diabetes mellitus ( dm ) is one of the common metabolic diseases characterized by hyperglycemia .
global prevalence of dm was approximately 2.8% in 2000 and is estimated to reach 4.4% in 2030 .
sustained hyperglycemia can lead to complications in multiple organs , such as neuropathy , retinopathy , nephropathy , male impotency , and cardiovascular diseases .
several studies from experimental animals and the diabetic men demonstrated that sustained hyperglycemia results in reproductive complications , as high glucose possibly lead to oxidative stress and cell apoptosis , which result in structure and functions impairments and finally contribute to infertility .
diabetes has been recently diagnosed in younger persons and the age barrier has been broken .
therefore , diabetes - induced reproductive dysfunction is emerging as a new and urgent challenge . increased oxidative stress in diabetes has been considered to contribute to the damage of reproductive system .
oxidative stress has been used to describe the disturbance between the production of reactive oxygen species ( ros ) and the ability to detoxify reactive intermediates in biological system .
imbalance in the redox state of cells can increase the level of free radicals and peroxides which can impair the components of the cell , such as protein and dna , and result in toxic effects .
recent study showed that increased oxidative stress in diabetic rats induced damage of testicular dna , depletion of sperm cells , and delay of spermatogenesis , resulting in infertility .
dm leads to vascular complications , associating with the appearance of macroangiopathy , microangiopathy , and diabetic thrombophilia in multiple organs .
actually , studies have demonstrated that diabetes can bring about oxidative stress - induced microvascular complications . vascular endothelial growth factor ( vegf ) ,
a signal protein that controls angiogenesis and vasculogenesis , plays an important role in diabetes - induced vasculopathy . during the processes of diabetes , dyslipidemia ,
hypertension , and damage of no synthesis occurred and decreased the blood flow rate in the vessels , which can result in tissue hypoxia and unbalance expression of vegf , whereas little was known about the change of testicular microcirculation and the role of vegf in diabetes .
although big effort has been made for controlling the blood glucose , the complication of diabetes is still the major reason to cause organ dysfunction and death .
the effect of agents especially on alleviating reproductive complications in diabetes has been rarely discussed .
recent studies show that many natural products have antidiabetic activity   and significant antioxidant capacity .
some of the natural products have been used as alternative and complementary medicine for diabetes .
scutellarin ( 40,5,6-trihydroxyflavone-7-glucuronide , linear formula : c21h18o12 , scu ) extracted from erigeron breviscapus ( vant . ) is one of the flavone glycosides .
preclinical data showed that scu can ameliorate diabetic nephropathy through reducing ros generation   and reduce cardiomyocyte apoptosis by upgrading the expression of proapoptotic genes in bcl-2 family .
acute and toxicological study showed that ld50 value of scu could not be measured , and its maximum tolerance was 10  g / kg .
continuous oral intake of scu ( 100 and 500  mg / kg ) up to one month did not lead to any death or organ damage in rats .
because of its satisfactory results , safety , and tolerance , scu has been widely utilized in treatment of diseases associated with diabetes . in this study
, we first evaluated the effect of scu on improving testicular dysfunction and structure and studied the mechanisms of its actions using diabetic model in rat .
all animal protocols were approved by china council on animal care and sun yet - san university committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments .
adult male wistar rats ( 200  g250  g ; 6 weeks of age ) were purchased from animal facility of sun yat - sen university and housed in stainless steel cages .
rats were randomly divided into three groups : control group ( n = 8) , diabetes group ( n = 8) , and diabetes + scu group ( n
= 8) . rats were fed under controlled environment at 25  3c , humidity of 4065% and 12  h light / dark cycle , and free access to food and drink water .
normal food ( animal facility of sun yat - sen university , guangzhou , china ) was given to the control group .
high - fat diet ( hfd , guangdong medical laboratory animal center , guangzhou , china ) , containing 30% fat , 15% protein , and 55% carbohydrate , was used in diabetes group and diabetes + scu group to induce insulin resistance . at week 8
, a single dose of streptozotocin ( stz , sigma - aldrich , mo , usa , s0130 ; 65  mg / kg ) dissolved in 0.1  m citrate - phosphate buffer was freshly prepared and intraperitoneally administered into diabetes group and diabetes + scu group to induce the type ii diabetes mellitus ( hfd feeding was continued ) .
