nafld ) .
the pathogenesis of nash is multifactorial , indicating a significant association with inflammation . in its severe form
, nash can lead to cirrhosis , with permanent liver damage ( 1 , 2 ) . patients with nash show significantly higher serum levels of tumor necrosis factor - alpha ( tnf- ) and interleukin-6 ( il-6 ) , compared with patients with simple steatosis ( 3 ) .
tumor necrosis factor - alpha is produced by several types of inflammatory cells and tissues , such as monocytes , macrophages , neutrophils , endothelial cells , neuronal tissue , t cells , hepatocytes , and kupffer cells ( 4 ) .
the tnf- is significantly involved in the development of nash and nafld in animal models and humans ( 5 - 8 ) .
interleukin-6 plays a pivotal role in liver pathology and synthesis of acute phase proteins , such as c - reactive protein and serum amyloid a ( 9 ) .
on one hand , il-6 can improve liver regeneration in il-6 knockout mice with diet - induced nash and , on the other hand , it can promote hepatocyte apoptosis in patients with nash ( 10 - 12 ) .
interleukin 8 ( il-8 ) is produced by peripheral blood mononuclear cells , macrophages , and infiltrated lymphocytes .
( 13 ) reported that il-8 , alanine aminotransferase ( alt ) , age , and adiponectin were independently associated with nash .
pentoxifylline ( ptx ) is a methylxanthine derivative and an antitnf- agent , which is reportedly administered for the treatment of nash .
it can decrease the production of several pro - inflammatory cytokines , including tnf- and shows anti - inflammatory properties , through the inhibition of nuclear factor - kappa b ( nf-b ) ( 14 - 16 ) .
the results of several studies , involving the use of ptx in patients with nash , have shown the effect of ptx in nash .
published studies have several limitations , due to variation in characterization of patients , such as those with biopsy - proven nash or nash based on sonography and alt and aspartate transaminase ( ast ) levels .
differences in study designs , such as prospective cohort studies , with control group , or randomized and controlled trials and , also , difference in sample size are other reasons for different findings of studies on the effects of ptx , in patients with nash ( 17 - 24 ) .
the primary aim of this study was to evaluate the effectiveness of ptx for 6 months , in a randomized , double - blind , placebo - controlled clinical trial , in patients with nash , in the city of kerman , in south - eastern iran .
the other aims were to assess the effect of ptx on the serum levels of alt , ast , tnf- , il-8 , and il-6 in patients with nash .
thirty patients with nash were recruited at the kerman university of medical sciences , besat clinic , kerman , south - eastern iran .
written informed consent was obtained from each participant in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki .
patients were considered for the study if they had a diagnosis of nash , based on sonography , and a 1.5-fold mean change from baseline of ast and alt levels .
patients were excluded if they had a past history of excessive alcohol consumption or any cigarette smoking or narcotics use .
patients with hypertension (  140/90 mmhg ) and those who tested positive for hepatitis c virus or hepatitis b virus infection were also excluded .
additionally , patients were excluded if they had high serum levels of alpha-1-antitrypsin , ceruloplasmin , anti - smooth muscle antibody , and antinuclear antibody .
patients were also excluded if they were taking anti - hypertensive , anti - hyperlipidemic , anti - hyperglycemic medications , or amiodarone or oral contraceptive pills .
patients who were suffering from acute or chronic liver disease were excluded before and during the intervention .
the study was designed as a randomized , double - blind , placebo - controlled trial .
the study protocol was approved by the ethics committee of kerman university of medical sciences , kerman , iran .
the study was designed for 30 patients ; 15 patients ( frequency : male = 80% , female = 20% , age = 35.7  6.6 years ) in the intervention group , who received a ptx dose of 400 mg thrice a day , for 6 months , and 15 patients ( male = 80% , female = 20% , age = 39.4  9.4 years ) in the placebo group , who received a placebo for 6 months .
all patients attended the gastroenterology clinic , at afzalipour hospital , and were given advice concerning low calorie diet and the importance of daily exercise by a resident in gastroenterology .
there were no differences with regard to shape , taste , and color between the placebo and ptx .
the placebo and ptx were labeled by an unblinded pharmacist , as a and b , respectively , before random allocation to the patient .
patients , the medical examiner , laboratory and pharmacy staff did not know the nature of the drugs . on completion of data collection ,
statistical analysis was performed by a statistician who was unaware of the nature of the compounds represented by a and b. fifteen untreated healthy subjects were enrolled to determine the serum levels of cytokines , to present a cut - off point when comparing with that of the patients .
demographic data for the 15 controls were as follows : males = 36% , females = 64%
the healthy controls , selected from kerman blood transfusion centre , kerman , iran , had no history of systemic or organ specific autoimmune diseases , allergy , infectious diseases , cancer , and metabolic disorders .
