Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido:18/03/2025 Aceptado:31/05/2025 Publicado: 30/06/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Revista Cienﬁca, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35657 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Eﬀects of Clitoria Ternatea on renal ischemia and reperfusion injury in rats Efectos de la Clitoria Ternatea en la lesión renal por isquemia y reperfusión en ratas ¹Bingol Universıty, Faculty of Veterinary Medicine, Department of Surgery. Bingöl, Turkey. ²Hatay Mustafa Kemal University, Faculty of Veterinary Medicine Department of Pathology. Hatay, Turkey. ³Siirt University, Faculty of Veterinary Medicine, Department of Physiology. Siirt, Turkiye. ⁴Ataturk University, Faculty of Veterinary Medicine, Department of Pathology. Erzurum, Turkiye. ⁵Siirt University, Faculty of Veterinary Medicine, Department of Biochemistry, Siirt, Turkiye. ⁶Dokuz Eylul University, Faculty of Veterinary Medicine, Department of Anımal Nutrıon and Nutrional Diseases. İzmir, Turkiye. ⁷Van Yüzüncü Yıl University, Faculty of Science, Department of Chemistry. Van, Turkiye. ⁸Bingöl University, Vocaonal School of Health Services. Bingöl, Turkiye. ⁹Erciyes Universıty, Faculty of Veterinary Medicine, Department of Surgery. Kayseri, Turkiye. 10 YYU University, Faculty of Sciences, Department of Chemistry. Van, Turkiye. *Corresponding author: sulemelek@gmail.com ABSTRACT Ischemia-reperfusion (I/R) injury refers to damage caused by the temporary interrupon of blood ﬂow into an organ/ ssue and the restoraon of circulaon. This injury, which aﬀects many vital organs, is sll an important clinical and surgical problem. This study aims to invesgate the eﬀect of anoxidant and an-inﬂammatory eﬀects of Clitoria ternatea on renal I/R injury in rats. Twenty-four male Wistar albino rats were clustered into 4 groups, 6 rats in each (Control, I/R group, Clitoria ternatea group, I/R group + Clitoria ternatea group). At the end of the experimental procedures, all groups were euthanised in accordance with ethical rules. Blood and ssue samples were taken and subsequent biochemical (MDA, SOD, CAT, GSH-Px, BUN, Na, K, creane), histopathological and immunohistochemical analyses demonstrated that Clitoria ternatea eﬀecvely prevented I/R-induced renal injury in rats. Based on these ﬁndings, Clitoria ternatea may be considered a potenal therapeuc agent for the prevenson of I/R-induced kidney damage. Key words: Kidney; ischemia-reperfusion; rat; clitoria ternatea; therapeuc RESUMEN La lesión por isquemia-reperfusión (I/R) se reﬁere al daño causado por la interrupción temporal del ﬂujo sanguíneo en un órgano/tejido y el restablecimiento de la circulación. Esta lesión, que afecta a muchos órganos vitales, sigue siendo un importante problema clínico y quirúrgico. Este estudio pretende invesgar el efecto anoxidante y aninﬂamatorio de la Clitoria ternatea sobre la lesión renal por I/R en ratas. Se agruparon 24 ratas albinas Wistar macho en 4 grupos, 6 ratas en cada uno (Control, grupo I/R, grupo Clitoria ternatea, grupo I/R + grupo Clitoria ternatea). Al ﬁnal de los procedimientos experimentales, se praccó la eutanasia a todos los grupos de acuerdo con las normas écas. Se tomaron muestras de sangre y tejidos y los posteriores analisis bioquímicas (MDA, SOD, CAT, GSH-Px, BUN, Na, K, creana), histopatológicas e inmunohistoquímicas demostraron que la Clitoria ternatea prevenia eﬁcazmente la lesión renal inducida por I/R en ratas. Basandose en estos resultados, la Clitoria ternatea puede considerarse un agenta terapeuco potenscial para la prevencion del dano renal inducido por I/R. Palabras clave: Riñón; isquemia-reperfusión; rata; Clitoria ternatea; terapeuco 1 of 8 Şule Melek 1 * , Ahmet Uyar 2 , Hurrem Turan Akkoyun 3 , Serdar Altun 4 , Mahire Bayramoğlu Akkoyun 5 , Fatma Karagözoğlu 6 , Suat Ekin 7 , Aydın Şükrü Bengü 8 , Gültekin Atalan 9 , Ahmet Bakır 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Injury renal I/R refers to the damage resulng from the temporary cessaon of blood