Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 26/02/2025 Aceptado:20/05/2025 Publicado: 12/06/2025 hps://doi.org/10.52973/rcfcv-e35641 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 10 Eﬀect of sea buckthorn (Hippophae rhamnoides) on kidney and tescular damage, sperm quality and expression of Irisin and Asprosin in Streptozotocin-Induced diabec rats Efecto del espino amarillo (Hippophae Rhamnoides) sobre el daño renal y tescular, la calidad del esperma y la expresión de Irisina y Asprosina en ratas diabécas inducidas por Estreptozotocina Merve Pekince-Özöner 1 * , Sema Timurkaan 2 , Fatih Mehmet Gür 3 , Berrin Tarakçi-Gençer 2 , Hatice Eröksüz 4 , İbrahim Halil Güngör 5 . ¹Department of Histology and Embryology, Faculty of Veterinary Medicine, Siirt University, Siirt, Turkey. ²Department of Histology and Embryology, Faculty of Veterinary Medicine, Fırat University, Elâzığ, Turkey. ³Department of Histology and Embryology, Faculty of Medicine, Niğde Ömer Halisdemir University, Nigde, Turkey. ⁴Department of Pathology, Faculty of Veterinary Medicine, Fırat University, Elazığ, Turkey. 5Department of Reproducon and Arﬁcial Inseminaon, Faculty of Veterinary Medicine, Firat University, Elazig, Turkey. *Correspondence author: merve.pekince@siirt.edu.tr, mervepknc2323@gmail.com. ABSTRACT In recent years, many anoxidants have been used against hyperglycemia and oxidave damage in diabetes. The purpose of this study is to invesgate the protecve eﬀects of Sea buckthorn (Hippophae rhamnoides) against adverse eﬀects of diabetes on tescular and renal ssues. A total of 39 male Sprague-Dawley rats were assigned to 5 groups as follows: control, citrate, SBT, diabetes, and diabetes+SBT. Diabetes inducon was made by streptozotocin (50 mg/kg intraperitonally) to diabetes and diabetes+SBT group. SBT oil was administered to SBT and diabetes+SBT group (50mg/ kg/48h by oral gavage). At the end of study, tescular and kidney samples for histochemical and immunohistochemical examinaons, serum samples for biochemical examinaons and sperm samples for spermatogenic examinaons were collected. The results of the analysis showed that SBT reduced body weight loss and lowered blood glucose levels by reducing the harmful eﬀects of diabetes-induced oxidave stress. When serum TAS and TOS levels were evaluated, it was determined that TAS level was the highest in the SBT group and TAS level increased in the diabetes + SBT group compared to diabetes and the other groups. While TOS level increased in the diabetes group, it decreased in the diabetes+SBT group. SBT also increased sperm density and molity and reduced total abnormality (head-tail) in diabec rats. In SBT-treated diabec rats, histopathological changes during the course of diabetes signiﬁcantly reduced. In addion, decreased irisin expression in renal ssue and decreased irisin and asprosin expression in tescular ssue in the diabetes group were signiﬁcantly normalized in the diabetes+SBT group. In this study, it was found that the applicaon of SBT oil signiﬁcantly prevented hyperglycemia and oxidave stress in diabetes, and protected tescular and renal ssues from the funconal and histopathological changes in these organs caused by hyperglycemia and oxidave stress in diabec animals. These results showed that SBT oil was an eﬀecve nutrional supplement that can be used to protect against the adverse eﬀects of diabetes. Key words: Asprosin; diabetes; Irisin; oxidave Stress; sea buckthorn; sperm quality RESUMEN En los úlmos años, se han ulizado muchos anoxidantes contra la hiperglucemia y el daño oxidavo en la diabetes. El objevo de este estudio es invesgar los efectos protectores del espino amarillo (Hippophae rhamnoides) contra los efectos adversos de la diabetes en los tejidos tescular y renal. Se asignaron 39 ratas macho Sprague-Dawley a 5 grupos: control, citrato, SBT, diabetes y diabetes+SBT. Se indujo diabetes mediante estreptozotocina (50 mg/kg por vía intraperitoneal) a los grupos diabetes y diabetes+SBT. Se administró aceite SBT al grupo SBT y diabetes+SBT (50mg/kg/48h por sonda oral). Al ﬁnal del estudio, se recogieron muestras tesculares y renales para exámenes histoquímicos e inmunohistoquímicos, muestras de suero para exámenes bioquímicos y muestras de esperma para exámenes espermatogénicos. Los resultados de los análisis mostraron que el SBT reducía la pérdida de peso corporal y disminuía los niveles de glucosa en sangre al reducir los efectos nocivos del estrés oxidavo inducido por la diabetes. Cuando se evaluaron los niveles séricos de TAS y TOS, se determinó que el nivel de TAS era el más alto en el grupo SBT y el nivel de TAS aumentó en el grupo diabetes + SBT en comparación con la diabetes y los otros grupos. Mientras que el nivel de TOS aumentó en el grupo de diabetes, disminuyó en el grupo de diabetes + SBT. El SBT también aumentó la densidad y la molidad espermácas y redujo la anormalidad total (cabeza-cola) en las ratas diabécas. En las ratas diabécas tratadas con SBT, los cambios histopatológicos tras la diabetes se redujeron signiﬁcavamente. Además, la disminución de la expresión de irisina en el tejido renal y la disminución de la expresión de irisina y asprosina en el tejido tescular en el grupo diabéco se normalizaron signiﬁcavamente en el grupo diabetes+SBT.En este estudio, se descubrió que la aplicación de aceite SBT prevenía signiﬁcavamente la hiperglucemia y el estrés oxidavo en la diabetes, y protegía los tejidos tescular y renal de los cambios funcionales e histopatológicos en estos órganos causados por la hiperglucemia y el estrés oxidavo en animales diabécos.Estos resultados demostraron que el aceite de SBT era un suplemento nutricional eﬁcaz que puede ulizarse para proteger contra los efectos adversos de la diabetes. Palabras clave: Asprosina; diabetes; Irisina; estrés oxidavo; espino amarillo; calidad del esperma.

