Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 17/10/2024 Aceptado: 02/01/2025 Publicado: 11/03/2025 hps://doi.org/10.52973/rcfcv-e35539 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 8 Eﬀects of Silymarin on immunohistochemical Bax and 8-Ohdg expression, biochemical markers and sperm parameters in an experimental varicocele model in rats Efectos de la Silimarina sobre la expresión inmunohistoquímica de los marcadores Bax y 8-OHDG sobre los parámetros espermácos en un modelo experimental de varicocele en ratas 1* Van Yuzuncu Yil University, Faculty of Veterinary Medicine, Department of Reproducon and Arﬁcial Inseminaon, Van, Türkiye. 2 Van Yuzuncu Yil University, Faculty of Veterinary Medicine, Department of Surgery, Türkiye. ³Van Yuzuncu Yil University, Vocaonal School of Health Services, Van, Türkiye. ⁴Kyrgyz-Turkish Manas University, Faculty of Veterinary Medicine, Department of Pathology, Bishkek / Kyrgyzstan. *Corresponding author: saadetbelhan@yyu.edu.tr; Phone +905546993339 ABSTRACT In this study, the eﬀects of silymarin on immunohistochemical Bcl-2-associated X protein (Bax) and 8-hydroxy-2’- deoxyguanosine (8-OHdG) expression, biochemical markers and sperm parameters were invesgated with an experimentally induced varicocele model in rats. The study was conducted on 36 Wistar albino rats. The distribuon of rats within the group was made in an equal number. Rats in the control group were administered physiological saline daily via oral gavage. In the sham group, an incision was made on the midline and the renal vein (located on the leﬅ) was made visible. A probe was placed on this vein. The probe was coiled with the vein but not ligated. In the silymarin group, silymarin was administered by oral gavage at a dose of 75 mg/kg 3 mes a week for 8 weeks. Ligaon was performed on rats in the varicocele group, unlike the sham group. Varicocele was created in the varicocele+silymarin groups (50 mg/kg, 75 mg/ kg). Silymarin applicaon was started 8 weeks aﬅer varicocele inducon and was applied 3 days a week for 8 weeks. Aﬅer the analysis, it was seen that sperm parameters were negavely aﬀected in the varicocele group. Addionally, severe caspase 3, 8-OHdG and Bax expressions were detected. Silymarin administraon reduced the intensity of expression and had posive eﬀects on spermatology. These posive eﬀects were even more pronounced with the 75 mg dose. Based on the results obtained, silymarin may have the potenal to reduce both clinical and pathological symptoms in varicocele cases. Key words: Heat Shock Protein (HSP); silymarin; varicocele; transforming growth factor Alpha (TGF-α); vascular endothelial growth factor-A (VEGF-A) RESUMEN En este estudio se invesgó los efectos de la silimarina sobre la expresión inmunohistoquímica de la proteína X asociada a Bcl-2 (Bax) y la 8-hidroxi-2’-desoxiguanosina (8-OHdG), los marcadores bioquímicos y los parámetros espermácos con un modelo de varicocele inducido experimentalmente en ratas. El estudio se realizó en 36 ratas albinas Wistar. La distribución de ratas dentro del grupo se realizó en igual número. A las ratas del grupo de control se les administró diariamente solución salina ﬁsiológica por sonda oral. En el grupo simulado, se praccó una incisión en la línea media y se hizo visible la vena renal (situada a la izquierda). Se colocó una sonda en esta vena. La sonda se enrolló con la vena pero no se ligó. En el grupo de la silimarina, ésta se administró por sonda oral a una dosis de 75 mg/kg 3 veces por semana durante 8 semanas. La ligadura se realizó en las ratas del grupo varicocele, a diferencia del grupo simulado. Se creó varicocele en los grupos varicocele+silimarina (50 mg/ kg, 75 mg/kg). La aplicación de silimarina se inició 8 semanas después de la inducción del varicocele y se aplicó 3 días a la semana durante 8 semanas. Tras el análisis, se observó que los parámetros espermácos se veían afectados negavamente en el grupo con varicocele. Además, se detectaron expresiones graves de caspasa 3, 8-OHdG y Bax. La administración de silimarina redujo la intensidad de la expresión y tuvo efectos posivos en la espermatología. Estos efectos posivos fueron aún más pronunciados con la dosis de 75 mg. Según los resultados obtenidos, la silimarina puede tener el potencial de reducir tanto los síntomas clínicos como los patológicos en los casos de varicocele. Palabras clave: Proteína de choque térmico (HSP); silimarina; varicocele; factor de crecimiento transformante alfa (TGF-α); factor de crecimiento endotelial vascular A (VEGF-A) Saadet Belhan 1 (*) , Caner Kayikci 2 , Ahmet Ufuk Kömüroğlu 3 , Uğur Özdek 3 , Serkan Yildirim 4