ﬂow into an organ/ssue and then the restoraon of circulaon. This injury is sll a signiﬁcant clinical and surgical concern, aﬀecng several organs including the kidneys, liver, heart, lungs, brain, and intesnes. Renal I/R injury may originate from condions such as sepsis, hydronephrosis, open kidney surgeries, shock, kidney transplantaon, paral nephrectomy, hemorrhage, and resuscitaon eﬀorts [1 , 2]. During kidney transplantaon, various degrees of I/R injury may develop on the graﬅ, potenally resulng in postoperave graﬅ dysfuncon or loss. Similarly, vascular clamping, commonly performed in tumor resecon surgeries, facilitates a bloodless operave ﬁeld but can also induce ischemic damage in healthy renal parenchyma due to temporary arterial occlusion. Therefore, implemenng eﬀecve prophylacc treatments to prevent I/R injury in such surgical scenarios may increase success rates [3]. Therefore, there are ongoing studies on pharmacological agents protecng kidney ssue from I/R injury. In addion to immunosuppressants and corcosteroids, anoxidants have been increasingly ulized. Recent studies emphasized the eﬀects of anoxidants on scavenging free radicals generated by I/R and reducing the resultant damage [1 ,4]. Clitoria ternatea is widely recognized in tradional Indian medicine for its mulfaceted pharmacological properes. It has anbacterial, an- inﬂammatory, diurec, anpyrec, analgesic, local anesthec, platelet aggregaon inhibitory, andiabec, and smooth muscle relaxant eﬀects [5 ,6]. Sai et al. [6] reported the an- inﬂammatory and anoxidant properes of Clitoria ternatea and its ability to prevent kidney damage and dysfuncon in rats induced by L-NG-Nitro-Arginin-Melester (l-NAME). Given this informaon, this study aims to invesgate the eﬀect of anoxidant and an-inﬂammatory eﬀects of Clitoria ternatea on renal I/R injury in rats. MATERIALS AND METHODS Preparaon of Clitoria ternatea extract In the present study, Clitoria ternatea extract was prepared by following the guidelines established by Vidana-Gamage et al. [7] and Saraç [8]. Experimental animals The approval was granted by Bingöl University’s Animal Experiments Local Ethics Commiee (25.04.2022, No: 2022/02) and carried out at the Animal Experimentaon Center of the same University. Twenty-four Wistar albino rats (Raus norvegicus) with weights of 200-280g (Denver Instrument SI- 234, Germany) were involved [3 , 9] The rats were given a 12-h light/dark cycle and ad libitum access to pellet feed and water. They underwent a one-week acclimazaon period in their cages before experiments were started. They were not fed for 12 h (h) before experiments, but there was water available during the enre experiment process. Experimental design Control (n=6): Only right nephrectomy, I/R group (n=6): right nephrectomy, followed by 30min. ischemia and 2h reperfusion [9], Clitoria ternatea group (n=6): rats were administered 200 mg/kg Clitoria ternatea ﬂower extract orally for 7 d [10], I/R group + Clitoria ternatea group (n=6): rats received 200 mg/kg Clitoria ternatea extract for 7 d. On the ﬁnal d, right nephrectomy and leﬅ kidney I/R (30 min ischemia, 2 h reperfusion) were performed. All groups were anaesthezed 10mg/kg Xylazine hydrochloride (Rompun 2% Bayer Türk Kimya San. Ltd. Ş., İstanbul, Turkey) + 60mg/kg Ketamine hydrochloride (Ketasol 10% Richter Pharma AG, Wels, Austria) by intraperitoneal injecon and ﬁxed in the supine posion. Right kidneys were removed in all groups. In order to induce ischaemia in the leﬅ kidney (in I/R and I/R + Clitoria ternatea groups), the renal arteries and veins of this kidney were ligated for 30 min. and this condion was maintained unl discolouraon was observed in the leﬅ kidney. The kidneys were reperfused for 2 h aﬅer the ligature was released. At the end of reperfusion, while anesthezed 10mg/kg Xylazine hydrochloride (Rompun 2% Bayer Türk Kimya San. Ltd. Ş., İstanbul, Turkey) + 60mg/ kg Ketamine hydrochloride (Ketasol 10% Richter Pharma AG, Wels, Austria), a cardiac puncture was performed to collect blood samples, and kidney ssue samples