the same volume of 0.1  m citrate - phosphate buffer was used as vehicle control for control group .
the rats with blood glucose levels higher than 13  mmol / l for three times were considered as type ii diabetes mellitus ( t2 dm ) .
blood obtained from rat tail - vein was evaluated to determine the levels of blood glucose using automated blood glucose analyzer ( roche , model gc , switzerland ) .
after one week of the injection of stz , rats in diabetes + scu group were intragastrically administrated with scu ( purity > 98.5% , yunnan plant pharmaceutical , yunnan , china ; 100  mg / kg / day ) dissolved in a phosphate buffer ( 0.1  m , ph 7.3 at 20  mg / ml ) every day for 2 months .
the dose of stz and scu was according to previous published work [ 17 , 22 ] . the same volume of phosphate buffer was used as vehicle control for control group and diabetes group . at week 16 , all rats were sacrificed with injection of chloral hydrate as anesthetic , and then testes and epididymis were removed and weighed .
apoptotic cells in testicular were measured through tunel assay with the in situ cell death detection kit , pod ( roche , basel , switzerland ) following the manufacturer 's instructions .
testicular tissue was fixed in 4% paraformaldehyde , embedded in paraffin , and then sectioned at 5  m .
briefly , slides were dewaxed and rehydrated in xylene and ethanol followed by incubation with proteinase k working solution at 37c for 20  min . after rinsing with pbs
, the samples reacted with tunel reaction mixture ( 50  l ) at 37c for 1 hour followed by rinsing the slides with phosphate - buffered saline to stop the reaction .
apoptotic cells were analyzed by randomly counting the tunel positive cells from sixty cross - sections of seminiferous tubule / slide under the fluorescence microscope at 200 and 400 magnification ( olympus , th4 - 200 , tokyo , japan ) .
results were quantitative analysis as tunel positive cells per 100 cells ( image - pro plus 6.0 ) .
after dewaxing in xylene and dehydrating in graded concentrations of alcohol , sections were stained by haematoxylin and eosin ( h&e ) used to evaluate testicular morphology .
immunohistochemistry was performed according to sisman et al .   and the manufacturer 's instructions .
the slides were prepared as described in tunel assay , immersed in citrate buffer , and heated in a microwave oven for 15  min .
after three times washing with tbs , the slides reacted with anti - vegf primary antibody ( abcam , britain , dilution 1  :  200 ) or anti - vwf primary antibody ( abcam , britain , dilution 1  :  200 ) and then reacted with anti - rabbit secondary antibody at 37c for 30  mins followed by three washes with pbs , respectively .
diaminobenzidine ( dab kit ; beijing zhongshan biotechnology co. , china ) was used to visualize immunoreactive proteins .
the protein levels were detected using polink-2 plus ihc detection system ( beijing zhongshan biotechnology co. , beijing , china ) after staining with diaminobenzidine ( dab kit ; beijing zhongshan biotechnology co. , china ) to visualize immunoreactive proteins following the manufacture instruction .
diaminobenzidine ( dab ) was added to the slides for 3  min as a chromogen .
followed by rinsing in running tap water , slides were counterstained with hematoxylin ( blue ) . finally , slides were dehydrated in absolute ethanol and mounted .
all of them were observed under the light microscope at 200 magnification and 400 magnification ( olympus , japan ) .
testes were taken from rats under ethylether anaesthesia and were grinded by tube mill control ( ika , germany ) .
we diluted homogenates to 5  mg  tissue / ml in ice - cold locke 's buffer and then transferred samples into 24-well plates ( 0.45  ml / well ) .
after that , 5  l of dcfh - da ( 10  m final concentration ) was dropped into each sample . during 15  min incubation at room temperature
, dcfh - da can incorporate into any membrane - bound vesicles and esterases can cleave the diacetate group . at that time
, 40  l of the fe was put into the samples , which can transform the dcfh to fluorescent product dcf measured by fluorescence spectrophotometer ( f-7000 , hitachi , japan ) with excitation at 485  nm and emission at 530  nm .