Eﬀect of Silymarin in an experimental model of varicocele / Belhan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Varicocele, deﬁned as pathological dilataon of the veins in the spermac cord, is considered one of the factors that cause reproducve problems in men. While it is seen in 15-22% of the adult male populaon, the rate of varicocele among inferle men reaches 30-40% [1 , 2]. Many condions occur in varicocele that negavely aﬀect ferlity. These include many negave situaons such as decreased spermatozoa concentraon and viability and increased malondialdehyde (MDA) level [3 , 4]. Addionally, pathological levels of reacve oxygen species develop in varicocele. Reacve oxygen species (ROS) at pathological levels can cause many undesirable consequences such as defecve sperm funcon, deterioraon of sperm morphology, damage to sperm DNA and inadequate sperm-oocyte fusion [5 , 6]. Varicocele negavely aﬀects reproducve physiology if leﬅ untreated or if treatment is delayed. The most appropriate treatment in varicocele cases is varicocelectomy. However, there are some unclear situaons regarding varicocelectomy. In addion to the lack of pre- and postnatal data, there is sll controversy regarding the methodology and the criteria applied. Addionally, there is informaon that inferlity connues signiﬁcantly aﬅer varicocelectomy [3 , 7 , 8 , 9]. Since the negave impact on reproducve funcon also aﬀects psychology negavely, ﬁnding new alternave agents to surgical intervenon is of great importance. For these reasons and because they present lower risks than surgery, many studies invesgated the beneﬁts of treatment with anoxidants. Silymarin is a ﬂavonoid complex obtained from the seeds of milk thistle (Silybum marianum). Silymarin was reported to have many biological acvies such as hepatoprotecve [10], an-cancer [11], and an-inﬂammatory eﬀects [12]. It acts as a powerful anoxidant by reacng with ROS and also potenates the eﬀects of physiological anoxidants such as glutathione and superoxide dismutase [13 , 14]. A review of the literature reveals the existence of studies examining silymarin and varicocele. However, it has been determined that the eﬀects of silymarin on varicocele have not been comprehensively invesgated within the framework of the methodology and markers ulized in this study. Therefore, an experimental varicocele model was established, and the eﬀects of silymarin on biochemical markers (HSP-90, HSP-70, Inhibin B, VEGF-A, TGF-α, Testosterone), histopathological ﬁndings, immunohistochemical markers (Caspase 3), and immunoﬂuorescent markers (8-OHdG, Bax), as well as its inﬂuence on spermatological parameters (Spermatozoa concentraon, Molity, Abnormal spermatozoa rate), were evaluated. MATERIALS AND METHODS Animals and creaon of experimental groups The study was conducted using 36 male rats (Raus norvegicus) (Wistar albino, 300-370 g). A total of 6 groups were arranged and groups included 6 rats. Rats were kept in individual cages in a suitable living space (21 ± 1 °C temperature, 55 ± 15% humidity and 12 hours of daylight). In the study, extreme care was taken about hygiene and surgical intervenons were performed under xylazine/ketamine anesthesia. Blood was taken from all rats at the end of the study, including the control group, and orchiectomy was performed. The study received the necessary approval from the Van Yuzuncu Yil University Animal Experiments Ethics Commiee with the number “2020/07-09”. In addion, the study was carried out by taking into account the instrucons of the relevant board. Control group: Physiological saline was administered daily by oral gavage throughout the study period. Sham group: Aﬅer an incision was made on the midline, the renal vein on the leﬅ side was made visible. A probe was placed on this vein. This vein and the probe were wrapped around it with the help of a ligature without being ed. The midline incision was closed. Aﬅer 60 days (d), blood was taken under anesthesia and orchiectomy was performed. Silymarin group (75 mg/kg): Silymarin was administered by gavage 3 mes a week for 8 weeks (75 mg/kg). Varicocele group: Aﬅer an incision was made on the midline, the renal vein on the leﬅ side was made visible. A probe was placed on this vein. This vein and the probe were connected with the help of a ligature. The midline incision was then closed. Aﬅer 60 d, blood was taken under anesthesia and orchiectomy was performed. Varicocele+silymarin group (50 mg/kg): Varicocele was induced. Silymarin administraon was started 8 weeks aﬅer varicocele inducon and applied 3 d a week for 8 weeks. Varicocele+silymarin group (75 mg/kg): Varicocele was created. Silymarin administraon was started 8 weeks aﬅer varicocele inducon and applied 3 d a week for 8 weeks. Varicocele inducon An incision was made at the top of the midline of the abdomen. Aﬅer the renal vein was made visible, a probe was placed on it and ed with a ligature. The probe was removed. The vessel was allowed to expand within the boundaries of the ligature. The abdominal wall and anterior abdominal muscles were sutured separately and the incision area was closed. As a result of this narrowing process, intravascular pressure increased and this pressure was transferred to the spermac vein, varicocele was formed. Overdoing the narrowing process may lead to kidney necrosis. If the narrowing process is inadequate, it causes insuﬃcient intrarenal vein pressure. For this reason, great care was taken in the narrowing process [15]. Semen collecon and spermatological evaluaon While the rats were under anesthesia, one tess was removed immediately aﬅer blood collecon and before the body cooled. The cauda epididymis of the excised tess was inially used to assess sperm molity under a light microscope (ECLIPSE E 400 Nikon JAPAN), set at 37°C. During the evaluaon of molity, the dense rat semen were diluted in saline at 37°C. The