laboratory studies were performed to measure levels of ast and alt , in patients with nash , at the baseline , 3 months , and 6 months .
the serum levels of il-6 , il-8 , and tnf- were also measured in patients at baseline and 6 months .
weight and height of the patients were measured , to determine body mass index ( bmi ) at baseline and during the clinical trial at 3 and 6 months .
the height of placebo and intervention groups at baseline , 3 months and 6 months was 171.2  9.8 and 172.8  7.2 , respectively .
the weight of the placebo group at baseline , 3 months and 6 months was 82.2  13 , 80.2  13 and 77.7  13.1 kg , respectively , and the weight of the intervention group at baseline , 3 months and 6 months was 80.8 12.5 , 78.2  13.1 and 73.9  12.7 kg , respectively .
sampling lasted 6 months . during this period , five patients in the intervention group stopped treatment , four of which because of adverse effects and one for personal reasons .
nine patients in the placebo group stopped treatment , five of which because of problems similar to adverse effects of ptx and four for personal reasons .
liver aminotransferases , including alt and ast , were measured for patients with nash by enzymatic photometry .
serum levels of il-6 , il-8 , and tnf- were determined for patients with nash and healthy controls by an enzyme - linked immunosorbent assay ( elisa ) technique , according to the manufacturer s instructions ( bosterbio , pleasanton , ca , usa ) .
according to the previous study ( 18 ) , by considering alpha error of 5% and the power of 90% and also employing the formula for two - sample comparison of means , the sample size was calculated to be nine , for each group .
statistical analyses , such as descriptive statistics , repeated measurement test , one - way anova , and independent t - test were performed by spss software version 17.0 ( spss inc . ,
thirty patients with nash were recruited at the kerman university of medical sciences , besat clinic , kerman , south - eastern iran .
written informed consent was obtained from each participant in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki .
patients were considered for the study if they had a diagnosis of nash , based on sonography , and a 1.5-fold mean change from baseline of ast and alt levels .
patients were excluded if they had a past history of excessive alcohol consumption or any cigarette smoking or narcotics use .
patients with hypertension (  140/90 mmhg ) and those who tested positive for hepatitis c virus or hepatitis b virus infection were also excluded .
additionally , patients were excluded if they had high serum levels of alpha-1-antitrypsin , ceruloplasmin , anti - smooth muscle antibody , and antinuclear antibody .
patients were also excluded if they were taking anti - hypertensive , anti - hyperlipidemic , anti - hyperglycemic medications , or amiodarone or oral contraceptive pills .
patients who were suffering from acute or chronic liver disease were excluded before and during the intervention .
the study was designed as a randomized , double - blind , placebo - controlled trial .
the study protocol was approved by the ethics committee of kerman university of medical sciences , kerman , iran .
the study was designed for 30 patients ; 15 patients ( frequency : male = 80% , female = 20% , age = 35.7  6.6 years ) in the intervention group , who received a ptx dose of 400 mg thrice a day , for 6 months , and 15 patients ( male = 80% , female = 20% , age = 39.4  9.4 years ) in the placebo group , who received a placebo for 6 months .
all patients attended the gastroenterology clinic , at afzalipour hospital , and were given advice concerning low calorie diet and the importance of daily exercise by a resident in gastroenterology .
there were no differences with regard to shape , taste , and color between the placebo and ptx .
the placebo and ptx were labeled by an unblinded pharmacist , as a and b , respectively , before random allocation to the patient .
patients , the medical examiner , laboratory and pharmacy staff did not know the nature of the drugs . on completion of data collection ,
statistical analysis was performed by a statistician who was unaware of the nature of the compounds represented by a and b. fifteen untreated healthy subjects were enrolled to determine the serum levels of cytokines , to present a cut - off point when comparing with that of the patients .
demographic data for the 15 controls were as follows : males = 36% , females = 64% .
the healthy controls , selected from kerman blood transfusion centre , kerman , iran , had no history of systemic or organ specific autoimmune diseases , allergy , infectious diseases , cancer , and metabolic disorders .
laboratory studies were performed to measure levels of ast and alt , in patients with nash , at the baseline , 3 months , and 6 months .
the serum levels of il-6 , il-8 , and tnf- were also measured in patients at baseline and 6 months .
weight and height of the patients were measured , to determine body mass index ( bmi ) at baseline and during the clinical trial at 3 and 6 months .
the height of placebo and intervention groups at baseline , 3 months and 6 months was 171.2  9.8 and 172.8  7.2 , respectively .