the concentration of mda was considered as a marker of lipid peroxidation , which can reveal the oxidative damage as a result of diabetes .
testes were removed from rats under ethylether anaesthesia and were grinded by tube mill control ( ika , germany ) .
the whole blood samples were taken from the abdominal vein of rats , and then they were spun to get serum .
we detected the mad level of testes and serum by lipid peroxidation ( mda ) assay kit ( abcam , ab118970 ; cambridge , uk ) , following the manufacturer 's instructions .
after anaesthesia by chloral hydrate , the testicular vasculature was localized with a ms 400 probe ( 1838  mhz ) for color doppler sonography ( visualsonics
the control steps were carried out following the manufacturer 's instructions and previous published paper .
the red color indicated blood flow towards the transducer and the blue color indicated blood flow away from the transducer .
color images were shown in real time and doppler spectral analyses were done . at least three measurements from each recording were carried out and the mean of the three readings was taken as the representative .  
testis samples were immersed in lysed buffer and centrifuged at 13,000  rpm for 15  min at 4c .
bicinchoninic acid ( bca ) kit ( pierce , rockford , il , usa ) was used to measure the protein concentration in the supernatants with steps according to the manufacturer 's protocol . total protein
( 50  g ) was added to each lane onto 10% sds - polyacrylamide gels .
after electrophoresis , we used tris - buffered saline containing 0.1% tween-20 to wash the polyvinylidene fluoride ( pvdf ) membranes ( millipore , billerica , ma , usa ) and then incubated them with primary antibody : anti - bcl-2 ( diluted 1  :  1000 , cell signaling technology , beverly , ma , usa ) , anti - bax ( diluted 1  :  1000 , cell signaling technology , beverly , ma , usa ) , anti - vwf ( diluted 1  :  1000 , abcam , britain ) , and anti - vegf ( diluted 1  :  1000 , abcam , britain ) at 4c overnight .
secondary antibody ( 1  :  5000 , pierce , rockford , il , usa ) was added to the membrane for 2-hour incubation .
protein was visualized with immobilon western chemiluminescent hrp substrate ( millipore , millipore corp . ,
band intensity was quantified by densitometry using the image j software ( national institutes of health , bethesda , md , usa ) .
blood samples ( 5  ml ) were taken from the inferior vena cava before sacrifice at the week 16 .
serum was spun at 1500  rpm for 30 minutes and stored at 20c until used .
these sera were sent to clinical laboratory of the first affiliated hospital , sun yat - sen university , analyzed for triglyceride ( tg ) , total cholesterol ( tc ) , high density lipoprotein ( hdl ) , and low density lipoprotein ( ldl ) .
significant differences between the groups were determined using a one - way anova through the spss 19.0 software .
after injection of stz , body weights of diabetes group and diabetes + scu group showed a decreased tendency .
serum glucose suddenly exceeded the normal level in diabetes group and diabetes + scu group after stz injection ( figure 1(b ) ) .
serum glucose in diabetes + scu group was lower than diabetes group , but there were no statistically significant differences between them ( p > 0.05 ) .
the data of body weight and serum glucose from week 1 to week 16 were present as mean  sem ( see supplemental table 1 of the supplementary material available online at  http://dx.doi.org/10.1155/2015/252530 ) . the testicular weights and epididymal weights of rats were presented in table 1 . comparing with the control group ,
decreased testicular weights and epididymal weights were shown in the diabetes group ( p  0.05 ) . in the diabetes + scu group , both of these weights were heavier than those of diabetes group ( p  0.05 ) .
mda and ros concentrations in testes , epididymis , and serum were shown in figure 2 .
compared with control , obviously higher concentrations of mda and ros in testes , epididymis , and serum were shown in diabetes group ( p < 0.05 ) .
the increased mda and ros can be effectively suppressed by scu , while concentrations of mda and ros have significant difference between diabetes group and diabetes + scu group ( p < 0.05 ) .