percentage of sperm molity was evaluated at 200x magniﬁcaon using a heated-stage light microscope as described by Sonmez et al. [16]. Sperm density and abnormal sperm rates were determined from the suspension mixture obtained by slicing the same cauda epididymis secon (aﬅer molity assessment) in 2 ml of saline. Sperm density was determined using a slightly modiﬁed method 2 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico described by Sonmez et al. [16] The semen sample was drawn into an Eppendorf tube using a 10 µL automac pipee, and 990 µL of eosin soluon was added. Approximately 10 µL of the diluted sperm suspension was transferred to the counng chambers of a Thoma slide (Germany) and leﬅ for 5 min. The sperm count was performed under a phase-contrast microscope at 200x magniﬁcaon, and the sperm density was calculated using the appropriate formula. The mean of three consecuve evaluaons was used as the ﬁnal molity score. To assess abnormal sperm cells, the method reported by Turk et al. [17] was applied. Brieﬂy, a drop of the semen sample was mixed with an equal amount of eosin-nigrosin stain (1.67% eosin, 10% nigrosin, and 0.1 M sodium citrate), and a thin smear was prepared. A total of 300 sperm cells were examined under a light microscope (Olympus BX 51, JAPAN) at 400x magniﬁcaon. Histopathological examinaon of tescles Tescular ssue samples were ﬁrst detected (10% formaldehyde). Then, the secons prepared in paraﬃn blocks were stained with hematoxylin-eosin (HE) and microscopic evaluaon was performed (Olympus BX 51, JAPAN). Secons were evaluated as none (-), mild (+), moderate (++) and severe (+++) according to their histopathological ﬁndings Immunohistochemical examinaon For immunohistochemical examinaon, ssue secons placed on adhesive (poly-L-Lysin) slides were deparaﬃnized and dehydrated. Then, endogenous peroxidase was inacvated by keeping in 3% H2O2 for 10 minutes. Then, the ssues were boiled in 1% angen retrieval (citrate buﬀer (pH+6.1) 100X) soluon and leﬅ to cool at room temperature. To prevent nonspeciﬁc background staining in the ssues, the secons were incubated with protein block for 5 minutes. Then, primary anbody (Caspase 3 Cat No: sc-56053, Diluon Rao: 1/100, US) was dropped onto the ssues and incubated according to the instrucons for use. 3-3’ Diaminobenzidine (DAB) chromogen was used as the chromogen in the ssues. Stained secons were examined with a light microscope (Zeiss AXIO GERMANY). Double immunoﬂuorescence staining method The 4-μm secons were taken on adhesive slides, deparaﬃnized, dehydrated, and washed with PBS. Endogenous peroxidase was inacvated in 3% H2O2 for 10 min. Then, the samples were boiled in 1% angen retrieval (citrate buﬀer (pH + 6.1) 100X) soluon and cooled to room temperature. Secons were incubated with protein block for 5 min to abolish nonspeciﬁc background staining. Then, the primary anbody (8 OHdG cat no: sc-66036, diluon rao: 1/100, US) was added according to the manufacturer’s instrucons. This was followed by the immunoﬂuorescence 2nd anbody marker (FITC cat no. ab6785, diluent rao 1/500, UK) and dark incubaon for 45 min. Then, the primary anbody (Bax cat no. sc-7480, diluon rao 1/100, US) was dripped onto the secons and incubated according to the manufacturer’s instrucons. A secondary immunoﬂuorescence anbody was used as a secondary marker (Texas Red cat no. ab6719, diluent rao 1/500, UK) and kept in the dark for 45 min. Then, DAPI with mounng medium (cat no. D1306, diluon rao 1/200, UK) was dripped onto the preparaons and kept in the dark for 5 min. Aﬅerward, the stained secons were covered with a coverslip and examined under a ﬂuorescence microscope (Zeiss AXIO, Germany). Biochemical analyses The rats were euthanized using xylazine 10 mg·kg-1 IP (2% Rompun® Bayer) and Ketamine (HCl) (10% Alfamine® Atafen) 75 mg·kg-1 IP injectable anesthecs, and the animals were sacriﬁced by high volume blood collecon under anesthesia and tescular ssue was taken. The blood taken was centrifuged (Nuve, NF 800R, Turkey) at 4000 G for 10 min and the serum was separated. Serum samples were stored (Dt, Fydl– 268, Turkey) at -40 °C unl the day of study. Phosphate buﬀer (pH: 7.2–7.4) was added to the tescular ssues at 10 mes their weight. It was homogenized with the aid of a homogenizer. Care was taken to ensure the cold chain at all stages of the experiment. The homogenates were centrifuged at 2000–3000 rpm, +4°C for 20 minutes and the supernatant was separated. In serum and tescular ssue, HSP-90 (Catalog No: SG-20499, SinoGeneClon Biotech, China), HSP-70 (Catalog No: SG-20498, SinoGeneClon Biotech, China), inhibin B (Catalog No: SG-20735, SinoGeneClon Biotech, China), transforming growth factor-alpha (TGF-α) (Catalog No: SG-20059, SinoGeneClon Biotech, China), and VEGF-A (Catalog No: SG-21042, SinoGeneClon Biotech, China) levels were detected with species-speciﬁc ELISA kits. In addion, serum testosterone level was measured with the Abo Architect ci16200 (Germany) device using a commercial kit. Stascal analysis For stascal analysis of sperm parameters, one-way Analysis of Variance (ANOVA) was used to compare group means and post-hoc Tukey test was used to determine the diﬀerence between groups following analysis of variance. In order to determine the intensity