Sea buckthorn ameliorates renal and tescular damage in diabec rats / Pekince et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Diabetes mellitus is a metabolic disorder which results in impaired glucose homeostasis aﬅer the failure of the synthesis of the hormone insulin and/or defects in the secreon of the hormone insulin. Impaired glucose balance leads to macrovascular (peripheral vascular disease, ischemic stroke and coronary heart disease) and microvascular (nephropathy, neuropathy, renopathy and erecle dysfuncon) disturbances in the body [1]. Previous studies have also reported that hyperglycemia together with diabetes causes damage to the urinary system due to disorders in germ cells and disorders in kidney funcons and kidney histology due to disorders in the pituitary-hypothalamic system [2 , 3]. Today, the pathogenesis of diabetes is sll not fully determined. However, the studies have indicated that the impaired glucose balance during the course of diabetes causes oxidave stress by increasing the amount of free radicals [4]. In recent years, there have been many studies invesgang the eﬀecveness of anoxidant substances against free radicals that occur during the course of diabetes [5]. Sea Buckthorn (Hippophae rhamnoides L. Sea Buckthorn, SBT) is a shrub which can grow in diﬀerent climates and belongs to the family Elaeagnaceae, and the color of its fruits varies from yellow to orange [6]. The SBT plant contains high concentraon of unsaturated fay acids, and also high levels of omega-7, omega-9, omega-6, and omega-3, which are abundant in its seeds [6 , 7]. Numerous studies have shown that this plant has andiabec, anulcerogenic, anoxidant, cardioprotecve and anatherogenic eﬀects [5 , 8 ,9]. Irisin, ﬁrst discovered by Boström et al. [10], a hormone-like polypepde called as ﬁbronecn type III domain 5 in skeletal muscle. Irisin is a myokine that increases energy expenditure in the body by converng white adipose ssue into brown adipose ssue. It has been reported that irisin may have posive eﬀects on glucose homeostasis and insulin sensivity [10 , 11 ,[12]. Asprosin, discovered by Romere et al. [13] in studies conducted in individuals diagnosed with neonatal Progeroid Syndrome, is a glucogenic adipokine synthesized from white adipose ssue aﬅer fasng. Asprosin causes a rapid release of glucose from liver cells via the G protein-cAMP-PKA pathway, leading to elevated blood glucose levels. It has been revealed that serum asprosin levels of individuals with mutaons in hunger- related genes have decreased. In cases where asprosin is not supplied to the blood, a decrease in the amount of glucose and insulin is observed. It has been reported that the level of asprosin released into the circulaon increases in fasng and decreases aﬅer feeding (high glucose state) [13 , 14]. The aim of the present study was to invesgate the ameliorave eﬀects of Sea Buckthorn on the biochemical, spermatogenic and histopathological changes occurring in renal and tescular ssues aﬅer STZ-induced diabetes. In addion, it was aimed to determine the immunoreacvity of irisin and asprosin, whose relaonship with energy and glucose metabolism in tescular and renal ssues of diabetes and diabetes+SBT rats. MATERIALS AND METHODS The present study was conducted in accordance with Ethics Commiee Decision taken at the meeng of Fırat University Animal Experiments Local Ethics Commiee dated and numbered 2022/8. In the study, totaly 39, seven-week-old male Sprague Dawley rats (Raus norvegicus) weighing 200-250 g were used (Necklife FLY 500, Turkey). A total of 5 groups were randomly selected from these rats obtained as control, citrate, SBT, diabetes and diabetes+SBT groups. During the experiment, all animals were housed in standard cages in an environment at 22-25 °C and with 12 hours (h) of light/12 h of darkness and fed ad libitum with commercial rat feed Control group (n:7) was not subjected to any treatment for 8 weeks, the experimental period. Citrate buﬀer (SST) group (n:7) Single dose of 0.1 M SST was administered intraperitoneally (i.p.). SBT Group (n:7) SBT oil (70mg/kg/48 h dose) was given by oral gavage for two months. The nutrional supplement Sea Buckthorn Oil Capsules (Pharma Nord, Erfurt, Germany) was used as a source of SBT Oil. TABLE I shows the nutrional content of these capsules. Diabetes group (n:9) STZ 50 mg/kg dissolved in 0.1 M SST (pH:4.5) was administered to each rat i.p. as a single dose. Blood glucose level was measured 72 h aﬅer the injecon from the tail vein. Rats with blood glucose levels above 250 mg/dL were considered as diabec and included in the study. STZ- treated rats were given 5% dextrose for 12 h aﬅer the injecon to prevent sudden hypoglycemic shock. Diabetes+ SBT group (n:9) STZ 50 mg/kg was dissolved in 0.1 M SST (pH:4.5) and administered i.p. as a single dose to each rat. Rats with blood glucose levels above 250 mg/dL 72 h aﬅer injecon were considered diabec. SBT oil was administered to diabec rats via oral gavage catheter at a dose of 70 mg/kg every 48 h for two months. The body weights of the rats were recorded at the beginning and end of the experimental procedures. At the end of the eight-week experimental period, the rats were euthanized and blood, sperm, testes and kidney samples were taken. Testes and kidney samples were immersed in 10% buﬀered neutral formalin soluon and ﬁxed for 24 h for future histopathological examinaons. For biochemical analysi, serum samples were stored at -20°C (Vesrost Vf 4820 Nf, Turkey). Semen samples were sent to the laboratory for analysis. 2 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE I. Sea Buckthorn Oil Capsules Nutrional Informaon NUTRIONAL INFORMATION Per 2 capsules (1000 mg) Sea bucthorn oil contains: 1000 mg Saturated fay acids 210 mg Monounsaturated fay acids 480 mg Of which Omega 7 (palmiloic asit) 240 mg Omega 9 (oleic asit) 180 mg Omega 7 (cis vassenik asit) 60 mg Polyunsaturated fay acids 300 mg Of which Omega 6 (linoleic asit) 170 mg Omega 3 (alfa linoik asit) 130 mg Vitamin E ( as alpha tocopherol) 4,0 mg Vitamin A (from beta carotene) 400 ug Biochemical analysis At the beginning and end of the experimental procedures, blood-glucose values were measured with a glucometer (Rina Check AP 10, Germany) from the blood taken from the tail veins of the rats. Aﬅer the other blood samples were placed in serum extracon tubes and centrifuged (3400 g, 10 min, Hech EBA 200, Germany), TAS (Reel Assay Total Oxidant Status Test Kit, Gaziantep, Turkey) and TOS (Reel Assay Total Oxidant Status Test Kit, Gaziantep, Turkey) levels in these samples were measured using Erel’s method [15]. Spermatological analysis To examine sperm density in the study, the right cauda epididymis was separated from the testes and placed in a special glass petri dish with 1 mL of 0.9% NaCl. It was then cut into small pieces and incubated at room temperature for four h. The supernatant, which contained the spermatozoa, was combined with a 1:200 soluon of eosin. Approximately 10 µL of this mixture was transferred to the thoma slide. The mixture was then spread over both ﬁelds and leﬅ for ﬁve minutes. Aﬅer this period, the sperm density was evaluated under a 200x magniﬁcaon (Celestron 44348 Penta View LCD Digital Microscope, USA). To assess sperm molity, one drop of semen were collected from the leﬅ cauda epididymis, mixed it with a Tris buﬀer soluon on a slide and analyzed it under the microscope at 37 °C (Celestron 44348 Penta View LCD Digital Microscope, USA). Molity rate was calculated as a percentage at 400 magniﬁcaon in three diﬀerent ﬁelds. To determine the percentage of morphologically abnormal spermatozoa, 20 µL of the Tris buﬀer-spermatozoa mixture used for molity assessment was taken and dripped onto a microscope slide heated to 37°C. A thin smear of this mixture was applied to the slide and allowed to dry at room temperature. The slides were then examined under a light microscope at 400× magniﬁcaon (Celestron 44348 Penta View LCD Digital Microscope, USA). A total of 200 spermatozoa were examined on each slide, and the head, tail, and total abnormality rates of the spermatozoa were expressed as percentages [16]. Histopathological analysis Tissue processing and Haematoxylin and Eosin (H&E) staining. Fixed tescular and kidney ssues were subjected to roune ssue processing procedures. Subsequently, the ssue samples were embedded in paraﬃn blocks. Secons of 5 µm thickness were excised from the paraﬃn blocks using a rotary microtome (Thermo Scienﬁc, USA), and the slides were stained with H&E, Periodic Acid Schiﬀ (PAS) and Crossman methods to determine morphological and histopathological changes for histopathological analysis. Right and leﬅ tescular ssues stained with H&E were examined for histopathological changes such as intersal edema, congeson and subcapsular hemorrhage and scored as undamaged, -; slightly damaged, +; moderately damaged, ++ and very damaged, ++++. The degree of maturaon of germ cells was determined by evaluang at least 20 seminiferous tubules under a light microscope with a 40x objecve (Olympus BX50, Japan) according to the Johnsen scoring [17]. Aﬅer measuring the mean seminiferous tubule diameter (MSTD) and mean seminiferous epithelial thickness (MSET) in the seminiferous contor tubules closest to the round in diﬀerent regions using the Olympus Cell Sens Standard program (Version 1.1.17), the data obtained were stascally evaluated [18]. Haematoxylin and