an obvious increasing of testicular apoptosis in diabetic rats at 2 months after diabetes onset , measured by tunel staining ( figures 3(a ) , 3(b ) , and 3(c ) ) , was found along with a significant increase in bcl-2/bax ratio ( figure 3(e ) ) . in the control group ,
only a few apoptotic cells were present in testis ( figure 3(a ) ) , but they obviously increased in diabetic group ( figure 3(b ) ) .
scu treatment resulted in decrease of tunel - positive cells ( figure 3(c ) ) , though it was still larger than control group ( p < 0.05 ; figure 3(c ) ) .
the apoptosis index was 3.7% , 21.9% , and 11.8% in control group , diabetes group , and diabetes + scu group , respectively ( figure 3(d ) ) .
we also used western blot to confirm the antiapoptosis effects of scu on testes of diabetic rats ( figure 3(e ) ) .
rats in diabetes group showed a decreased expression of bcl-2 and increased expression of bax , while scu treatment led to increased expression of bcl-2 and decreased expression of bax ( figure 3(e ) with quantifications of ratio of bcl-2/bax in figure 3(f ) ) .
these data are consistent with our tunel results that diabetes induced testicular apoptosis , which can be alleviated by scu treatment .
control group showed the normal testicular structure and regular seminiferous tubular morphology with plenty germ cells and spermatozoa .
the blood vessel walls in the control group were thin and smooth ( figure 4(a ) ) . in the diabetes group , atrophic
also the thickness of blood vessel walls were significantly increased ( figure 4(b ) ) .
treatment with scu effectively increased the number of germ cells and spermatozoa , but germ cells were still disordered with some sloughing .
blood vessel walls were thinner than those in diabetes group ( figure 4(c ) ) .
the effect of scu on improving the testicular blood flow rate in diabetic rats was evaluated through analyzing the vti .
color - coded doppler ultrasound images of testicular blood were easily visualized ( figure 5(a ) ) .
vti was decreased from 243.57  mm / s in control group to 56.87  mm / s in diabetes group ( p < 0.05 ) . in the diabetes + scu group , vti
was obviously increased to 131.25  mm / s ( p < 0.05 ) ( figure 5(k ) ) .
cytoplasmic staining in the photographs indicated the expression of vegf which is more prominent in control group , compared to diabetes group ( figures 5(e ) and 5(f ) with quantifications in figure 5(l ) ) .
scu treatment brought about increasing expression of vegf in testes of diabetic rats , which were measured by immunohistochemical staining and western blot ( figures 5(g ) , 5(l ) , 5(n ) , and 5(o ) ) immunohistochemical staining and western blot indicated that the testicular blood vessel density did not show any differences in these three groups ( figures 5(h ) , 5(i ) , 5(j ) , and 5(n ) , with quantifications in figures 5(m ) and 5(p ) ) .
dm , a state of chronic hyperglycemia , is a serious global disease with multiple organ and system dysfunction , including reproductive system .
hyperglycemia - mediated apoptosis contributes to the damage of targeted organs [ 27 , 28 ] . as the downstream effectors of hyperglycemia , oxidative stress and microcirculation impairment
thus , the regimen that can both target oxidative stress and improve microcirculation is a promising strategy for prevention and treatment of diabetic complications , including reproductive disorder . in this study
, we evaluated the effect of scu , a traditional herbal medicine , which has been shown to diminish oxidative stress and improve microcirculation in some diabetic complications , on hyperglycemia - induced apoptosis in testes of stz - induced diabetic rats .
data showed that stz - induced diabetic rats exhibited increased levels of oxidative stress detected by higher levels of ros and lipid peroxidation , increased levels of apoptotic cells associated with upregulation of proapoptotic bax and downregulation of antiapoptotic bcl-2 in testes , and increased levels of microcirculation impairment demonstrated by decreased levels of blood flow velocity and vegf , whereas scu significantly reversed those hyperglycemia - induced actions .
our data , for the first time , suggest that scu is a potent agent for prevention and treatment of diabetes - associated reproductive disorder .