of posive staining on images obtained as a result of immunohistochemical and immunoﬂuorescence staining, 5 random areas were selected from each image and evaluated with the ZEISS Zen Imaging Soﬅware program. Data were stascally described as mean and standard deviaon (mean±SD) for % area. One-way ANOVA followed by Tukey’s test was performed to compare posive immunoreacve cells and immunoposive stained areas with healthy controls. One-way ANOVA was used to determine the diﬀerence between groups in terms of biochemical parameters, and the Duncan test, one of the mulple comparison tests, was used to determine which group caused the diﬀerences. RESULTS AND DISCUSSION Although varicocelectomy is known to be a suitable method to treat inferlity in varicocele, there is a trend towards some non-surgical techniques because inferlity connues from me to me aﬅer varicocelectomy. In the literature review, the eﬀecveness of many chemical agents was invesgated in this ﬁeld [18 , 19]. The values obtained regarding sperm ﬁndings are presented in TABLE I. A non-signiﬁcant diﬀerence was detected between the control group, sham group and silymarin group in terms of sperm parameters (spermatozoa concentraon, molity, abnormal spermatozoa rate) (P>0.05). However, in the other 3 groups (varicocele group, varicocele+50, varicocele+75), the diﬀerences detected in terms of all three sperm parameters were signiﬁcant (P˂0.05). In the current study, silymarin increased spermatozoa concentraon and molity, while signiﬁcantly reducing the abnormal spermatozoa rate and 3 of 8

Eﬀect of Silymarin in an experimental model of varicocele / Belhan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico leading to signiﬁcant improvements in sperm parameters. Similar signiﬁcant changes in sperm parameters (sperm vitality, molity and count) were reported in a diﬀerent varicocele studie evaluang the eﬀect of sodium selenite on tescular damage induced by experimental varicocele in male Wistar rats [20]. 4 of 8 TABLE I . Sperm parameters in diﬀerent treatment groups in an experimental varicocele model in rats Groups Spermatozoa concentraon (×10 6 ) Molity (%) Abnormal spermatozoa rate (%) Control 126.16 ± 0.98a 78.33 ± 0.51a 11.16 ± 0.40d Sham 125.16 ± 0.40a 77.50 ± 0.54a 11.50 ± 0.54d SMN75 126.50 ± 0.83a 78.50 ± 0.54a 10.50 ± 0.54d VC 72.16 ± 0.75d 30.83 ± 0.40d 43.50 ± 0.54a VC+SMN50 88.16 ± 0.98c 45.83 ± 0.98c 32.16 ± 0.40b VC+SMN75 96.50 ± 0.54b 53.16 ± 0.98b 27.83 ± 1.16c a b c d; Diﬀerent leers in the same column indicate a stascally signiﬁcant diﬀerence (P<0.05). SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/ kg). VC+SMN75: Silymarin (75 mg/kg). In histopathological examinaon, the tescular ssues of the control group, sham group and silymarin 75 group had normal histological appearance (FIG. 1). In the evaluaon performed in the varicocele group, severe degeneraon and necrosis of spermatocytes, thinning of the tubule wall, edema in the intertubular region and hyperemia in the vessels were detected (FIG. 1). The ﬁndings detected in the histopathological examinaon of the varicocele group were moderate in the varicocele+silymarin 50 group (FIG. 1). However, the hyperemia of vessels was severe. The ﬁndings detected in the varicocele group were at a mild level in the varicocele+silymarin 75 group (FIG. 1). However, a stascally signiﬁcant diﬀerence (P<0.05) was detected when the varicocele+silymarin 75 group was compared with the varicocele group. It is possible to see the histopathological ﬁndings in TABLE II. FIGURE 1. Tescular ssue in diﬀerent treatment groups in an experimental varicocele model in rats. Degeneraon of spermatocytes in the tubular wall (arrowheads), necrosis (arrows), thinning of the tubular wall, hyperemia in the vessels, edema in the intertubular spaces (stars), H&E, Bar: 20 µm. SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/kg). VC+SMN75: Silymarin (75 mg/kg). TABLE II . Histopathological ﬁndings and scoring for tescular ssues with experimental varicocele of the leﬅ spermac vein Parameters Control Sham SMN 75 VC VC+ SMN 50 VC+ SMN 75 Degeneraon in spermatocytes - - - +++ ++ + Necrosis in spermatocytes - - - +++ ++ - Hyperemia in vessels - - - +++ +++ + Thinning of the tubular wall - - - +++ ++ + Edema in the intertubular region - - - +++ ++ + SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/ kg). VC+SMN75: Silymarin (75 mg/kg) While caspase 3 expression was at a severe level in the varicocele group, it was at a moderate level in the varicocele+silymarin 50 group and at a mild level in the varicocele+silymarin 75 group (FIG. 2). A stascally signiﬁcant diﬀerence (P<0.05) was detected when the varicocele+silymarin 75 group was compared with the varicocele group. Data for immunohistochemical ﬁndings are presented in TABLE III. Oxidave stress resulng from increased Reacve oxygen species in varicocele was reported to increase apoptosis [21]. Acvated caspase 3 can induce spermatogenic cell apoptosis in varicocele [22]. In this regard, caspase 3 expression was evaluated immunohistochemically in the current study. The results obtained regarding caspase 3 in current study conﬁrm previous reports that caspase 3 expression was reported to be at severe levels in the varicocele group [23 , 24].

