Introduction

One of the emergency conditions in male infertility is testicular torsion (Taati et al. 2016). Testicular torsion needs early iden- tification and surgical operation to prevent further damage to the testis, subfertility and infertility (Ranade et al. 2011). It is report-  ed ischemia/reperfusion (I/R) injury leads to germ cells loss and disruption of the semi- niferous epithelium (Taati et al. 2012). The main treatment for correction of testicular torsion is surgery to detorsion spermatic cord and re-establishing testis blood circulation (Asghari et al. 2016). During the long testic- ular torsion oxidation damage affects testis  by production of the reactive oxygen species (ROS) (Asghari et al. 2016). Testis and sper- matozoa contain higher fatty acids levels which are vulnerable to the ROS (Wei et al. 2011). Excessive generation of the ROS in- teracts with lipids, proteins and nucleic acids which has adverse effect on cell function and damage (Yuluğ et al. 2013). Testis has high cell metabolism such that excessive ROS production weakens antioxidant capacity (Tuglu et al. 2015). Malondialdehyde (MDA) is the end product of lipid peroxidation and increased MDA level has adverse effect on sperm fertility (Ghiasi Ghalehkandi, 2014). Glutathione peroxidase (GPx) is peroxidase enzyme and protects sperm from lipid per- oxidation and oxidative damage (Hsieh et al. 2006; Lee et al. 2012).

Today, there is growing interest in the application of medical plants due to their medicinal properties (Mansouri and Ab- dennour, 2011). Echinacea  purpurea  (EP) is a herbal medicine belonging to the As- teraceae (Compositae) family and contains bioactive metabolites including lipophilic, alkamides,  caffeic  acid  and polysaccharides

(Bayramoglu et al. 2011). Caffeic acid is the main bioactive component of the EP which has anti-inflammatory, antivirus,  antican- cer and antiandrogenic activities (Rezaie et al. 2013). Also, it is used for pain relief and wound healing (Rezaie et al. 2013). In folk medicine, it is used in in bacterial and viral infections (Barnes et al. 2005).  Echinacea has antioxidant and free radical scavenging properties (Bayramoglu et al. 2011). It is re- ported, administration of the 50 and 100 mg/ kg EP reduced MDA and amplified SOD and CAT levels on experimental renal I/R injury in the rats (Bayramoglu et al. 2011). Recently, Awaad et al. (2017) reported  administration of the hinacea purpurea extract (EPE) (30 mg/kg) protects against magnetic nanoparti- cles intra-testicular injection-induced toxici- ty. Also, EPE (100 mg/kg) has protective role against Gama-irradiation on hepatic and tes- ticular in rat (Ahmed et al. 2017). Also, the EP stimulates T-cell, lymphocytic and cy‌to- kine production in Arsenic-induced hepatic toxicity (Rezaie et al. 2013).  Even  though the correlationwith antioxidant activity  of  the EP has been reported, there is no previ- ous research on effect of the EPE on testicu- lar IR injury in rats. So, the main purpose of this reseach was to investigate effects of the EPE on testicular ischemia/reperfusion (I/R) injury in rat.

Material & Methods

Animals

Fifty healthy mature male Wistar rats  (250

± 20 g) were obtained from  Razi  Vaccine and Serum Research Institute (Tehran, Iran). Rats were provided commercial chow pellets and fresh water. Animals were kept in lab- oratory  one  week  prior  to experiments. All

experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals to Investigate Experimental Pain in Animals (Zimmermann 1983). Each animal was used only once and killed immediately after the  experiment.

Drugs

Pure sample of the EPE (Sigma Aldrich, UK; CAS Number 90028-20-9)  and  as-  say kits of MDA, SOD and GPx (Randox Laboratories Ltd., Crumlin, Antrim, United Kingdom) were purchased. The doses  for  the EPE were selected based on the pilot study (un-published data) and previous re- port (Rezaie et al. 2013; Ahmed et al. 2017; Motamedi et al. 2017).

Experimental protocol

Intraperitoneal injection of ketamine hy- drochloride (60 mg/kg) and xylazine hydro- chloride (10 mg/kg) was used for surgical procedures under anesthesia, then experi- mental testicular IR was created (Koksal  et al. 2013). A midline longitudinal incision was made for access to both testes. Torsion was created through twistings the left testes 720° in counter clockwise direction and pre- served through fixing the testes to scrotum with a 6-0 nylon suture passing by the tunica albuginea and dartos. Two hours later, suture was removed, left testes were detorted and replaced with scrotum reperfusion contin- ued for 24 h (Sahin et al. 2005). During the surgery, heating pad was used to keep body temperature connstant, then after surgery, the incision was closed. Group 1 was kept as con- trol with no surgery. Group 2 was subjected to 2h I /24 h R period. Group 3 was subjected to 2 h I which after 1 h of ischemia, rat   was

i.p. injected with 25 mg/kg EPE and isch- emia continued for an hour, then followed by 24 h R period. In group 4 rat was subjected to 2 h I which after 1 h of ischemia, rat was i.p.