thus , hyperglycemia combined with body weight loss is the common marker for a success in induction of diabetes .
data in figures 1(a ) and 1(b ) show the significant body weight loss and increase in hyperglycemia level in diabetes group , indicating a successful induction of t2 dm in our study .
data also show that scu has effect on recovery of body weight loss but has no effect on glucose levels , demonstrating that scu targets downstream effectors of hyperglycemia .
body weight loss is a common symptom in stz - induced diabetic rats , which is combined with organ weight loss .
the reason for weight loss is that stz possibly prevents the secretion of testosterone and growth hormone , resulting in disorder of anabolic activities .
data in table 1 show a significant decrease in the weights of testes and epididymis in diabetes group in comparison with control group , suggesting that the weight loss of reproductive organs may be one of the reasons to cause its dysfunction .
diabetes - induced weight loss of reproductive organs in male has been reported to be induced by oxidative stress , leading to atrophy of sex organs .
scu totally reversed the weight loss of testes and epididymis to control level ( table 1 , p  0.05 ) , suggesting that downstream effector of hyperglycemia plays major role in the weight loss of reproductive organs in diabetic rat . in our study ,
ros - induced apoptotic cell death and organ atrophy may be the major factor for the organ weight loss .
it is noted that scu totally reversed the weight loss of testes and epididymis but only partially blocked body weight loss ( figure 1(a ) ) , suggesting that different mechanisms are applying for body weight loss and organ weight loss , respectively , in diabetes .
growing evidence indicates that oxidative stress is increased in diabetes due to overproduction of ros and decreased efficiency of antioxidant defences .
ros , as an effector of hyperglycemia , occurs very early during diabetes development and plays major role in organ damage .
the dominant lethal - type mutation in male , a genetic alteration in a gamete , can be induced under high oxidative stress , which kills the conceptus early in development .
as early as 5 days of stz - induced rats , enhanced ros and lipid peroxidation can be measured in testes .
male - mediated dominant lethal - type mutations , a genetic alteration in a gamete , can be induced under high oxidative stress , which kills the conceptus early in development .
ros has been reported to induce apoptosis of testicular germ cells , leading to degeneration of testes and infertility in diabetic rats .
ros - induced apoptosis is associated with ros - induced mitochondrial dna damage , leading to activation of dna damage - mediated p53 signaling pathway , involving upregulation of proapoptotic bax and downregulation of antiapoptotic bcl-2 in testes of diabetic rats .
our data showed that diabetes - induced obvious apoptotic morphological changes ( figure 4 ) and increased tunel positive cells ( figure 3 ) of testicular cells , as well as increased levels of proapoptotic bax and decreased levels of antiapoptotic bcl-2 in testes , were associated with increased levels of ros and lipid peroxidation ( figures 2(a ) and 2(b ) ) in diabetes group ( figure 2 ) , suggesting that ros induces apoptosis of testicular cells in our diabetic mode .
recent study showed that sulforaphane , a controller of detoxification response , targeted antioxidative factor , nrf2 , to inhibit diabetes - induced cell apoptosis pathway in testes .
actually , our research has a similar result that scu , an antioxidant , blocks both hyperglycemia - induced ros and apoptosis in testes further confirming that ros contributes to hyperglycemia - induced apoptosis in testes .
although researches have proven that sulforaphane has effect on suppressing oxidative stress and cell death , but little evidence indicates that it can improve diabetes - induced microcirculatory disturbance of testes [ 38 , 39 ] .
microcirculatory disturbance is a common feature in diabetes , which plays critical role in organ damage .
blood flow velocity is used to evaluate the change of bloodstream , which indicates the microcirculatory situation of organs
. the lower level of blood flow velocity possibly results in lack of nutrients and deoxygenation of organs , in which apoptosis is triggered .
the mechanism underlying the blood flow abnormalities possibly relates to oxidative - induced endothelial dysfunction , leading to vasospasm and elevated blood viscosity .
there is no change of testicular blood vessel density in diabetic group ( figure 6 ) ; therefore the low expression of vegf is manifested by endothelial dysfunction which can result in microcirculatory impairment .