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 2. Tess ssue caspase 3 expression, IHC, Bar: 20 µm. SMN75: Silymarin (75mg/ kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/kg). VC+SMN75: Silymarin (75 mg/kg). When the tescular ssues stained with the immunoﬂuorescence staining method were examined, 8-OHdG and Bax expressions were negave in the control group, sham group and silymarin 75 group (FIG. 3). While 8-OHdG and Bax expressions were at severe levels in the varicocele group, they were at moderate levels in the varicocele+silymarin 50 group and at mild levels in the varicocele+silymarin 75 group (FIG. 3). A stascally signiﬁcant diﬀerence (P<0.05) was detected when the varicocele+silymarin 75 group was compared with the varicocele group. Data for immunohistochemical ﬁndings are presented in TABLE III. In current study, silymarin signiﬁcantly reduced the expression levels of both 8-OHdG and Bax. The high levels of 8-OHdG and Bax expressions detected in the varicocele group are compable with the results of previous studies [25]. TABLE III . Data and stascal analysis of immunohistochemical and immuno- ﬂuorescence ﬁndings for tescular ssues with experimental varico- cele of the leﬅ spermac vein. Groups Caspase 3 8-OHdG Bax Control 18.49 ± 0.56 a 24.61 ± 0.75 a 20.03 ± 0.91 a Sham 19.16 ± 0.41 a 24.52 ± 0.39 a 21.18 ± 0.43 a SMN75 19.03 ± 0.7 a 24.63 ± 0.26 a 20.84 ± 0.51 a VC 64.95 ± 1.83 b 80.66 ± 1.54 b 71.5 ± 1.55 b VC+SMN50 43.38 ± 1.26 c 53.16 ± 1.43 c 48.66 ± 1.4 c VC+SMN75 31.54 ± 1.33 d 35.7 ± 0.87 d 32.15 ± 0.93 d a b c d; Diﬀerent leers in the same column indicate a stascally signiﬁcant diﬀerence (p<0.05). SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/kg). VC+SMN75: Silymarin (75 mg/kg). FIGURE 3. Tess ssue 8-OHdG expression (FITC) and Bax expression (Texas Red), IF, Bar: 50 µm. SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/kg). VC+SMN75: Silymarin (75 mg/kg). Serum biochemical parameters of all groups are presented in TABLE IV, and tescular ssue biochemical parameters are presented in TABLE V. There was no signiﬁcant diﬀerence between the groups in terms of serum HSP-90 levels (P>0.05). Although the serum HSP-70 level in the varicocele group was lower than the control group, this decrease was not signiﬁcant (P>0.05). Serum HSP-70 level was signiﬁcantly lower in the varicocele+silymarin 50 group (P<0.05). Tescular ssue HSP- 90 levels were similar in all groups. The highest HSP-90 level belonged to the varicocele group. Although the HSP-90 level in the varicocele groups treated with silymarin was lower than in the varicocele group, this decrease was not signiﬁcant. (P>0.05). Varicocele group tescular ssue HSP-70 level was higher than the control and silymarin groups. Although tescular ssue HSP- 70 levels in the varicocele groups treated with silymarin were lower than those in the varicocele group, this decrease was not signiﬁcant (P>0.05). When the varicocele group was compared with the control group, serum HSP-70 and HSP-90 levels were found to decrease in the varicocele group. Silymarin (75 mg) administered to the varicocele group was able to cause an increase in serum HSP-70 level. The results obtained for serum HSP-70 in the present study were obtained by administering testosterone and VitE in a previous study [26]. In current study, HSP-70 and HSP-90 levels in tescular ssue increased in the varicocele group, and this increase was consistent with a previous study [27]. The upregulaon of HSPs detected in the varicocele group may be a cellular response to scrotal hyperthermia occurring in varicocele. Serum inhibin B level in the varicocele group was lower than the other two groups (control and sham groups) (P<0.05). Although serum inhibin B levels in the varicocele groups treated with silymarin were higher than the varicocele group, this increase was not signiﬁcant (P>0.05). Tescular ssue inhibin B levels were similar in all groups. The lowest inhibin B level was detected in the varicocele group. Findings with inhibin B are similar to previous studies [25 , 28]. Both doses of silymarin 5 of 8