were taken. Aﬅer the samples collecon the animals were euthanized by decapitaon technique in accordance with ethical rules. Anoxidant enzyme acvies in kidney ssue Kidney ssue preparaon The samples were homogenized ulizing an IKA (Ultra-Turrax T25, Germany) and a glass-porcelain homogenizer at 20000 rpmfor 5 min. Following this process, the centrifuge (Hech Rona 420 R, Germany) was performed at 9692 G for 30 min at +4°C [11]. Anoxidant enzyme acvity analyses were conducted using clear supernatants [12]. Anoxidant enzyme esmaon The clear supernatant was ulized to analyze SOD, catalase, and GSH-Px. Tissue SOD acvity was invesgated ulizing the technique introduced by Sun et al. [13], while that of GSH-Px was examined employing the procedure introduced by Gallo and Marno [14]. Esmaon of catalase acvity was performed following the technique reported by Aebi [15]. MDA analysis MDA level measurement was performed using the technique introduced by Ohkawa et al. [16]. The determinaon of total protein content was performed employing the standard method introduced by Lowry et al. [17] with bovine serum albumin. Serum Analysis The serum was separated and kept in covered polypropylene tubes. Sodium (Na), Potassium (K), Creane, Urea, and BUN levels were measured following standard procedures with the Roche Modular P 800 auto-analyzer. (Roche Diagnoscs, Model P 800, Germany). 2 of 8

Clitoria ternatea on renal ischemia injury in rats / Melek et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Histopathological and immunohistochemical invesgaons Kidney ssue samples aﬅer necropsy were ﬁxed at 10% neutral formalin for 72 h. The samples were processed through alcohol and xylol series in an automac machine (Leica ASP6025, Leica Biosystems, Germany) according to roune ssue tracing procedures and then embedded in paraﬃn. Secons of 5 μm thick were taken from the blocked ssues in a microtome (Leica RM 2255, Germany). Haematoxylin eosin (H&E) staining process was conducted following the roune process. The evaluaon criteria included degeneraon and necrosis in the tubular epithelium, desquamaon, tubular atrophy, intratubular hyaline casts, inﬂammaon, and congeson. The results were (BX51 dp72 camera, Olympus, Tokyo, Japan) as none (0 lesions), mild: + (degeneraon), moderate: ++ (degeneraon+necrosis), and severe: +++ (degeneraon+ diﬀuse necrosis) [18], 19]. For immunohistochemical analysis, samples prepared at 5 µm and put on poly-L-lysine coated slides were subjected to standard avidin–bion–peroxidase method (ABC) by the manufacturer’s protocol against an-caspase-3 anbody (sc- 56053). All the secons were incubated with DAB (Histostain- Kit, Invitrogen, Camarillo, CA, USA) and counterstained using Mayer’s hematoxylin. The staining intensity was independently analyzed by a pathologist. The an-caspase-3 staining intensity was rated as no staining (0), poor staining (+1), moderate staining (+2), and strong staining (+3) [20]. Stascal analyses This process was conducted ulizing SPSS (SPSS for Windows, version 20.0). Diﬀerences between the groups were examined employing a nonparametric test (Kruskal–Wallis). Paired comparisons in case of signiﬁcant diﬀerences were conducted ulizing the Mann–Whitney U-test (P<0.05). RESULTS AND DISCUSSION The level of MDA, a lipid peroxidaon product, was found to be signiﬁcantly higher in I/R group in comparison to control (P<0.05). This increase indicates lipid peroxidaon and cellular damage. However, the MDA level of I/R + Clitoria ternatea group was observed to decrease in comparison to the I/R group (FIG. 1). Examining the SOD enzyme acvity, it was observed that acvity signiﬁcantly decreased in the I/R group in comparison to control (P<0.01). However, comparing the I/R+Clitoria ternatea group to the I/R group, there was an increase in enzyme acvity (P>0.05) (FIG. 1). CAT enzyme acvity, one of the key anoxidant enzymes, was determined to signiﬁcantly decrease in the I/R group in comparison to control (P<0.05). In the I/R+Clitoria ternatea group, enzyme acvity showed a non-signiﬁcant increase in