type 1 diabetes ( t1d ) results from the destruction of insulin producing pancreatic  cells by a  cell specific autoimmune process ( 1 ) . chronic pancreatic inflammation ( insulitis ) and destruction of islet -cells in type 1 diabetes
is mediated by the immune cells , particularly autoreactive cd4 and cd8 t lymphocytes , b cells , macrophages and dendritic cells ( 2 ) . in order to obtain insight into type 1 diabetes pathogenic mechanisms in humans and to test novel therapeutic approaches for its treatment , different preclinical models of the disease such as spontaneous and accelerated diabetes in the non - obese diabetic ( nod ) mice ,
biobreeding rats , or diabetes induced in susceptible rodent strains by multiple low doses of streptozotocin ( mlds ) are now available ( 3 ) . in the pathogenesis of t1d , several proinflamma - tory cytokines including ifn -  , tnf- , il-1 , as well as il-17 ( 4 - 6 ) , have been implicated .
it is also thought that the production of anti - inflammatory cytokines such as il-4 , il-10 and tgf- correlates with protection from t1d ( 6 ) .
it has been shown that some drugs such as pentoxifylline ( ptx ) have immunomodulatory and anti - inflammatory activity , which might represent a potential preventive therapy for autoimmune diseases .
ptx is a methyl xanthine - derived general phosphodiesterase ( pde ) inhibitor that has been available for many years to treat vascular disorder ( 7 ) .
phosphodiesterases ( pdes ) are a family of enzymes that regulate intracellular levels of the cyclic nucleotides cyclic adenosine monophosphate ( camp ) and cyclic guanosine monophosphate ( cgmp ) by catalyzing their breakdown to inactive metabolites .
drugs that increase the levels of camp , tend to reduce the production of proinflammatory mediators and increase the production of anti - inflammatory mediators by immune cells ( 8) . in recent years , the potential of ptx as an immunomodulatory and anti - inflammatory agent gained interest as it has been shown to effectively suppress the synthesis of tnf- and other pro inflammatory cytokines such as ifn -  and il-12 in vitro and in vivo ( 9 - 10 ) .
ptx has been successfully used for the treatment of experimental autoimmune diseases including experimental autoimmune myocarditis ( eam ) ( 11 ) , experimental autoimmune encephalomyelitis ( 12 ) , and adjuvant arthritis ( 13 ) . in the present study
, we hypothesized that pentoxifylline , due to its anti - inflammatory and immunosuppressive activity , may affect autoimmune diabetes .
consequently , we decided to investigate whether pentoxifylline treatment could prevent the development of mlds - induced diabetes in mice .
streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) .
male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle .
diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice .
stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days .
the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) .
mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) .
subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip )
plasma , separated from blood by centrifugation , was stored at -80c until insulin assay .
mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test .
non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) . before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes .
mice were euthanized and their spleens were removed on day 21 for cytokine production assay .
after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle .
a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris  hcl and 1% nh4cl ( ph 7.2 ) .
subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes .
remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2
the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn -  were measured by elisa kits according to the manufacturers instructions .
mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr .
after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a. one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups .
streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) .
male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle .
diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice .
stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days .
the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) .
mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) .
subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip ) was initiated in treatment group when they were considered diabetic . at the same time , the control groups received saline vehicle alone with the same schedule .
plasma , separated from blood by centrifugation , was stored at -80c until insulin assay .
mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test .
non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) .
before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes .
mice were euthanized and their spleens were removed on day 21 for cytokine production assay .
after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle .
a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris  hcl and 1% nh4cl ( ph 7.2 ) .
subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes .
remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2 mmol / l l - glutamine .
the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn -  were measured by elisa kits according to the manufacturers instructions .
mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr . the plate was then centrifuged and followed by removal of medium .
after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a.
one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups .
all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group .
however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) .
effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) .
ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage .
effect of pentoxifylline treatment on plasma levels of insulin ( * p<0.05 ) treatment of mice with ptx significantly decreased mlds - induced production of ifn-  and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) .
effect of pentoxifylline on productions of ifn  il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group ) the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) .
effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 )
all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group .
however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) .
effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) .
ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage .
treatment of mice with ptx significantly decreased mlds - induced production of ifn-  and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) .
effect of pentoxifylline on productions of ifn  il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group )
the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) .
effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 )
in rodents , experimental insulin - dependent diabetes can be induced by multiple low doses of streptozotocin ( mlds ) ( 15 ) .
this model is a commonly used animal model that has many histological and clinical features similar to those of human type 1 diabetes and involves the participation of macrophages and t cells .
streptozotocin ( stz ) is a pancreatic  cell toxin that induces inflammation of the islets by immune cells when it is given in multiple low doses ( 16 ) .
this model of diabetes offers some advantages , such as simultaneous appearance of diabetes mellitus in all animals and not having immune abnormalities that may complicate studies in spontaneous diabetes in non - obese diabetic ( nod ) mice or bio breeding ( bb ) rats .
therefore , this model as a good model has been applied extensively to investigate the type 1 diabetes mellitus , especially study the immune pathways and modulations of autoimmune insulitis and  cell death ( 17 - 18 ) .
c57bl/6 mice , also called  c57 black 6  or simply  black 6  has the advantage of strain stability and easy breeding .