Eﬀect of Silymarin in an experimental model of varicocele / Belhan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico administered in the current study signiﬁcantly regulated the inhibin B levels reduced by varicocele. A similar eﬀect to silymarin on inhibin B was obtained with berberine in another study [28]. Serum VEGF-A level in the varicocele group was signiﬁcantly higher than all other groups except the control (P<0.05). Serum VEGF-A levels in the varicocele+silymarin groups were found to be signiﬁcantly lower than the varicocele group (P<0.05). Tissue hypoxia, which is likely to occur in varicoceles, increases VEGF-A expression and increased VEGF-A causes damage to the seminiferous tubules [29]. Serum and tescular VEGF-A levels evaluated in the current study increased in the varicocele group compared to the control group, consistent with previous studies [30]. This condion expression may have been elevated in the varicocele group as a compensatory mechanism to counteract the hypoxic state that can impair tescular funcon. The group with the lowest serum TGF-α level was the varicocele group. Serum TGF-α level in the varicocele group was signiﬁcantly lower than in only one group (varicocele+silymarin 50 group) (P<0.05). Varicocele+silymarin 50 group tescular ssue TGF-α level was found to be signiﬁcantly higher than all other groups (P<0.05). Although the tescular ssue TGF-α level of the varicocele group was lower than the control group, this decrease was not signiﬁcant (P>0.05). In the current study, both serum and ssue TGF-α levels were signiﬁcantly lower in the varicocele group compared to the other groups. This detected situaon supports the ﬁndings of previous studies [31]. Both doses of silymarin administered in the current study increased serum and ssue TGF-α levels. Testosterone levels in the varicocele group were signiﬁcantly lower than the other 3 groups (control, sham and silymarin groups) (P<0.05). Although the serum testosterone level in the varicocele groups treated with silymarin was higher than the varicocele group, it was not signiﬁcant (P>0.05). The serum level of testosterone hormone, which is considered a clinical marker for detecng physiological acvity in Leydig cells, was signiﬁcantly decreased in the varicocele group compared to the control and sham groups, and this ﬁnding is similar to previous varicocele studies [25 , 28 , 32]. In the study found that silymarin increased testosterone levels, but this increase was not signiﬁcant. TABLE IV . Serum biochemical parameters with experimental varicocele of the leﬅ spermac vein Groups HSP-90 (pg/ml) HSP-70 (pg/ml) Inhibin B (pg/ml) VEGF-A (pg/ml) TGF-α (pg/ml) Testosteronee (ng/ml) Control 512.48 ± 51.3 a* 423.98 ± 12.58 a 52.90 ± 4.38 a 20.09 ± 4.11 a,b 53.73 ± 3.78 a,b 4.79 ± 0.47 a Sham 480.24 ± 33.22 a 424.89 ± 19.97 a 50.63 ± 4.14 a 16.73 ± 3.14 b,c 50.61 ± 4.85 a,b 4.72 ± 0.45 a SMN75 453.37 ± 39.83 a 425.51 ± 15.90 a 49.09 ± 4.66 a,b 15.97 ± 3.77 b,c 52.16 ± 3.32 a,b 4.40 ± 0.73 a VC 497.48 ± 39.87 a 409.24 ± 30.43 a 45.20 ± 2.41 b 23.06 ± 4.78 a 47.86 ± 3.98 b 3.43 ± 0.48 b VC+SMN 50 482.02 ± 51.99 a 373.53 ± 18.52 b 49.40 ± 5.14 a,b 14.54 ± 1.67 c 54.68 ± 6.64 a 3.67 ± 0.62 b VC+SMN 75 463.37 ± 54.77 a 413.47 ± 11.93 a 47.83 ± 2.92 a,b 15.81 ± 3.16 b,c 50.15 ± 3.02 a,b 3.62 ± 0.63 b *; Diﬀerent leers in the same column indicate a stascally signiﬁcant diﬀerence (P<0.05). SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/kg). VC+SMN75: Silymarin (75 mg/kg). TABLE V . Biochemical parameters for tescular ssue with experimental varicocele of the leﬅ spermac vein Groups HSP-90 HSP-70 Inhibin B VEGF-A TGF-α Control 363.36 ± 36.81 a 306.21 ± 44.17 b 40.51 ± 5.78 a 11.76 ± 1.01 c 40.75 ± 3.78 b Sham 354.09 ± 30.30 a 324.93 ± 19.91 a,b 40.35 ± 4.79 a 12.57 ± 1.69 c 37.81 ± 4.48 b SMN75 361.56 ± 27.27 a 305.94 ± 24.13 b 38.68 ± 3.16 a 12.79 ± 1.05 c 41.39 ± 6.79 b VC 414.72 ± 59.20 a 350.78 ± 22.64 a 35.63 ± 3.37 a 13.57 ± 1.98 b,c 34.82 ± 4.26 b VC+SMN 50 386.38 ± 10.25 a 318.18 ± 24.54 a,b 41.41 ± 4.44 a 15.44 ± 1.81 a,b 48.37 ± 6.88 a VC+SMN 75 404.11 ± 107.10 a 337.94 ± 48.80 a,b 41.38 ± 4.38 a 16.31 ± 3.51 a 40.99 ± 8.04 b *; Diﬀerent leers in the same column indicate a stascally signiﬁcant diﬀerence (P<0.05). SMN75: Silymarin (75mg/kg). VC: Varicocele. VC+SMN50: Varococele + Silymarin (50 mg/kg). VC+SMN75: Silymarin (75 mg/kg). CONCLUSIONS AND IMPLICATIONS Varicocele negavely aﬀects sperm parameters, creates some changes in biochemical parameters and exacerbates apoptosis by