comparison to the I/R group (FIG. 1). The GSH-Px enzyme catalyzes the oxidaon of GSH to GSSG. GSH-Px acvity decreased signiﬁcantly in the I/R group in comparison to control due to the damage induced (P<0.001). Moreover, the enzyme acvity values of the Clitoria ternatea and controls were found to be similar (P>0.05). Enzyme acvity increased in the Clitoria ternatea (P<0.001) and I/R+Clitoria ternatea (P>0.05) groups in comparison to I/R (FIG. 1). FIGURE 1. CAT, SOD, GSH-Px (EU/mg protein), and MDA in the kidney ssues by groups. Signiﬁcant diﬀerences between the groups due to diﬀerent leers (a:p<0.001, b:p<0.01, c:p<0.05). CAT (Catalase), GSH-Px (Glutathione Peroxidase), SOD (Superoxide Dismutase), MDA (Malondialdehyde) Ischemic injuries in the kidneys occur in various condions such as sepsis, hydronephrosis, open renal surgeries, kidney transplantaon, shock, paral nephrectomy, and renal tumors [4 , 21 ,[22]. Consequently, many studies are now being carried out to migate or prevent renal I/R injury. These studies employed various approaches, ranging from altering I/R duraon to administering therapeuc agents either before or aﬅer the I/R process, to achieve eﬀecve results [23]. One such approach involves the use of anoxidants. It was reported that oxidave stress can be directly or indirectly migated by the external administraon of anoxidants when endogenous anoxidants are insuﬃcient. Accordingly, several agents were employed both experimentally and clinically to protect ssues and organs from I/R injury. These include pentoxifylline, melatonin, silymarin, astaxanthin, prostaglandin E1, vitamin E, L-carnine, quercen, vitamin C, and deoxycoformycin [4 , 24 , 25 , 26 , 27]. The ﬂower of Clitoria ternatea contains ﬂavonols like quercen, myricen, and kaempferol derivaves, as well as anthocyanins. Extracts of Clitoria ternatea were reported to exhibit various therapeuc potenals, including anoxidant and anmicrobial acvies. Moreover, it also has various pharmacological properes [28], 29]. Previous studies indicated that MDA levels increase due to kidney cell membrane damage during IR and that MDA measurement is a crical parameter for diagnosing I/R or subsequent renal failure [30 , 31]. Examining the acvity of Clitoria ternatea in treang cisplan-induced acute kidney injury, cisplan administraon was shown to elevate MDA levels, whereas groups treated with both cisplan and Clitoria ternatea exhibited signiﬁcant reducons in MDA levels [6]. In this study, the MDA level in I/R group was higher in comparison to control (P<0.05). Similar to the previous results [30 , 31], this increase conﬁrms the successful inducon of I/R injury and the associated rise in MDA levels. Furthermore, the reducon observed in MDA level following the administraon of Clitoria ternatea supports the results reported by Sai et al. [6]. The decrease in MDA level may be related to the anoxidant properes of ﬂavonols and anthocyanins present in Clitoria ternatea. The kidneys are among the organs aﬀected by I/R injury the most. Inﬂammatory cell inﬁltraon and oxidave stress are key mechanisms in the pathogenesis of I/R injury [32 ,[33]. It 3 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico has been reported that oxidants in ischemic ssue accelerate the inacvaon of CAT, GPx, and SOD, rendering cells more vulnerable to oxygen radicals generated during reperfusion Several I/R studies reported signiﬁcant reducons in SOD, CAT, and GPx acvies, accompanied by increased lipid peroxidaon levels [34 , 35]. In a study invesgang the inﬂuence of Clitoria ternatea ethanol extract on acetaminophen-induced toxicity in rats, signiﬁcant reducons (P<0.05) were observed in renal CAT, GPx, SOD, and GSH levels in the acetaminophen-administered group. However, the extract-treated group exhibited remarkable increases (P<0.05) in the levels of these enzymes [36]. Similarly, in this study, the I/R group exhibited decreases in SOD (p<0.01), CAT (P<0.05), and GSH-Px (P<0.001) acvies in comparison to the control, corroborang the results reported in previous studies [34]. Moreover, enzyme acvies in the I/R + Clitoria ternatea group were higher in comparison to the I/R group, but this increase was not signiﬁcant. This outcome may be related to the higher doses (250 and 500 mg/kg) of Clitoria ternatea used in other studies [36]. The analysis of serum sodium levels revealed an increase in the Clitoria ternatea group in comparison to control (P<0.01). Furthermore, a paral increase in sodium levels in the I/ R+Clitoria ternatea group in comparison to the I/R group was found to be signiﬁcant (P<0.05). No signiﬁcant diﬀerence was found between the other groups (FIG. 2). Serum potassium levels were signiﬁcantly increased in the I/R (P<0.01) and Clitoria ternatea (P<0.05) groups in comparison to the control (FIG. 2). BUN and serum creanine levels increased in the I/R (P<0.01) and I/R+Clitoria ternatea (P<0.05) groups in comparison to control. There was a reducon in BUN and creanine levels only in the Clitoria ternatea group (P<0.01) in comparison to the I/R group. Besides, BUN and creanine levels increased in the I/ R+Clitoria ternatea group (P<0.05) in comparison to the Clitoria ternatea group (FIG. 2). FIGURE 2. Na, K, BUN, Creanin levels in serum samples of groups. Signiﬁcant diﬀerences between the groups due to diﬀerent leers (b:p<0.01, c:p<0.05)..Na (Sodium), K (Potassi- um), BUN (Blood Urea Nitrogen) I/R was reported to lead to increased producon of ROS, loss of selecve permeability of cell membranes, and disrupon of cellular ion balance [37]. Loss of kidney funcon can result in disturbances in blood electrolyte levels and acid-base balance [38]. In a study on the eﬀect of hydralazine using a renal ischemia-reperfusion model in rats, it was reported that the diﬀerence in sodium (Na) levels between the control and I/R groups was not signiﬁcant, whereas sodium excreon via urine decreased in hydralazine-treated rats in comparison to the I/R group [39]. In the present study, while no diﬀerence was found in Na levels between the control and I/R groups (P>0.05), an increase was determined in the Clitoria ternatea (Clitoria t.) and I/R + Clitoria t. groups when compared to control (P<0.05). This increase may be due to reduced urinary excreon in the groups treated with Clitoria t. Renal I/R injury was reported to elevate serum potassium (K) levels [40 , 41]. I/R injury leads to direct damage to the cells responsible for potassium secreon in the distal tubules and collecng ducts [42]. As reported in previous studies [40 , 41], serum K levels in the I/R group were signiﬁcantly elevated (P<0.05) in the present study. Furthermore, K levels in the Clitoria t. and I/R + Clitoria t. groups exhibited an increase in comparison to the control (P<0.05) but a numerical decrease in comparison to the I/R group (P>0.05). Acute kidney injury (AKI) is characterized by the sudden loss of kidney funcon, accompanied by a rise in BUN and serum creanine levels. Condions that impede adequate renal perfusion, such as renal ischemia, are among the pre-renal causes of AKI [43]. It was reported that a rise in serum creanine levels is a beer indicator of AKI than tracking BUN levels [44]. In a study invesgang the eﬃcacy of calcium dobesilate administered prophylaccally for renal I/R injury, rats in the sham and I/R groups were observed without any treatment, whereas rats in the treatment group were administered 100 mg/kg daily dose of calcium dobesilate dissolved in 0.5 mL of drinking water for 10 days (d). It was concluded that there was no signiﬁcant diﬀerence in urea levels among the experimental groups [45]. A rise in serum creanine levels, a marker of renal (glomerular) dysfuncon, aﬅer I/R injury was reported to indicate funconal impairment in renal proximal tubule cells [46]. In a study on the acvity of silymarin on oxidave stress- induced damage in the kidneys of rats with renal I/R injury, serum creanine levels in the I/R group were higher than control (P<0.05), whereas there was no signiﬁcant diﬀerence between the I/R group and the groups given 50 mg/kg and 100 mg/kg silymarin (P>0.05) [26]. Sai