the weight of the placebo group at baseline , 3 months and 6 months was 82.2  13 , 80.2  13 and 77.7  13.1 kg , respectively , and the weight of the intervention group at baseline , 3 months and 6 months was 80.8 12.5 , 78.2  13.1 and 73.9  12.7 kg , respectively .
sampling lasted 6 months . during this period , five patients in the intervention group stopped treatment , four of which because of adverse effects and one for personal reasons .
nine patients in the placebo group stopped treatment , five of which because of problems similar to adverse effects of ptx and four for personal reasons .
liver aminotransferases , including alt and ast , were measured for patients with nash by enzymatic photometry .
serum levels of il-6 , il-8 , and tnf- were determined for patients with nash and healthy controls by an enzyme - linked immunosorbent assay ( elisa ) technique , according to the manufacturer s instructions ( bosterbio , pleasanton , ca , usa ) .
according to the previous study ( 18 ) , by considering alpha error of 5% and the power of 90% and also employing the formula for two - sample comparison of means , the sample size was calculated to be nine , for each group .
statistical analyses , such as descriptive statistics , repeated measurement test , one - way anova , and independent t - test were performed by spss software version 17.0 ( spss inc . ,
the mean ages of the patients in the intervention and placebo groups were 35.7 and 39.4 years , respectively .
the difference between the two groups , concerning age , was insignificant ( p = 0.220 ) .
the means of bmi in the intervention and placebo groups were 26.9 and 26.6 , respectively , and the difference was statistically insignificant ( p = 0.912 ) .
data regarding laboratory parameters and ultrasonography of the liver , for patients with nash are presented in table 1 .
the levels of ast and alt , measured at the baseline , 3 and 6 months , are presented in table 2 .
results in table 2 were presented after repeated measure anova with greenhouse - geisser correction .
post - hoc tests were performed to obtain mean studied parameters between time points and results were statistically significant after bonferroni correction .
abbreviations : alt , alanine transaminase ; ast , aspartate transaminase ; bmi , body mass index .
significant difference was detected from comparison between the baseline and 3 months and also between the baseline and 6 months .
there was no significant difference with regard to the serum level of alt , between the placebo and intervention groups , at baseline ( p = 0.217 ) , at 3 months ( p = 0.446 ) or at 6 months ( p = 0.236 ) .
similarly , no significant difference was observed in the serum level of ast between the groups , at baseline ( p = 0.701 ) , at 3 months ( p = 0.857 ) or at 6 months ( p = 0.125 ) .
the bmis obtained at baseline , 3 and 6 months , are presented in table 2 .
there was no significant difference in term of bmi between the groups , at baseline ( p = 0.912 ) , at 3 months ( p = 0.346 ) , and at 6 months ( p = 0.391 ) .
the serum levels of il-6 , il-8 , and tnf- , as mean  sd , in healthy controls , were 6.3  3.9 , 18.0  7.5 , and 16.8  11.3 pg / ml , respectively . levels of these cytokines were significantly higher in patients with nash , compared with healthy controls .
serum levels of these cytokines were unchanged between baseline and 6 months in the intervention and placebo groups , with the exception of il-8 , where a statistically lower level in the intervention group at baseline had increased at 6 months ( table 3 ) .
abbreviations : il-6 , interleukin 6 ; il-8 , interleukin 8 ; tnf , tumor necrosis factor .
most clinical trials on the effect of ptx in patients with nash have sufficed to compare the cytokine levels in the intervention and placebo groups , without including the healthy controls . in the present study ,
patients with nash had significantly higher serum levels of il-6 , il-8 , and tnf- , as compared to healthy individuals , which confirmed the important role of inflammation in the pathogenesis and progression of nash ( 11 , 12 ) . in the present study ,
ptx decreased the serum levels of liver transferases in patients with nash , without any significant differences between the intervention and placebo groups .
the results of a study by van wagner et al . which measured and compared the serum levels of alt , ast , tnf- , and il-6 , in patients with nash during 12 months , shows that their intervention versus placebo clinical trial is in accordance with our findings ( 25 ) .
reported serum levels of liver transferases , as well as serum levels of tnf- and il-6 in patients with nash , which were decreased in intervention and placebo groups in a 3-month , randomized , double - blind , placebo - controlled trial ( 17 ) .
they recommended daily exercise and a low - calorie diet to both the groups and reported no significant difference between the groups , with regard to the serum levels of tnf- , il-6 , ast , and alt at the end of the clinical trial .
their results are also in agreement with our findings . however , our findings are in disagreement with the results of another randomized , double - blind , placebo - controlled trial , which was performed by buranawui et al . on 32 patients with nash ( 26 ) .
they reported a significant decrease of alt and ast in the intervention group , as compared to that in the placebo group .