injected with 50 mg/kg EPE and ischemia continued for an hour,  then followed by 24   h R period. In group 4 rat was subjected to 2 h I which after 1 h of ischemia, rat was i.p. injected with 100 mg/kg EPE and ischemia continued for an hour,  then followed by 24   h R period. The doses for EPE were selected based on the pilot study (un-published data) and previous report (Awaad et al. 2018). Af- ter 2 h of I, the suture was removed and left testis was detorted and replaced in scrotum for 24 h of reperfusion. At the end of the study, rats were euthanized (pentobarbital  300 mg/kg, i.p.), peritoneum opened and left testis was removed. The testicle was divided into two halves by a sagittal section, one half was fixed in Bouin’s solution, the  second half was stored at -80 °C for the biochemical analysis (Fakouri et al. 2017). The right tes- tis was removed as control for histological investigations.

Tissue processing

The tissue was fixed in Bouin’s solution (2.5 mL 7% formaldehyde, 2.65 mL glacial acetic acid and 7.5 mL saturated picric acid), post-fixed in 70% alcohol and fixed in par- affin blocks. A 5µm tissue section was ob- tained, deparaffinized and stained using he- matoxyline eosin. The testicular tissue was observed with standard light microscopy by  a sole observer 14. A 5µm thickness tissue section was taken and stained with hematox- ylin and eosin [H & E]. The testis sections were graded numerically to assess the degree of seminiferous tubule injury according  to the method of Johnsen ( 1971) as (1) neither germ cells nor Sertoli cells present, (2) no germ cells present, (3) only spermatogonia present, (4) only a few spermatocytes pres- ent, (5) no spermatozoa or spermatids pres- ent but many spermatocytes present, (6) only a few spermatids present, (7) no spermatozoa

but many spermatids present, (8) only a few spermatozoa present, (9) many spermatozoa present but disorganized spermatogenesis and (10) complete spermatogenesis and per- fect tubules.

Antioxidant activity

The tissue MDA level was determined with a maximum absorption at 532 nm (Plac- er et al. 1966). The GPx level was measured in absorbance of 340nm (Paglia and Valen- tine, 1967). The GPx activity was expressed as U/mg tissue. Tissue SOD activity was measured according to the method of Paolet- ti and Mocali (Paoletti and Mocali, 1990). The SOD activity was expressed as nmol/g tissue. Nicotinamide adenine dinucleotide oxidation was measured at 340nm and ex- pressed as U/mg tissue. The total antioxidant status detecting kit was obtained on the ba- sis of suppression in color production which was measured at 600nm and expressed as mmol/ml (Miller et al. 1993).

Statistical analysis

The parametric data was analyzed by one- way analysis of variance (ANOVA) using SPSS  24.0  and expressed  as mean  values ±

Figure 1. Histological score for assessing testis associat- ed with seminiferous tubules injury in EPE injection fol- lowed by I/R rat. Different letters (a-d) indicate significant differences between treatments (P<0.05).EPE: Echinacea purpurae extract.

standard  error  of  mean  (SEM).  The differences between groups were analysed using Duncan Multiple Range Test. The histo- pathological scores were analysed by Kru- skaleWallis test. P

Results

As seen in Fig. 1, I/R group had higher testis damage compared to the other groups (P<0.05). The control and sham groups have the least testis damage (P>0.05). A dose de- pendent difference was detected on testis damage grade in EPE treated groups in com-

Figure 2. Testis section of left testis in control rats showing normal seminiferous tubules (Arrow) and interstitial cells (Ar- row head) between tubules (Left). Testis section of right testis in control rats showing normal seminiferous tubules with spermatogonia (black arrow), spermatocyte (black arrow head) and many spermatozoa (white arrow) (Right) (H&E). H & E: hematoxylin and eosin.

Figure 3. Testis section of left testis in I/R rats showing degenerated seminiferous tubules (arrow) and loss of spermatogen- esis (H&E) (Left) and testis section of right testis in I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (H&E) (Right). H & E: hematoxylin and eosin.

parision with I/R group (P<0.05). No differ- ence was observed between 25 and 50 mg/kg of the EPE (P>0.05).

Effect of various EPE on tissue MDA, SOD and GPx levels in experimental testic- ular I/R-induced rat is presented in Table 1. As seen, testicular MDA levels significantly increased in I/R rat (P<0.05) while i.p injec- tion of the EPE (25, 50 and 100 mg/kg) nor- malized I/R-induced MDA (P<0.05). Exper- imental I/R significantly decreased SOD and GPx activity in comparision to control group

(P<0.05). Injection  of the of the EPE (25,  50 and 100 mg/kg) significantly increased SOD and GPx activity (P<0.05). No signifi- cant difference was detected on TAS in EPE treated groups compared to the control group (P>0.05).