Eosin stained renal ssues were examined for histopathological changes such as glomerular atrophy, glomerular lobulaon, tubular damage, vacuolizaon and the ﬁndings were rated as follows: No damage, 0; less than 10% damaged, 1; 25% damaged, 2; 26-50% damaged, 3; more than 75% damaged, 4. In addion, PAS stain was applied to determine the basement membrane thickness in both tescular and renal ssues. These ssues were photographed aﬅer staining and evaluated. Immunohistochemical analysis The immuno-peroxidase method was used for immunohistochemical analysis according to kit (Ultravision Detecon System, Thermo Fisher Scienﬁc, USA). Brieﬂy, 5-µm thick secons of renal ssues were deparaﬃnized in xylol and passed through a series of alcohols. Angen retrieval procedure was performed by boiling the ssue secons in 0.01 M citrate buﬀer (pH: 6) at 90-95°C for 30 min. Then, ssue secons were treated with H2O2 soluon to inhibit endogenous peroxidase acvity for 15 min and then treated with Ultra V Block soluon for 10 min and incubated overnight at +4°C in slide tray (eBioscience StainTray, Thermo Scienﬁc, USA) with primary anbodies (Irisin, 1/100 diluon, rabbit polyclonal, 201r-0335, Sun Red, China; Asprosin 1/200 diluon, rabbit polyclonal, 201r- 5721, Sun Red, China). The ssue secons were incubated with secondary anbody following streptavidin peroxidase for 30 min in a in slide tray (eBioscience StainTray, Thermo Scienﬁc, USA) at 37°C. Hematoxylin was used as the counterstain. Immunohistochemical staining intensity was graded in the range of 0-3. 0, negave; 1 light; 2, medium; and 3 were rated as intense. Also, negave ﬁelds are 0; <25% stained areas as 0.1; 26–50% stained areas 0.4; Areas 51–75 stained 0.6; Areas stained between 76–100% were rated as 0.9. Aﬅer obtaining these data, the histoscore was determined using the area x density formula [19 ,[20]. 3 of 10

Sea buckthorn ameliorates renal and tescular damage in diabec rats / Pekince et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Stascal analysis The data were determined as mean ± standard deviaon and stascal analyses were performed using SPSS 22 soﬅware (IBM Corp., Armonk, NY, USA). Kruskal Wallis and Posthoc Dunn test, were used for the evaluaon between groups. The value of P<0.05 was considered as stascally signiﬁcant. RESULTS AND DISCUSSION Diabetes has many systemic eﬀects on the body. Findings from several studies have shown observable changes in body composion in experimental diabec rats, including decreased tescular weight [21]. In contrast, a diﬀerent study by Scarano et al. [22] reported that the while body weight of diabec rats decreased signiﬁcantly, there was no diﬀerence in tescular weight and as a result relave tescular weight increased. In the present study, although the relave tescular weights of the rats in the diabetes group increased compared to the control, citrate, and SBT groups, this increase was not stascally signiﬁcant (TABLE II). This can be explained by the preservaon of tescular weight in conjuncon with a decrease in body weight among diabec animals. In nephropathy caused by diabetes, kidney weight increases due to cell proliferaon and hypertrophy. In studies, it has been observed that nephropathy occurs in diabec animals due to tubulointersal ﬁbrosis and increased expression of angiogenic factors [23 , 24]. In the study, relave kidney weights were similar in the control, citrate, and SBT groups. Although there was an increase in the diabetes+SBT and diabetes groups compared to other groups, the relave kidney weights in the diabetes+SBT group were closer to control and citrate groups. However, these changes were not stascally signiﬁcant (P>0.05) (TABLE II). This may be explained by the preservaon of kidney weight with a decrease in body weight when calculang relave kidney weight in diabec animals. The fact that the kidney weight of SBT supplemented diabec animals was close to the control group suggests that SBT may prevent the increase in kidney weight caused by hyperglycaemia due to its anoxidant content. However, it was hypothesised that the reason for the lack of stascal diﬀerence may be aributable to the individual diﬀerences observed in the animals. There are numerous studies showing that diabetes causes a decrease in body weight as a result of lipid and protein degradaon [2 , 3]. However, in contrast to this decrease, it has been shown that administering diﬀerent doses of SBT to diabec animals causes an increase in body weight [25]. In this study, the post-experimental body weights of the Control, citrate, SBT and diabetes+SBT groups were similar (P>0.05). It was found that the body weights of diabec rats decreased signiﬁcantly compared to the other groups (P<0.05) (TABLE