they also stated no significant differences in tnf- and bmi between the groups , at the end of a 6-month clinical trial , which is in agreement with our findings .
our results are in disagreement with the findings of zein et al . , who showed that liver fibrosis and the histological features of nash could be improved by ptx administration for one year ( 27 ) .
georgescu ef and georgescu m carried out a non - randomized and uncontrolled prospective study on an outpatient database to determine the efficiency of several agents , including ptx , for the treatment of nash ( 19 ) .
the bmi , serum levels of alt , glutamyl transpeptidase , alkaline phosphatase , total cholestrol , triglycerides and liver biopsies were determined at baseline and at the end of the treatment .
they concluded that ptx showed efficacy in treating non - hypertensive / non - dyslipidemic patients with nash .
however , this study did not control for the confounding effect of dietary and lifestyle interventions .
performed a systematic review on the treatment with ptx in patients with nash until june 2010 ( 28 ) .
they extracted their results from six studies , including two randomized , double - blind , placebo - controlled trials and four prospective cohort studies .
they showed that the serum levels of ast and alt were significantly decreased in ptx - treated patients .
however , there were no significant differences between the serum levels of il-6 and tnf- , in the intervention group , as compared to that in the placebo group .
they have suggested performing more well - designed , randomized , controlled clinical trials , with a larger sample size , as well as more prospective observational studies , to investigate the effect of ptx in reducing levels of cytokines .
they reviewed all relevant controlled trials of the effect of ptx in patients with nafld / nash from 1997 to july 2013 .
two prospective cohort studies with controls and three randomized , double - blind , placebo - controlled trials were included in their meta - analysis .
their findings indicated that ptx administration results in improved liver function and histological changes , as well as weight loss , in patients with nafld / nash .
one study , which was included in their meta - analysis , had a limitation , as the researchers ignored to evaluate the influence of exercise and a low - calorie diet in ptx - treated patients with nash .
the small sample size of several trials and low number of trials were other limitations of the meta - analysis .
recently performed a clinical trial to evaluate the effect of ptx on liver aminotransferases , in patients with nafld ( 30 ) .
although the levels of liver aminotransferases were reduced during the trial in intervention and control groups , their results did not show significant differences between the groups , which are in agreement with our results .
the lack of biopsies from the liver of patients with nash , as a gold standard confirmation method , was a limitation in our study .
satapathy et al . showed that five out of six patients with nash improved histologically and with improved liver aminotransferases , particularly alt , after 12 months of ptx ( 21 ) .
therefore , they suggested that measurement of liver transferases can be an acceptable criterion for evaluating patients with nash . in general , our results suggest that ptx administration does not significantly help in the treatment of patients with nash
. however , other studies show different results and it might be premature to draw conclusions about the efficacy of ptx in the treatment of nash .
hence , we suggest that a further randomized , double - blind , placebo - controlled trial should be performed , with the enrolment of more patients with nash from different races , to confirm the effect of ptx on liver aminotransferases , histological findings , and inflammatory cytokines , in patients with nash .
employment of modern techniques , such as microarray to evaluate and define more inflammatory cytokines genes , which are potentially involved in nash , may help scientists and clinicians to address the efficacy of ptx therapy on inflammation in the future .
 background : non - alcoholic steatohepatitis ( nash ) is a progressive form of nonalcoholic fatty liver disease . 
 several studies suggest that pentoxifylline ( ptx ) can improve the disease outcome.objectives:we aimed to compare the effect of pentoxifylline with placebo on liver aminotransferases and cytokines , including interleukin 6 ( il-6 ) , tumor necrosis factor alpha ( tnf- ) , and interleukin 8 ( il-8 ) in patients with nash.patients and methods : thirty patients with nash were included in the study , based on ultrasonography and 1.5-fold mean change from baseline serum levels of liver aminotransferases . 
 patients with nash were randomized to receive 1200 mg ptx ( the intervention group ) or placebo ( the placebo group ) for 6 months . 
 the serum levels of liver aminotransferases and cytokines were compared between the intervention and placebo groups , at various time points.results:the serum levels of liver aminotransferases were significantly reduced at 3 months and at 6 months , compared with baseline , in both groups . 
 the serum levels of il-6 were significantly decreased , in both groups , only at 6 months , compared with baseline . compared to the placebo group , 
 the serum level of tnf- was significantly decreased in the intervention group , at 6 months . the serum level of il-8 was increased , in both groups , after 6 months , without reaching clinical significance . 
 there was no significant difference in serum levels of liver aminotransferases and cytokines , between intervention and placebo groups.conclusions:decreases in the serum levels of liver aminotransferases and cytokines , in both groups , are related to low - calorie diets and exercise , rather than ptx . 