According to the data, left and right  tes- tis section of control rats had shown normal seminiferous tubules and spermatogenesis with spermatocytes, sertoli and spermatozoa (Fig. 2).

As seen in Fig. 3, seminiferous tubules de-

Figure 4. Testis section of left testis in the EPE (25 mg/kg) followed by I/R rats showing seminiferous tubules (Arrow) with few spermatocyte and interstitial cells (Arrow head) between tubules (Left) and testis section of right testis in the EPE (25 mg/kg) followed by I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (H&E) (Right). H & E: hematoxylin and eosin. EPE: Echinacea purpurae extract.

Figure 5. Testis section of left testis in the EPE (50 mg/kg) followed by I/R rats showing seminiferous tubules (Arrow) with few spermatocyte and interstitial cells (Arrow head) between tubules (Left) and testis section of right testis in the EPE (50 mg/kg) followed by I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (H&E). H & E: hematoxylin and eosin. EPE: Echinacea purpurae extract.

generation and loss of spermatogenesis with few spermatocytes were detected in left de- generated testis tubules in I/R injur rat. How- ever, no significant effect was observed on right testis (Fig. 3, right).

Seminiferous tubules degenerated and loss of spermatogenesis with few spermatocytes was detected in degenerated in i.p injection of the EPE (25 mg/kg) followed by I/R inju- ry rats (Fig. 4, left). However, no significant effect  was  observed  on  right  testis  (Fig. 4,right).

In this study, i.p administration of the EPE (50 mg/kg) followed by I/R improved testis characteristics with few normal seminiferous tubules and spermatocyte in seminiferous tu- bules in I/R injury rat (Fig. 5).

According to the Fig. 6, injection of the EPE (50 mg/kg) improved testis character- istics with few normal seminiferous tubules and spermatocyte in seminiferous tubules in experimental I/R-induced  rat.

Table 1. Effect of different levels EPE on tissue values of Malondialdehyde, Superoxide dismutase, Glu- tathione peroxidase and total antioxidant status in experimental testicular I/R-induced rat

EPE:  Echinacea  purpurae  extract,  MDA:  malondialdehyde,  SOD:  superoxide  dismutase,  GPx:    glutathione

peroxidase, TAS: total antioxidant status, I/R: ischemia/reperfusion. Different letters (a-d) indicate significant

  differences  between  treatments  (P<0.05).                                                                                                                                

Figure 6. Testis section of left testis in the EPE (100 mg/kg) followed by I/R rats showing many normal seminiferous tubules (arrow) (H&E) (Left) with few spermatocyte (Arrow head) and testis section of right testis in the EPE (100 mg/kg) followed by I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (H&E). H & E: hematoxylin and eosin. EPE: Echinacea purpurae extract.

Discussion

In the current study, untreated rats that were subjected to ischemia for 2 h followed by 24 h reperfusion and revealed testicular injury with apparent seminiferous tubular necrosis. In I/R rat seminiferous tubules degenerated and few spermatocytes were observed. The 50 and 100 mg/kg of the EPE improved testis characteristics with normal seminiferous tu- bules were observed in experimental I/R-in- duced rat.

Several medical properties were reported for the EP including antifungal, antibacteri- al, antiinflammatory, antioxidant and wound healing properties (Nematalla et al. 2011). Echinacea extract has protective effects on the liver against cyproterone acetate and mentioned antioxidant properties of the EP induced these effects (Nematalla et al. 2011). It is well documented I-followed by R has adverse effects on germ cell loss and disrup- tion of the seminiferous epithelium in the testis (Ranade et al. 2011). The ROS such as superoxide anions, singlet oxygen and hy- drogen peroxide has negative  adverse  role in the testicular  I/R injury  (Kheradmand   et

al. 2011). In the physiologic condition, anti- oxidant mechanisms scavenge produced free radicals while in the oxidative stress condi- tion, imbalance occurs between ROS and scavenge free antioxidants (Agarwal et al. 2014). Ischemia increases in intracellular hy- poxanthine as a result of ATP breakdown and during R, xanthine oxidase converts hypox- anthine and superoxide radicals (Agarwal et al. 2014). The GPx and CAT are the first line of cellular defense against oxidative stress (Agarwal et al. 2014). During testicular tor- sion and detorsion inversly enhanced ROS indicates lipid peroxidation. Testicular cell membranes are rich in polyunsaturated fatty acids and are vulnerable to oxidative injury (Ma et al. 2018). So, oxidative stress inhibi- tors or increase in anti-oxidant enzymes lev- el has beneficial effect on testicular IR injury (El-Shahat et al. 2012). In this regard, report- ed Caffeic Acid derivatives, and polysaccha- ride fractions from EP have strong antioxi- dative effects (Newair et al. 2017). Based on the literature, antioxidant protective effects have been reported for the EPE (Ahmed  et al. 2017). In our recent study, 50 and 100 mg/