II). When the data obtained and the literature presented above were evaluated together, it was concluded that SBT may be eﬀecve against body weight loss due to diabetes as a result of reducing cellular oxidave stress and prevenng lipid and protein degradaon. TABLE II. Results of Body Weight, Relave Kidney Weight, Relave Tescular Weight and Results of Spermatogenic Examinaon GROUPS Control Citrate SBT Diabetes Diabetes+ SBT Body Weight Relave Kidney Weight Relave Tescular Weight The amount of sperm molity Amount of Sperm Density Sperm Abnormality 322±22 a 0,83±0,059 a 0,92±0,11 a 63±16 a 84±12 a 4,8±1,8 a 313±77 ac 0,96±0,35 a 0,94±0,3 a 53±12 a 88±23 a 4,1±2,4 ac 345±35 a 0,82±0,063 a 0,90±069 a 58±8,1 a 90±27 a 1,9±0,58 bc 234±18 bc 1,3±0,093 a 1±0,11 a 43±24 a 71±9,7 a 5,1±1,8 a 276±58 a 1±0,16 a 0,90±0,24 a 51±19 a 84±14 a 5,1±2,3 a All values are in the form of mean ± standard deviaon. Diﬀerent leers in the same line show that the stascally diﬀerent from each other. In a previous study in which STZ-induced diabetes model was established, it was reported that administraon of SBT to rats decreased the blood-glucose value [5], 9 , 26]. In another study, it was reported that SBT administered to rats fed a high-fat diet decreased blood glucose levels [27]. In the present study, it was observed that blood sugar levels between the groups were similar in control, citrate and SBT groups (P>0.05), while blood sugar levels in the diabetes + SBT group were signiﬁcantly reduced compared to the diabetes group (P<0.05) (TABLE IV). In this study, consistent with other literature [5 , 9 , 26], it was observed that blood glucose levels decreased in diabec rats administered SBT oil. Sea buckthorn oil has been found to protect against oxidave stress caused by oxidizing lipids [25]. In the present study, it was determined that the serum TAS level was the highest in the SBT group, and that the TAS level increased in the diabetes + SBT group compared to the diabetes and other groups (P<0.05). The highest TOS value was observed in the diabetes group. In addion, while it was similar in the control, citrate and SBT groups, the TOS level decreased in the diabetes + SBT group compared to the diabetes group altough there was no signiﬁcant diﬀerence (P>0.05) (TABLE III). The data obtained suggested that sea buckthorn may have a protecve eﬀect against oxidave stress caused by diabetes. TABLE III. Results of Biochemical Blood-glucose, Serum TAS value, Serum TOS value GROUPS Control Citrate SBT Diabetes Diabetes+ SBT Blood-glucose value 82±23 a 69±11 a 67±12 a 430±66 b 326±69 c Serum TAS value 0,93±0,063 ac 0,87±0,11 ac 1.0±0,13 bc 0,85±0,044 a 0,90±0,091 ac Serum TOS value 8,7±1,4 a 8,6±1,2 a 8,6±0,72 a 10±0,82 a 9,5±2,3 a All values are in the form of mean ± standard deviaon. Diﬀerent leers in the same line show that the stascally diﬀerent from each other 4 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Oxidave stress in course of diabetes has been documented to reduce sperm count, sperm molity and increase abnormal sperm rate [28 , 29]. Navarro-Casado et al. [28] reported diabetes decreased sperm density and increased sperm total abnormality (head-tail) but did not aﬀect sperm molity. In another study, it was determined that sperm count, sperm viability and molity rates, which decreased during the course of diabetes, increased as a result of quercen administraon [29]. In this study, percentage molity, density and sperm total abnormality (head-tail) were similar in the control and citrate groups.There was a decrease in molity in the diabetes group. When sperm molity and density values were compared with the diabetes group, an increase was observed in the diabetes + SBT group altough there was no signiﬁcant diﬀerence (P>0.05). A slight increase in sperm density was observed in the SBT group. The rate of sperm anomaly (in the head and tail regions) was similar in the control, citrate, diabetes and diabetes + SBT groups and signiﬁcantly decreased in the SBT group compared to the other groups except citrate group (P<0.05) (TABLE II). This results are consistent with literature [28 , 29]. In summary in this study, although not stascally signiﬁcant, sperm density and molity decreased. Sperm anomalies was more evident in diabec rats. On the other hand, sperm molity and density increased and sperm anomalies decreased in rats treated with SBT. This result suggests that SBT oil can be used to improve sperm quality in normal and diabec animals. Previous studies reported diabetes lead to a decrease in MSTD and MSET parameters, an increase in intersal connecve ssue and seminiferous tubule basement membrane thickness, and histopathological changes such as atrophy and degeneraon in tunica