et al. [6] examined the role of Clitoria ternatea in conjuncon with mesenchymal stem cells in the treatment of cisplan-induced AKI in rats and reported that this procedure could adjust creanine, uric acid, and urea levels. In the present study, consistent with results reported by other studies, no stascal signiﬁcance was found in BUN levels across all experimental groups. However, serum creanine levels in the I/R and I/R + Clitoria t. groups were signiﬁcantly higher compared to the control and Clitoria t. groups. Moreover, even though the creanine level in the I/R + Clitoria t. group was lower than in the I/R group, the diﬀerence was not signiﬁcant. Histopathologically, the kidney ssues in the control and Clitoria ternatea groups exhibited a normal appearance in the tubular epithelial cells and glomeruli (FIGS. 3a and 3c). Diﬀuse degeneraon, necrosis, and desquamaon in the tubular epithelium cells, tubular dilataon, expansion of Bowman’s space, and atrophic glomerulus with inﬂammaon were observed in the I/R group (FIG. 3b). Comparing the I/R and I/R+ Clitoria ternatea groups, it was determined that the ﬁndings observed in the group using Clitoria ternatea were much milder in terms of severity and incidence. Overall, the tubules and glomeruli appeared close to normal (FIG. 3d). 4 of 8

Clitoria ternatea on renal ischemia injury in rats / Melek et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 3. a) Control, normal histological appearance of kidney ssue (H&E, 40 µm). b) I/R group, glomerular atrophy (arrows), necrosis, and desquamaon in the tubular epithelium cells (arrowhead), (H&E, 40 µm). c) Clitoria ternatea group, normal histological appearance of the glomerulus of kidney ssue (arrows), (H&E, 40 µm). d) I/R+ Clitoria ternatea group, enlarged bowman spaces (arrow), degenerated tubular epithelium (arrowhead) (H&E, 40 µm) The histopathological scores indicang the degree of kidney damage in diﬀerent groups and demonstrang the protecve inﬂuence of Clitoria ternatea on I/R are presented in TABLE I, while the microscopic results showing the distribuon of severity across groups, ranging from mild to severe, are detailed in TABLE II. TABLE I. Degree of kidney damage by groups No Control I/R Clitoria ternatea I/R+Clitoria ternatea 1 0 3 0 1 2 0 2 0 1 3 0 4 0 0 4 0 3 0 1 5 0 2 0 1 6 0 3 0 0 X±SD 0.00±0.00 b 2.83±0.75 a 0.00±0.00 b 0.67±0.52 b TABLE II. Distribuon of microscopic kidney ssue results by groups demonstrang the protecve eﬀect of Clitoria ternatea against ischemia-reperfusion Parameters Control I/R Clitoria ternatea I/R+ Clitoria ternatea Tubular dilataon and desquamaon Mild Moderate Severe 0/6 * * * 6/6 * 3 3 0/6 * * * 4/6 3 1 * Degeneraon Mild Moderate Severe 1/6 1 * * 6/6 * 2 4 0/6 * * * 3/6 2 1 * Tubular epithelial necrosis Mild Moderate Severe 0/6 * * * 6/6 1 3 2 0/6 * * * 2/6 1 1 * Inﬂammatory cell inﬁltraon Mild Moderate Severe 0/6 * * * 6/6 3 2 1 0/6 * * * 1/6 1 * * *: none P<0.0001 In the immunohistochemical analysis of caspase-3 immunoreacvity in the kidney ssue, no staining was detected in the control or the Clitoria ternatea group (FIGS. 4a and 4c). Strong and diﬀuse posive immunoreacvity of caspase-3 was found in I/R group (FIG. 4b). The frequency and intensity of posive reacons in the I/R + Clitoria ternatea group were signiﬁcantly lower than in I/R group. This indicates that the number of posive cells decreased proporonally with the administraon of Clitoria ternatea (FIG. 4d). FIGURE 4. a) Control kidney ssue, Caspase -3 negavity (IHC, 40 µm). b) I/R group. Strong Caspase -3 posivity (IHC, 40 µm). c) Clitoria ternatea group, Caspase -3 negavity (IHC, 40 µm). d) I/R+ Clitoria ternatea group, poor Caspase -3 posivity compared to I/R group (IHC, 40 µm) 5 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Ischemia-reperfusion injuries in the kidneys primarily occurs in the tubules due to tubular necrosis caused by ischemia [47]. Acute kidney failure may develop following ischemia, characterized by a reducon in glomerular ﬁltraon rate, edema in tubular epithelial cells, acute tubular