the 5-alpha reductase ( 5  ar ) inhibitor ( 5  ari ) was developed to treat the patients with symptomatic benign prostatic hyperplasia ( bph ) and decrease the frequency and risk of bph - related morbidities [ 1 , 2 ] . presently available orally administered 5  aris , finasteride and dutasteride , inhibit 5  ar , an enzyme that catalyzes the irreversible reduction of testosterone ( t ) to dihydrotestosterone ( dht ) , with nadph as the hydrogen donor . dutasteride is a selective inhibitor of 5  ar type 1 ( 5  ar1 ) and 5  ar type 2 ( 5  ar2 ) . finasteride is considered mainly an inhibitor of 5  ar2 and is approximately 50 times weaker in inhibiting 5  ar1 than 5  ar2 . the development of bph is an androgen - dependent process , and androgen suppression causes regression primarily of the epithelial elements of the prostate , resulting in a reduction in the size of the gland and improvement in symptoms [ 5 , 6 ] . as we know , androgen deprivation by surgical or medical castration is associated with penile cavernosal fibrosis resulting in penile tissue atrophy , alterations in dorsal nerve structure , alterations in endothelial morphology , reductions in trabecular smooth muscle content , increases in deposition of extracellular matrix , and increases in accumulation of adipocytes in the subtunical region of the corpus cavernosum . transforming growth factor- ( tgf- ) signaling pathway plays the most important role in the fibrotic process . tgf- reduces collagenase production and stimulates the expression of tissue inhibitor of metalloproteinases , resulting in an overall inhibition of extracellular matrix ( ecm ) degradation and leading to excessive matrix accumulation [ 8 , 9 ] . as androgen deprivation by surgical or medical castration induces cavernosal fibrosis , it may also affect the prostate tissue . in the present study , we investigated the effect of long - time treatment with 5  ari on tgf- signaling pathway in the prostate of bph patients . total 32  bph patients complaining of moderate - to - severe lower urinary tract symptoms ( luts ) who underwent transurethral resection of the prostate ( turp ) were enrolled in the present study . the patients were divided into two groups : group 1 , 16 patients underwent turp who had been treated with alpha blocker ( tamsulosin ) for 2 years ; group 2 , the other 16 patients were treated with combination of alpha blocker ( tamsulosin ) and 5  ari ( dutasteride ) for at least 1 year before the operation ( table 1 ) . approximately 1  g of each lateral and anterior portion of the prostate tissue resected from each patient was obtained during the turp . the tissues were immediately frozen at 80c . for western blot analysis , the removed tissues were placed in protease - inhibitor buffer containing 1% nonidet p-40 , 50  mm hepes , ph 7.5 , 100  mm nacl , 2  mm edta , 1  mm pyrophosphate , 10  mm sodium orthovanadate , 1  mm phenylmethylsulfonyl fluoride , and 100  mm sodium fluoride and were homogenized . the samples were normalized for total protein content with a pierce bca protein assay kit ( thermo fisher scientific , rockford , il , usa ) . 30  g aliquots were suspended in an equal volume of 2 laemmli buffer containing 2-mercaptoethanol and boiled for 5  min before performing sds - page . proteins were transferred to immobilon - p membranes ( millipore , billerica , ma , usa ) , blocked with skim milk ( 5% ) , and incubated with various primary antibodies recognizing nos ( santa cruz biotechnology , santa cruz , ca , usa ) , tgf- ( santa cruz biotechnology , santa cruz , ca , usa ) , p - smad2/3 ( santa cruz biotechnology , santa cruz , ca , usa ) , smad2/3 ( santa cruz biotechnology , santa cruz , ca , usa ) , e - cadherin ( cell signaling technology , danvers , ma , usa ) , n - cadherin ( cell signaling technology , danvers , ma , usa ) , -smooth muscle actin ( cell signaling technology , danvers , ma , usa ) , and actin ( santa cruz biotechnology , santa cruz , ca , usa ) . antibody detection was accomplished via horseradish peroxidase - conjugated protein a or goat anti - rabbit immunoglobulin g ( santa cruz biotechnology , santa cruz , ca , usa ) , a chemiluminescent substrate ( millipore , billerica , ma , usa ) , and exposure to x - ray film ( eastman kodak co. , rochester , ny , usa ) . the samples were normalized for total protein content with a bca kit and tested immediately or preserved at 80c . tgf- levels were analyzed using a commercially available elisa ( r&d systems , minneapolis , mn , usa ) per the manufacturer 's protocol . standard , controls , and samples are pipetted into the wells and any tgf-1 present is bound by the immobilized antibody . after washing away any unbound substances , an enzyme - linked polyclonal antibody specific for tgf-1 is added to the wells to sandwich the tgf-1 immobilized during the first incubation . following a wash to remove any unbound antibody - enzyme reagent , a substrate solution is added to the wells and color develops in proportion to the amount of tgf-1 bound in the initial step . the statistical significances of the differences were calculated by one - way anova followed by bonferroni 's multiple comparison . however , the expression of enos in lateral portion of prostate was decreased ( figure 1 , p < 0.05 ) . the result showed that lateral portion of prostate expressed more tgf-1 , tgf-2 than anterior wall . p - smad2/3 expression was also increased in group 2 , but there was no difference between the lateral portion and anterior wall ( figure 2 , p < 0.05 ) . the tgf- level was determined by elisa , and the results indicated that tgf- was significantly increased in group 2 ( figure 3 , p < 0.05 ) . the western blotting results of e - cadherin and n - cadherin were shown in figure 4 . the e - cadherin level decreased and n - cadherin increased significantly in group 2 . alpha smooth muscle actin was markedly increased in group 2 and expressed more in lateral portion of the prostate . tgf- participates in the pathogenesis of multiple cardiovascular diseases , including hypertension , atherosclerosis , cardiac hypertrophy , and heart failure [ 1012 ] . also , it has been shown to be a regulator of stromal proliferation and differentiation in the prostate . it is well known that androgen plays an important role in the pathogenesis of bph . the action of androgen is mediated through actions of a host of soluble growth factors , among which tgf- is the most versatile in its ability to regulate proliferation , growth arrest , differentiation , and apoptosis of prostatic stromal cells . it has been demonstrated that the effect of 5  ari on bph was associated with suppression of the insulin like growth factor 1 gene and with an increase in tgf- gene expression . tgf- predominantly transmits the signals through cytoplasmic proteins called smads , which translocates into the cell nucleus acting as transcription factors . eight different members of the smad family have been identified in mammals , in which smad2 and smad3 are specific mediators of tgf-/activin pathways [ 10 , 11 ] . in this study , the results showed that long - term treatment with 5  ari significantly increased the expression of tgf-1 , tgf-2 , and p - samd2/3 compared with the control group , suggesting 5  ari - induced cell death was regulated by tgf--smad pathway . decreased expression of e - cadherin accompanied by increased expression of n - cadherin resulted in a loss of epithelial characteristics and indicated that the epithelial cell underwent epithelial to mesenchymal transition ( emt ) . tgf--smad pathway is very important stream also involved in fibrotic process . in vascular smooth muscle cells , endothelial cells , and fibroblasts , tgf-1 increases the synthesis of ecm proteins , such as fibronectin , collagens , and activator inhibitor-1 ( pai-1 ) , even at low concentrations [ 8 , 9 ] . tgf- induces expression of fibronectin which is required for enhancement of -smooth muscle actin and collagen type i expression . pai-1 is a serpin class protease inhibitor , important in tissue remodeling by modulating thrombosis , inflammation , migration , and ecm . tgf- reduces collagenase production and stimulates the expression of tissue inhibitor of metalloproteinases ( timp ) , resulting in an overall inhibition of ecm degradation and leading to excessive matrix accumulation [ 8 , 9 ] . the mechanisms involved in tgf- mediated vascular fibrosis are complex , including