albuginea and seminiferous tubules in tescular ssue [30 , 31]. In the present study, Hematoxylin-Eosin, Crossman trichrome and PAS staining results of rats in control, citrate and SBT groups showed normal histological structure in tescular ssue (FIG. 1). Histopathological changes such as decreased spermatogenic acvity, atrophy in some seminiferous tubules, intersal edema, hyperemia in blood vessels and increased basement membrane thickness were found in the tescular ssues of rats in the diabetes group. The diabetes + SBT group exhibited enhanced morphologic abnormalies in comparison to the diabetes group. In the tescular ssues of the rats in this group, the capillaries in the intersal region were full and hyperemic (FIG. 1). FIGURE 1. H-E, Crossman Triple and PAS staining of tescular ssue. A, control group. B, citrate buﬀer group. C, SBT group. D, diabetes group. E, diabetes+SBT group. Spermatogo- nium (sg), spermatocyte (sp), spermad (st), spermatozoa (sz), blood vessel (kd), Leydig cell (le), sertoli cell (se), spermatogenic separang from basement membrane cells (orange star), intersal edema (red arrow), hyperemia in capillaries (green arrow), vacuolizaon in tubules (yellow arrow), degenerated tubules (blue star), intersal edema (black arrow). TABLE IV. Scoring of histopathological changes in tescular ssue and kidney ssue Histopathological changes Control Citrate SBT Diabetes Diabetes+ SBT TESTIS Intersal Edema 0,71±0,76 a 0,86±0,69 a 0,57±0,53 a 1,2±0,83 a 1,1±0,93 a Congeson 0,86±0,69 ac 0,29±0,49 bc 0,86±0,38 ac 1,2 ±0,67 a 0,44±0,53 bc Subcapsular Hemorrhage 0±0 a 0±0 a 0±0 a 0,11±0,33 a 0±0 a Johnsen Score 9,1±0,69 a 9,3±0,96 a 9,6±0,79 a 7,8±1,3 b 8,1±0,86 b Spermatogenic Acvity 2,8±0,41 a 2,6±0,79 ac 2,1±0,6 b 2,3±0,76 b 2,8±0,41 bc MSET 98±9,3 ac 90±4,4 bc 93±4,8 ac 85±4,3 b 91±2,4 bc MSTD 285±9,3 ac 271±13 b 290±10 a 270±15 ab 270±5,8 bcd KIDNEY Glomerular Atrophy 0±0 a 0±0 a 0±0 a 0,67±0,71 b 0,22±0,44 a Glomerular Lobulaon 0±0 a 0,14±0,38 a 0,14±0,38 a 0,56±0,53 b 0,11±0,33 a Tubular Damage 1,3±0,52 a 0,71±0,49 a 1,4±0,53 a 3,7±0,87 b 2,8±1,2 b Vacuolizaon 1,4±1,2 a 1,1±0,83 a 0,14±0,38 a 0±0 b 0,14±0,38 b Expansion in Bowman’s capsule 1,6±0,53 a 1,2±0,83 a 0,9±0,9 ac 0,43±0,53 bc 0,71±0,49 a All values are in the form of mean ± standard deviaon. Diﬀerent leers in the same line show that the stascally diﬀerent from each other 5 of 10

Sea buckthorn ameliorates renal and tescular damage in diabec rats / Pekince et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico It was established that the Johnsen scores of the rats in the control, citrate, and SBT groups were closely aligned, while the Johnsen score of the rats in the diabetes group exhibited a substanal decline in comparison to these groups (P < 0.05). Despite an increase in the Johnsen score of the rats in the diabetes + SBT group compared to the diabetes group, this increase was not stascally signiﬁcant (P > 0.05) TABLE IV). When MSET values were compared between groups, it was found that MSET in the diabetes group decreased stascally signiﬁcantly compared to the control, citrate, and SBT groups (P<0.05), whereas, MSET in the diabetes + SBT group increased and approached the control group (P>0.05) (TABLE IV). When evaluang the MSTD results, it was found that MSTD values decreased in the citrate, diabetes, and diabetes + SBT groups compared to the control and SBT groups, while there was no signiﬁcant diﬀerence between the diabetes and diabetes + SBT groups (P>0.05) (TABLE IV). In accordance with the ﬁndings, it was noted that the diameter and thickness of the seminiferous tubules decreased in diabetes. These results indicates that diabetes had a harmful eﬀect on the histological structure of seminiferous tubules. In this study, results of the diabetes + SBT group exhibited a greater similarity to those of the control group suggests that SBT reduced tescular damage caused by diabetes. In diabec kidney ssues, it causes damage to many histopathological changes including atrophy of molar glomeruli, expansion of Bowman’s capsule and thickening of basement membrane, shedding and vacuolizaon of tubular epithelium, atrophy of tubules and proteinosis ﬁltrate [32 , 33]. A literature review revealed that a study on the eﬀects of SBT on the kidney showed that SBT ameliorated renal damage [32]. In histochemical staining of secons of kidney ssue, it was observed that the histological structure of the cortex and medulla regions of the kidneys of control, citrate and SBT groups was normal. In H&E stained kidney ssues of the diabec group, histopathological changes such as atrophy of some glomeruli, expansion of Bowman’s capsule, intense bleeding in some areas, vacuolizaon of tubules (Armani-Ebstein