increased ros triggers parp / nf-b pathway , leading to vasospasm , enhanced coagulation of platelet and blood , and ischemic episodes [ 10 , 43 ] .
thus , ros induces apoptosis in diabetic organ via at least two mechanisms ( figure 6 ) : ( i ) ros directly triggers apoptosis via activation of ros - mediated apoptotic signaling pathways , such as p53/bcl-2 pathway , and ( ii ) ros indirectly triggers apoptosis via microcirculatory disturbance , leading to lack of nutrients in organs , in which starving apoptotic pathway can be activated , such as mtor pathway .
our data clearly showed lower levels of blood flow velocity in diabetic rats , suggesting that the testes lacked blood supply and the cells were in starving condition .
it will be interesting to know if starving apoptotic pathway is involved in hyperglycemia - induced apoptosis in testes .
scu , a well - known polyphenolic flavonoid extracted from the chinese traditional herb , fleabane compositae , erigeron breviscapus , is the major effective ingredient of breviscapine , which is widely used in the treatment of cardiovascular diseases .
it is also being used in the treatment of cerebral ischemia and infantile acute viral myocarditis [ 44 , 45 ] .
scu exhibits antioxidant property which has been studied for its protective effects on neuro , blood circulation , and nephropathy .
scu has been reported to be effective in dilating blood vessels , increasing blood flow rate , improving microcirculation and hemodynamics , decreasing blood viscosity , and preventing platelet conglomeration [ 47 , 48 ] .
due to its antioxidant property and its ability to improve microcirculation , scu has been studied for prevention and treatment of diabetic complications , such as diabetes associated cardiovascular disease , diabetic nephropathy , and diabetic retinopathy . however , whether scu can prevent and treat diabetes associated productive disorder is elusive .
present study , for the first time , demonstrated that scu has the ability to block hyperglycemia - induced apoptosis , ros , and microcirculation impairment in testes of stz - induced diabetes rats . since ros are the major upstream event of microcirculation impairment and germ cells apoptosis in diabetes , in this study we could not clarify if scu improves microcirculation via its antioxidant property , its ability to directly improve microcirculation , or both .
in addition , we could not clarify if scu blocks hyperglycemia - induced apoptosis via other mechanisms than ros , such as its direct effect on improving microcirculation .
further study is needed to better understand the mechanisms of scu 's actions and try to answer the question : is scu better than other antioxidants in improving diabetes associated reproductive disorder ?
in the future , we can compare the effect between sulforaphane and scu in protecting against t2dm - induced reproductive problems .
taken together , our data suggest that scu possesses the potential to reverse diabetes associated male reproductive disorder , providing first in vivo evidence to support further clinic study to use scu as a nontoxic drug to prevent and treat diabetes - mediated reproductive disorder .
data also suggest that targeting ros is one of the mechanisms whereby scu blocks hyperglycemia diabetes - induced apoptosis of germ cells and improves hyperglycemia - induced microcirculation impairment in testes .
 the goal of our study is to evaluate the effect of scutellarin on type ii diabetes - induced testicular disorder and show the mechanism of scutellarin 's action . 
 we used streptozotocin and high - fat diet to establish type ii diabetic rat model . 
 tunel and haematoxylin and eosin staining were used to evaluate the testicular apoptotic cells and morphologic changes . 
 immunohistochemical staining was used to measure the expression level of vascular endothelial growth factor and blood vessel density in testes . 
 oxidative stress in testes and epididymis was tested by fluorescence spectrophotometer and elisa . 
 the expression of bcl-2/bax and blood flow rate in testicular vessels were measured by western blot and doppler . 
 our results for the first time showed that hyperglycemia induced apoptotic cells and morphologic impairments in testes of rats , while administration of scutellarin can significantly inhibit these damages . 
 this effect of scutellarin is controlled by two apoptotic triggers : ros / bcl-2/bax and ros / microcirculation / starving pathway . 
1. Introduction
2. Material and Method
3. Results
4. Discussion
5. Conclusion