necrosis, inﬁltraon of inﬂammatory cells, congeson, and increased vascular resistance in the kidneys [48]. Reperfusion of ischemic ssue is necessary for cellular regeneraon and removal of toxic metabolites. However, reperfusion can cause more severe damage than the inial ischemic injury. I/R injury triggers a series of pathological events, including the producon of ROS, apoptosis, inﬁltraon of inﬂammatory cells, necrosis, and release of acve mediators that cause ssue damage [49]. Histopathological studies using roune hematoxylin and eosin staining revealed that I/R-induced renal damage includes degeneraon and necrosis in the tubules and glomeruli, hemorrhage, tubuler precipitates, endothelial and epithelial cell damage, vascular damage, and inﬂammatory cell inﬁltraon. These results vary depending on the cause, duraon, and severity of I/R injury. Furthermore, the extent and impact of ischemia- reperfusion injury are also examined immunohistochemically with a focus on apoptosis [50]. In their study involving rats, where 1-hour ischemia followed by 1-hour reperfusion was applied to the leﬅ renal artery, Ozturk et al. [50] reported signiﬁcant renal lesions in the I/R group, including tubular dilaon, vascular congeson, marked necrosis, and intratubular cast accumulaon. Mousavi [21], who applied 1-h ischemia followed by 24-h reperfusion, addionally reported inﬂammaon and cellular vacuolizaon in the I/R group, extending the results reported by Ozturk et al. [50]. Another study reported ﬂaened tubular epithelial cells, marked necrosis, vacuolizaon, and dilaon, along with diﬀuse tubuler precipitates in tubular lumens and congeson in glomeruli and vascular structures. These results were noted to be consistent with previous studies [22]. In this study, consistent with the results menoned above, degeneraon and necrosis were predominantly observed in the tubules and glomeruli, while vascular damage and inﬂammaon were mild. Melatonin, as studied by Sahna et al. [25] and Rodríguez- Reynoso et al. [51], signiﬁcantly reduced tubuler precipitates, tubular epithelial degeneraon, and necrosis in eﬀorts to protect kidneys from I/R injury. Similarly, Sener et al. [52] highlighted that melatonin applicaon notably reduced inﬂammatory cell inﬁltraon and other renal lesions related with I/R injury. In their experimental kidney ischemia study, Demirtaş et al. [26] reported a signiﬁcant histopathological improvement in a group treated with pentoxifylline. Şentürk et al. [27] invesgated the anoxidant properes of silymarin to determine whether exogenous anoxidant compounds have protecve acvies against renal I/R injury. Their results emphasized that silymarin, at doses of 50 and 100 mg/kg, yielded results comparable to the control and eﬀecvely inhibited ROS eﬀects. However, studies carried out by Aktoz et al. [53] and Unal et al. [24], which evaluated the protecve eﬀects of exogenous anoxidant vitamins like vitamin E and combined vitamin C+E against I/R injury, noted that while some degree of renal protecon was observed, these eﬀects were not signiﬁcant. The results of histopathological evaluaon of renal ssues were consistent with previous studies, except for those carried out by Aktoz et al. [53] and Unal et al. [24]. CONCLUSION Reviewing the literature, it was determined that the inﬂuence of Clitoria ternatea extract on experimental renal I/R injury in rats has not been previously studied. To our knowledge, this is the ﬁrst experimental study invesgang the prevenve eﬀects of Clitoria ternatea extract in renal I/R injury. This study demonstrated that Clitoria ternatea administraon showed a protecve eﬀect against renal I/R injury in rats. A decrease in oxidave stress markers, histopathological and immunohistochemical improvement in renal ssue and a signiﬁcant decrease in serum creanine and BUN levels were observed aﬅer administraon. These ﬁndings suggest that Clitoria ternatea may protect the kidney ssue against I/R injury thanks to its anoxidant and an-inlammatory properes. 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