lesions), atrophy and proteinosis ﬁltrate were observed. Although tubular degeneraon, vacuolizaon, hemorrhage, mononuclear cell inﬁltraon and clear structures were observed in the kidney ssues of the Diabetes + SBT group, pathological changes were reduced compared to the diabetes group (FIG. 2). TABLE IV shows the scoring of histopathological changes in the kidney ssue. Examinaon of Crossman triple stained secons showed a slight increase in collagen and ﬁbrosis in the intersal space in the diabetes group (FIG. 2). Examinaon of PAS stained secons showed shedding of the tubular epithelium and thickening of the basement membrane of the Bowman capsule in the diabetes group (FIG. 2). Shedding of the tubular epithelium was observed in the diabetes + SBT group, but other histopathological changes observed in the diabetes group were signiﬁcantly decreased (FIG. 2). The ﬁndings of the present study are consistent with the ﬁndings of previous studies conﬁrming the pathological eﬀects of diabetes on the kidneys. In this study, SBT, which has anoxidant eﬀects, decreased the histopathological changes menoned above in the kidney ssues of the diabec group. Irisin is a hormone-like polypepde which discovered by Boström et al. [10] in skeletal muscles. In their study, Eser et al. [34] observed that irisin level decreased signiﬁcantly aﬅer STZ-induced diabetes [34]. In a study exploring the relaonship between oxidave stress and irisin during the course of diabetes, it was found that irisin level decreased as oxidave stress increased [35]. Immunohistochemical expression of irisin has been shown in ssues such as brain, heart, stomach, pancreas, tess, epididymis, liver and tess [19 , 36]. Aydın et al. [19] reported that irisin-posive immunostaining was detected in spermatogenic and Leydig cells in human fetus, strong immunostaining was detected in Leydig cells and weak immunostaining was detected in spermatogenic cells in adult human tescular ssue. In the present study, the immunohistochemical staining paern of irisin in the tescular ssues of the control, citrate and SBT group rats was similar and weak immunostaining was observed in Leydig cells. The lowest Irisin expression was observed in the diabetes group, and the highest in the diabetes + SBT group. Irisin immunoreacvity was stascally diﬀerent between Diabetes and in favor of diabetes+SBT groups (P<0.05). This result may be due to anoxidant content of SBT to reduce oxidave stress [36]. No posivity was observed in the negave control preparaons (FIG. 3). Histoscore values between the groups are given in TABLE V. When the immunoreacvity of Irisin in kidney ssue was examined, posive cells with weak/medium staining intensity were found in the proximal and distal tubules in the cortex of the kidney ssue of the control, citrate and SBT groups. Although the expression decreased in the diabetes group compared to the control group, it was not stascally signiﬁcant (P>0.05). Intense posive Irisin immunostaining was seen in the proximal tubules of the diabetes + SBT group rats and a stascal diﬀerence was detected compared to other groups (P<0.05). Negave control staining did not show posivity (FIG. 3). TABLE V presents the intergroup histoscore values of irisin in kidney ssue. In this study, it was determined the intensity of irisin-posive staining in Leydig cells in both tescular and kidney ssues of diabec rats was weaker than in other groups, while the staining intensity in the diabetes + SBT group was signiﬁcantly increased compared to the diabetes group. It is hypothesised that posive enhancing eﬀect of SBT on irisin expression is by its intense anoxidant content and therefore its eﬀect of reducing oxidave stress. 6 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 2. H-E, Crosman Triple and PAS staining of kidney ssue. A, control group. B, citrate buﬀer group. C, SBT group. D, diabetes group. E, diabetes+SBT group. Glomerulus (G), proximal tubule (pt), distal tubule (dt), macula densa (md), hyperemia (green arrow) tubular dilataon (blue arrow), tubular vacuolizaon (Armani Ebstein lesions) (red arrow), narrowing of Bowman’s space (white arrow), mononuclear cell inﬁltraon (blue star), exfoliaon of tubular epithelium (orange arrow), proteinous ﬁltrate (black arrow), connecve ssue increase (yellow star), thickening of basement membrane (green arrow) FIGURE 3. Irisin and Asprosin immunoreacvity in tescular and kidney ssue. A, control group. B, citrate buﬀer group. C, SBT group. D, diabetes group. E, diabetes+SBT group. Weak Leydig cell immunoposivity (white arrow), moderate Leydig cell immunoposivity (green arrow, pink arrow), strong Leydig cell immunoposivity (yellow arrow) TABLE V. Evaluaon of Irisin and Asprosin immunoreacvity