increasing the expression levels of caspase-3, Bax and 8-OHdG. Silymarin administraon had posive eﬀects, especially on sperm quality parameters, biochemical parameters and enhancement of apoptosis by reducing the expression levels of caspase-3, Bax and 8-OHdG. Both doses of silymarin had posive eﬀects on TGF-α levels. Based on these results, we can aﬃrm that silymarin can be used therapeucally in varicocele, as it reduces the level of deterioraon resulng from tescular pathology. ACKNOWLADGEMENT This study was supported by the project numbered “TSA- 2021-9269” of Van Yuzuncu Yil University Scienﬁc Research Projects Coordinaon Unit. Conﬂict of interest The authors declare no compeng interests. 6 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico BIBLIOGRAPHIC REFERENCES [1] Lipshultz LI, Corriere Jr JN. Progressive tescular atrophy in the varicocele paent. J. Urol. [Internet]. 1977; 117(2):175–176. doi: hps://doi.org/n8g9 [2] Hargreave TB. Varicocele - a clinical enigma. Br. J. Urol. [Internet]. 1993; 72(4):401-408. doi: hps://doi.org/ frjqx7 [3] Asadi N, Kheradmand A, Gholami M, Saidi SH, Mirhadi SA. Eﬀect of royal jelly on tescular anoxidant enzymes acvity, MDA level and spermatogenesis in rat experimental varicocele model. Tissue Cell. [Internet]. 2019; 57:70–77. doi: hps://doi.org/gmzdwt [4] Soni KK, Zhang LT, Choi BR, Karna KK, You JH, Shin YS, Lee SW, Kim CY, Zhao C, Chae HJ, Kim HK, Park JK. Protecve eﬀect of MOTILIPERM in varicocele-induced oxidave injury in rat tess by acvang phosphorylated inositol requiring kinase 1α (p-IRE1α) and phosphorylated c-Jun N-terminal kinase (p-JNK) pathways. Pharm. Biol. [Internet]. 2018; 56(1):94–103. doi: hps://doi.org/n8hb [5] Aitken RJ, Clarson JS. Cellular bais of defecve sperm funcon and its associaon with the genesis of reacve oxygen species by human spermatozoa. J. Reprod. Ferl. [Internet]. 1987; 81(2):459–469. doi: hps://doi.org/ b33prx [6] Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidaon and producon of hydrogene peroxide and superoxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl. [Internet]. 1987; 8(5):330–348. doi: hps://doi.org/n8hc [7] Fretz PC, Sandlow JI. Varicocele: current concepts in pathophysiology, diagnosis, and treatment. Urol. Clin. North. Am. [Internet]. 2002; 9(4):921–37. doi: hps:// doi.org/br3q28x| [8] Ozbek E, Yurekli M, Soylu A, Davarci M, Balbay MD. The role of adrenomedullin in varicocele and impotence. BJU Int. [Internet]. 2000; 86(6):694–698. doi: hps://doi. org/b3xh4b [9] Nöske HD, Weidner W. Varicocele historical perspecve. World J. Urol. [Internet]. 1999; 17(3):151–157. doi: hps://doi.org/dd79vb [10] Bosisio E, Benelli C, Pirola O. Eﬀect of the ﬂavanolignans of Silybum marianum L. on lipid peroxidaon in rat liver microsomes and freshly isolated hepatocytes. Pharmacol. Res. [Internet]. 1992; 25(2):147–54. doi: hps://doi.org/ cqzww3 [11] Agrawal R, Agrawal C, Ichikawa H, Singh RP, Aggarwal BB. Ancancer potenal of silymarin:from bench to bed side. Ancancer Res. [Internet]. 2006 [cited July 12, 2024]; 26(6B):4457–4498. PMID: 17201169. Available in: hps://goo.su/1GoZBh [12] Dehmlow C, Erhard J, de Groot H. Inhibion of Kupﬀer cell funcons as an explanaon for the hepatoprotecve properes of silibinin. Hepatology. [Internet]. 1996; 23(4):749–754. doi: hps://doi.org/dwrdpr [13] El-Shitany NA, El-Haggar S, El-Desoky K. Silymarin prevents adriamycin-induced cardiotoxicity and nephrotoxicity in rats. Food. Chem. Toxicol. [Internet]. (2008); 46(7):2422– 2428. doi: hps://doi.org/dz5dqg [14] Nencini C, Giorgi G, Micheli L. Protecve eﬀect of silymarin on oxidave stress in rat brain. Phytomedicine. [Internet]. 2007; 14(2-3):129–135. doi: hps://doi.org/ bm8tvp [15] Pasqualoo FF, Lucon AM, Hallak J, Góes PM, Saldanha LB, Arap S. Inducon of spermatogenesis in azoospermic men aﬅer varicocele repair. Hum. Reprod. [Internet]. 2003; 18(1):108–112. doi: hps://doi.org/d2jx [16] Sonmez M, Türk G, Yüce A. The eﬀect of ascorbic acid supplementaon on sperm quality, lipid peroxidaon and testosterone levels of male Wistar rats. Theriogenology. [Internet]. 2005; 63(7): 2063–2072. doi: hps://doi.org/ cn6kn5 [17] Türk G, Ateşşahin A, Sönmez M, Ceribaşi AO, Yüce A. Improvement of cisplan-induced injuries to sperm quality, the oxidant–anoxidant system, and the histologic structure of the rat tess by ellagic acid. Ferl. Steril. [Internet]. 