activation of smad proteins , protein kinases , production of mediators , and crosstalk between pathways . tgf- also acts as a mediator of vascular fibrosis induced by several agents involved in cardiovascular diseases , including mechanical stress , angiotensin ii , high glucose , and advanced glycation products [ 1618 ] . in peyronie 's disease , the fibrosis is characterized by an increase in collagen over the intracellular compartment and is associated with the production of profibrotic factors , such as tgf-1 , pai-1 , and reactive oxygen species during oxidative stress [ 1921 ] . this fibrosis is accompanied by the induction of inos , which acts as an endogenous antifibrotic mechanism in response to the profibrotic processes [ 19 , 22 ] . the presence of nos has been demonstrated in the human prostate that the enos is related to the maintenance of local vascular perfusion , whereas the nnos is mainly involved in the control of smooth muscle tone and glandular function , including proliferation of epithelial and subepithelial cells [ 23 , 24 ] . until today , the inducible form of nos , the inos , has not been found in normal prostate tissue ; however , there are hints that the enzyme is expressed in hyperplastic and malignant tissue [ 25 , 26 ] . the reduction in glandular tissue by 5  ari is achieved by the induction of apoptosis , which is histologically manifested by ductal atrophy . inhibition also diminishes the number of blood vessels in the prostate because of a reduction in vascular - derived endothelial growth factor which downregulates the enos expression in lateral portion of prostate . the overexpression of nnos may contribute to prostate smooth muscle relaxation and improve the symptoms ; however , long - time treatment with 5  ari increases the risk of fibrosis by the tgf--p - smad signaling pathway . the 5  aris have been successfully used for treatment of bph by inhibiting the conversion of t to dht , resulting in a reduction in the size of the gland and improvement in symptoms.androgen deprivation by surgical or medical castration is associated with penile cavernosal fibrosis . the 5  aris have been successfully used for treatment of bph by inhibiting the conversion of t to dht , resulting in a reduction in the size of the gland and improvement in symptoms . androgen deprivation induced by long - term treatment with 5  ari increases the risk of fibrosis via the tgf--p - smad signaling pathway . the upregulated nnos expression may contribute to prostate smooth muscle relaxation and improve the symptoms . androgen deprivation induced by long - term treatment with 5  ari increases the risk of fibrosis via the tgf--p - smad signaling pathway . the upregulated nnos expression may contribute to prostate smooth muscle relaxation and improve the symptoms .
background and aim . 
 it is well known that androgen deprivation relates to penile fibrosis , so we hypothesize that long - term treatment with 5-alphareductase inhibitors ( 5aris ) may increase the risk of fibrosis of prostate . 
 patients and methods . 
 thirty - two bph patients who underwent transurethral resection of the prostate were enrolled . 
 the patients were divided into two groups : group one , 16 patients underwent turp who had been treated with tamsulosin for 2 years ; group two , 16 patients underwent turp who had been treated with combination of tamsulosin and dutasteride for at least 1 year . 
 we evaluated the expressions of nnos , inos , enos , tgf-1 , tgf-2 , phosphorylated - smad2/3 ( p - smad2/3 ) , e - cadherin , n - cadherin , and -smooth muscle actin in the resected prostate tissues by western blotting , and the tgf- concentration was determined by elisa kit . 
 results . 
 the expressions of 3 isoforms of nos were significantly increased in group 2 except of enos in lateral prostate , and the expressions of tgf-1 , tgf-2 , and p - smad2/3 increased about 2-fold compared with group 1 . in group 2 , 
 the e - cadherin expression decreased while n - cadherin expression increased significantly . 
 conclusions . 
 the overexpression of nnos may contribute to prostate smooth muscle relaxation ; however , long - time treatment with 5  ari increases the risk of fibrosis of prostate .