in tess and kidney ssue Immunohistochemical evaluaon Control Citrate SBT Diabetes Diabetes+ SBT Irisin immunoreacvity of tescular ssue 0,5±0,20 ac 0,8±0,46 ac 0,6±0,69 ac 0,35±0,41 a 1,0±0,23 bc Irisin immunoreacvity of kidney ssue 0,7±0,12 a 0,92±0,64 a 0,66±0,44 ac 0,58±0,26 a 1,5±0,63 bc Asprosin immunoreacvity of tescular ssue 0,90±0,14 a 0,6±0,28 a 0,6±0,28 a 0,47±0,12 a 0,76±0,41 a Asprosin immunoreacvity of kidney ssue 0,8±0 a 0,5±0,14 a 0,6±0,28 a 1,1±0,23 a 1,3±0,50 a All values are in the form of mean ± standard deviaon. Diﬀerent leers in the same line show that the stascally diﬀerent from each other. Asprosin is a recently discovered adipokine secreted from white adipose ssue [13]. In a previous study, asprosin administraon increased blood glucose concentraon in healthy rats; however, it was reported that it did not cause any changes in diabec rats [37]. One study showed immunohistochemical localisaon of asprosin in distal tubule cells in the kidney and Leydig cells in the testes of diabec rats. In the same study, when comparing control and diabetes in terms of expression, asprosin was found to be increased in the stomach and testes of diabec rats, decreased in liver, kidney and heart ssues, and there was no signiﬁcant change in brain ssue [38]. Another study found that serum asprosin levels were high in people with type 2 diabetes-induced nephropathy and that asprosin levels increased with the severity of nephropathy [39]. In a study determining the relaonship between asprosin and aging-related spermatogenec acvity, it was determined that asprosin level decreased in parallel with the decrease in testosterone level. The data obtained in the same study suggest that a decrease in asprosin level, which is associated with insulin sensivity and glucose transport, may cause regression in tescular development [40]. In a study by Keskin et al. [41], it was indicated that intracerebral administraon of asprosin 7 of 10

Sea buckthorn ameliorates renal and tescular damage in diabec rats / Pekince et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico increased hypothalamic GnRH, mRNA and protein levels in male rats, thus causing an increase in LH, FSH and testosterone levels. In addion, it was determined that sperm density and molity increased in the testes aﬅer asprosin administraon. These results suggest that asprosin has regulatory roles in the reproducve system and tescular funcons [41]. When asprosin immunoreacvity was examined in tescular ssue, the immunoreacon of tescular ssues of Control, Citrate and SBT groups was similar. The lowest asprosin immunoreacvity was observed in the diabetes group. The immunoreacon of asprosin was almost the same in control and diabetes + SBT groups. The ssues used as negave controls were asprosin negave (FIG. 3). No posivity was observed in the negave control preparaons Histoscore values between the groups are given in TABLE V. Kidney ssue asprosin expression was found to be similar in the control, citrate and SBT groups. The highest asprosin expression was seen in the diabetes + SBT group. It was detected that immunoreacvity increased in the diabetes and diabetes + SBT groups compared to the other groups, but it was not a signiﬁcant increase (P>0.05). Negave control staining did not show posivity (FIG. 3). These results may be related to severity of nephropathy [39]. Histoscore values between the groups are given in TABLE V. In the present study, it was found that asprosin expression in the tescular ssues of diabec rats decreased compared to the control group, while asprosin expression in the kidney ssues increased. The increase in asprosin expression in the kidney ssues of the diabec group was probably due to nephropathy. It was thought that the decrease of Asprosin immunoreacvity in tescular ssue may be due to insulin sensivity, glucose transport, and tescular development. CONCLUSION The study revealed that SBT oil administered to rats with experimental diabetes reduced diabetes-induced body weight loss, lowered blood glucose levels, and reduced the harmful eﬀects of oxidave stress during the course of diabetes. SBT administraon increased sperm density and molity, reduced abnormalies, signiﬁcantly reduced histopathological damage in tescular and renal ssues during the course of diabetes, and had posive eﬀects on irisin and asprosin expression related to glucose and energy metabolism in diabec rats. In order to determine more precisely the therapeuc eﬃcacy of SBT against the negave eﬀects of diabetes on tescular and renal ssues, it is thought that it would be beneﬁcial to plan studies in the future to invesgate the eﬀecveness of SBT at diﬀerent doses. 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