2008; 89(5):1474–1481. doi: hps:// doi.org/cxxqrg [18] Abo El Gheit RE, Soliman NA, Nagla SA, El-Sayed RM, Badawi GA, Emam MN, Abdel Ghafar MT, Ibrahim MAA, Elswaidy NRM, Radwan DA, Alshenawy HA, Khaled HE, Kamel S, El-Saka ME, Madi NM, Younis RL. Melatonin Epigenec Potenal on Tescular Funcons and Ferlity Proﬁle in Varicocele Rat Model Is Mediated by Silent Informaon Regulator 1. Br. J. Pharmacol. [Internet]. 2022; 179(13):3363–3381. doi: hps://doi.org/n8hg [19] Chen Q, Zhou R, Yang C, Jiang Q, Yuan H, Qiu X, Tian H, Zhou J, Liu C. Ergothioneine aenuates varicocele- induced tescular damage by upregulang HSP90AA1 in rats. J. Biochem. Mol. Toxicol. [Internet]. 2023; 37(4):e23301. doi: hps://doi.org/n8hh [20] Taghizadeh L, Eidi A, Mortazavi P, Rohani AH. Eﬀect of selenium on tescular damage induced by varicocele in adult male Wistar rats. J. Trace Elem. Med. Biol. [Internet]. 2017; 44:177–185. doi: hps://doi.org/gcfzht [21] Kang C, Punjani N, Lee RK, Li PS, Goldstein, M. Eﬀect of varicoceles on spermatogenesis. Semin. Cell. Dev. Biol. [Internet]. 2022; 121:114–124. doi: hps://doi.org/n8hj [22] Xu YW, Ou NJ, Song YX, Wang XH, Kang JQ, Yang YJ, Chen YG, Liu XQ. Seminal plasma miR-210- 3p induces spermatogenic cell apoptosis by acvang caspase-3 in paents with varicocele. Asian J. Androl. [Internet]. 2020; 22(5):513–518. doi: hps://doi.org/gk4wrm [23] Hosseini M, Shaygannia E, Rahmani M, Eskandari A, Golseﬁd AA, Tavalaee M, Gharagozloo P, Drevet JR, Nasr-Esfahani MH. Endoplasmic Reculum Stress (ER Stress) and Unfolded Protein Response (UPR) Occur in a Rat Varicocele Tess Model. Oxid. Med. Cell. Longev. [Internet]. 2020; 2020: 5909306. doi: hps://doi.org/ n8hk [24] Zhang J, Jin PP, Gong M, Guo JH, Fang K, Yi QT, Zhu RJ. Roles of Fas/FasL-mediated apoptosis and inhibin B in the tescular dysfuncon of rats with leﬅ-side varicocele. Andrologia. [Internet]. 2018; 50(2):e12850. doi: hps:// doi.org/gbngs4 [25] Abo El Gheit RE, Soliman NA, Nagla SA, El-Sayed RM, Badawi GA, Emam MN, Ghafar MTA, Ibrahim MAA, Elswaidy NRM, Radwan DA, Alshenawy HA, Khaled HE, Kamel S, El-Saka MH, Madi NM, Younis RL. Melatonin epigenec potenal on tescular funcons and ferlity 7 of 8

Eﬀect of Silymarin in an experimental model of varicocele / Belhan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico proﬁle in varicocele rat model is mediated by silent informaon regulator 1. Br. J. Pharmacol. [Internet]. 2022; 179(13):3363–3381. doi: hps://doi.org/n8hg [26] Khosravanian N, Razi M, Farokhi F, Khosravanian H. Testosterone and vitamin E administraon up-regulated varicocele-reduced Hsp70-2 protein expression and ameliorated biochemical alteraons. J. Assist. Reprod. Genet. [Internet]. 2014; 31(3):341–354. doi: hps://doi. org/f5vg5s [27] Chan CC, Sun GH, Shui HA, Wu GJ. Diﬀerenal spermatozoal protein expression proﬁles in men with varicocele compared to control subjects: upregulaon of heat shock proteins 70 and 90 in varicocele. Urology. [Internet]. 2013; 81(6):1379.e1–1379.e8. doi: hps:// doi.org/f2mgdv [28] Rashtbari H, Razi M, Hassani-Bafrani H, Najaran H. Berberine reinforces Sertoli cells niche and accelerates spermatogonial stem cells renewal in experimentally- induced varicocele condion in rats. Phytomedicine. [Internet]. 2018; 40:68–78. doi: hps://doi.org/n8hn [29] Velickovic LJ, Stefanovic V. Hypoxia and spermatogenesis. Int. Urol. Nephrol. [Internet]. 2014; 46(5):887–894. doi: hps://doi.org/f53x [30] Kilinç F, Kayaselcuk F, Aygun C, Guvel S, Egilmez T, Ozkardes H. Experimental varicocele induces hypoxia inducible factor-1a, vascular endothelial growth factor expression and angiogenesis in the rat tess. J. Urol. [Internet]. 2004; 172(3):1188–1191. doi: hps://doi. org/ﬅxbgm [31] Seval-Celik Y, Akkoyunlu G, Kocamaz E, Köksal IT, Özer S, Baykara M, Demir R. Expression of vascular endothelial growth factor and transforming growth factor alpha in rat tess during chronic renal failure. Folia Histochem. Cytobiol. [Internet]. 2014; 52(4):308–316. doi: hps:// doi.org/n8hp [32] Dolatkhah MA, Khezri S, Shokoohi M, Alihemma A. The eﬀect of Fumaria parviﬂora on the expression of sexual hormones along with their receptors in tescles of adult rats induced by varicocele. Andrologia. [Internet]. 2022; 54(9):e14512. doi: hps://doi.org/n8hq 8 of 8