the most common application of c57bl/6 mice is to serve as physiological or pathological models for in vivo experiments ( 19 ) . as such
, mlds induced c57bl/6j mice were chosen to evaluate the anti - hyperglycemic activity of ptx and investigate the immunotherapeutic molecular mechanisms of how ptx attenuated the development of t1d .
ptx is commonly used for the treatment of microcirculatory disorders and yields only minimal side effects in patients .
in addition , due to its varied immunomodulatory effects , ptx has been used in several autoimmune diseases including rheumatoid arthritis ( 20 ) , multiple sclerosis ( 21 ) , and systemic lupus erythematosus ( 22 ) .
drugs that increase the level of camp , including pde inhibitors , are known to prevent or attenuate inflammatory cell responses ( 20 ) .
previous studies have also shown that ptx lowers blood glucose levels in diabetic animals ( 23 ) , and the results from the present studies also indicated that ptx possessed an anti - hyperglycemic property .
more importantly , the present data demonstrated that ptx could increase serum insulin concentrations in type 1 diabetic mice .
it has been shown that ptx could reduce ( but not abrogate ) the insulin requirements or lengthen the  honeymoon  ( non - insulin - requiring ) period in a study of 21 children with new - onset type 1diabetes ( 24 ) .
t helper 1 cells produce proinflammatory cytokines ( tnf- , ifn -  , il-1 , il-6 , and il-12 ) , which activate macrophages and cytotoxic t cells to destroy -cells , whereas anti - inflammatory cytokines ( il-4 and il-10 ) that are produced by activated t helper 2 cells , prevent -cell destructive insulitis ( 25 ) .
il-10 has been shown to prevent the onset of diabetes in mice ( 26 ) . in response to cytokine stimulation , -cells generate reactive oxygen species ( ross ) and reactive nitrogen species such as nitric oxide ( no ) , which facilitate their destruction ( 27 ) .
no is also synthesized within cytokine - activated macrophages by inducible nitric oxide synthase ( inos ) ( 28 ) .
more recently , the role of il-17-producing t cells have been demonstrated in the development of several autoimmune diseases , including multiple sclerosis , rheumatoid arthritis and type 1 diabetes .
il-17 is a proinflammatory cytokine that is detrimental to pancreatic islet cells ( 29 ) .
il-17 promotes infiltration of neutrophils and macrophages , stimulates the production of other proinflammatory cytokines , including tnf- , il-1 , il-6 and il-12 , by activated macrophages and induces nitric oxide release from  cells that interferes with their function and cause -cell destruction ( 30 , 5 ) .
it has been assumed that ptx similar to camp elevating agents , selectively suppresses the production of t helper type 1(il-2 , ifn -  ) , but not t helper type 2(il-4 , il-6 , il-10 ) cytokines ( 31 ) .
however , it has also been demonstrated that the effect of ptx on cytokine production is cell - specific ( 32 ) .
the present study shows that ptx is not only able to inhibit the release of proinflammatory cytokines ( ifn -  and il-17 ) , but can also increase the production of anti - inflammatory cytokines ( il-10 ) . since ptx has been found to be able to inhibit the release of proinflammatory cytokines ( 33 ) , and favor th2 response ( 34 ) , it may indirectly interfere with the induction of inos .
it has been shown that pde inhibitors elevate camp , prevent or attenuate inflammatory cell responses ( 35 ) and suppress proliferation of lymphocytes ( 36 ) .
our findings are consistent with previous studies , showing that ptx treatment prevented lymphocyte proliferation .
in summary , results of the present work demonstrated that the suppressive effect of the ptx treatment on t1d was accompanied by decreased blood glucose level , increased plasma insulin level , suppression of t cell proliferation , down - regulation of th1 and th17 cytokines ( ifn -  and il-17 ) and increase in the production of il-10 in supernatant of splenic culture of the treated mice .
it seems that pentoxifylline treatment has a therapeutic effect against mlds - induced diabetes in mice .
 objective(s):pentoxifylline is an immunomodulatory and anti - inflammatory agent and is used in vascular disorders . 
 it has been shown that pentoxifylline inhibits proinflammatory cytokines production . 
 the purpose of this study was to investigate the therapeutic effects of pentoxifylline on the treatment of autoimmune diabetes in mice.materials and methods : diabetes was induced by multiple low dose of streptozotocin ( mlds ) injection ( 40 mg / kg / day for 5 consecutive days ) in male c57bl/6 mice . 
 after induction of diabetes , mice were treated with pentoxifylline ( 100 mg / kg / day ip ) for 21 days . 
 blood glucose levels and plasma levels of insulin were measured . 
 splenocytes were tested for proliferation by mtt test and cytokine production by elisa.results:pentoxifylline treatment prevented hyperglycemia and increased plasma insulin levels in the diabetic mice . aside from reducing lymphocyte proliferation , pentoxifylline significantly inhibited the production of proinflammatory interleukin 17 ( il-17 ) as well as interferon gamma ( ifn- ) , while increased anti - inflammatory cytokine il-10 as compared with those in mlds group ( diabetic control group).conclusion : these findings indicate that pentoxifylline may have therapeutic effect against the autoimmune destruction of the pancreatic beta - cells during the development of mlds - induced type 1 diabetes in mice . 