Revealing Sarcophyton extract's alleviating potential against gentamicin-induced renal and testicular toxicity in rat model

Abstract

Background

Gentamicin (GEN) is a potent antibiotic known for inducing oxidative stress and causing adverse effects in the kidneys and testes. Sarcophyton species possess significant antioxidant and anti-inflammatory properties, making them potential candidates for addressing oxidative stress and inflammation-related gentamicin toxicity. The study aims to explore the antioxidant and anti-inflammatory properties of Sarcophyton acutum methanol extract (SAME) to counteract gentamicin effects.

Methods

Sarcophyton acutum were collected and macerated with methanol, followed by phytochemical analysis of extract. Twenty-four adult male albino rats were separated into four equal groups: Control, SAME-treated (200 mg/kg/day), GEN-treated (100 mg/kg/day), and GEN + SAME-treated rats. Various parameters, including body weight, relative kidney and testes weight, differential white blood cell count, blood urea, creatinine, luteinizing hormone, testosterone, total antioxidant capacity, myeloperoxidase activity, and histopathological changes in kidney and testes tissue, were analyzed.

Results

Phytochemical analysis revealed SAME's composition, including alkaloids, saponins, phenolics, flavonoids, and tannins, with an average total antioxidant capacity of 10.503 ± 0.632 mg AAE/g extract. GEN treatment resulted in altered body and organ weights, changes in white blood cell percentages, elevated urea and creatinine levels, reduced luteinizing hormone and testosterone, decreased renal and testicular tissue total antioxidant capacity, and increased myeloperoxidase levels in both tissues. However, the administration of SAME with GEN attenuated these effects, restoring parameters closer to control levels. Histological evaluation showed that GEN treatment induced significant renal tissue damage characterized by enlarged renal corpuscles, glomerular tuft hypertrophy, tubular dilation, and necrosis, interstitial leukocyte infiltration, and tubular hyaline cast formation. Co-administration of SAME with GEN mitigated these effects, reducing renal corpuscle swelling, tubular vacuolization, and hypertrophy and preventing hyaline deposition and leukocyte infiltration. In testicular tissue, GEN injection caused seminiferous tubule atrophy, decreased spermatogenic layer thickness, and interstitial expansion and degeneration. However, SAME administration with GEN preserved normal tubular size and spermatogenic layer thickness, reduced vacuolization, and epithelial necrosis, and maintained spermatogenesis.

Conclusion

Sarcophyton acutum methanol extract demonstrates promising protective effects against gentamicin-induced renal and testicular toxicity in rats, signifying its potential as a therapeutic agent to mitigate antibiotic-induced oxidative damage in vital organs.

Background

Antibiotics attract attention as medical marvels for their pivotal role in combatting bacterial infections, are not without their complexities. The ubiquitous use of gentamicin (GEN), a potent aminoglycoside antibiotic, in clinical settings has undeniably revolutionized the treatment of severe bacterial infections (Hamad et al., 2023). However, its widespread application comes at a cost, as the drug is notorious for inducing adverse effects, particularly in the kidneys (Abdelrahman & Abdelmageed, 2020) and testes (Taha et al., 2022).

Gentamicin-induced nephrotoxicity is explained as a multifaceted cascade involving oxidative stress, inflammatory responses, and functional impairment of renal tissues (Nadeem et al., 2023). Concurrently, the impact of gentamicin on the testes is characterized by disruptions in spermatogenesis and alterations in reproductive parameters due to oxidative stress (El-Sayed et al., 2022). These effects underscore the need for novel therapeutic strategies to mitigate the collateral oxidative damage inflicted by this potent antibiotic. Innovative approaches to minimize harmful effects focus on marine organism's rich pharmacological reservoirs, with vast potential for new drug discoveries.

Marine natural compounds are abundant in exogenous antioxidants, effectively mitigating cellular oxidative stress and providing oxidative stress-suppressing properties (Wang et al., 2021). Oceans contain natural products and active components with biological activity, potentially aiding in developing novel drugs for treating human diseases, particularly sponges and corals (Byju et al., 2015; Tanod et al., 2019a). Cnidaria, a diverse group of marine invertebrates, comprises over 11,000 species, primarily found in aquatic environments, including hydroids, jellyfish, anemones, and corals (Rocha et al., 2015). In the vast spectrum of marine organisms, soft corals stand out for their ecological significance and their rich repertoire of bioactive compounds, demonstrating their adaptability to diverse marine environments (Tanod et al., 2019a).

Soft corals, classified within the family Alcyoniidae and order Alcyonacea, have long been revered for their ecological importance and intriguing biochemistry (Tammam et al., 2023). Within this taxonomy, Sarcophyton exhibits a unique morphology—encrusting or lobate forms and vibrant colors contribute to the aesthetic diversity of coral reef ecosystems (Riyadi et al., 2019). Beyond their ecological role, Sarcophyton have been recognized for their medicinal potential due to their richness in diverse secondary metabolites, including terpenoids, flavonoids, steroids, and alkaloids (Fouad et al., 2021; Tammam et al., 2023). These compounds have demonstrated anti-inflammatory, immunomodulatory, and antimicrobial activities, anticancer, neuroprotective, and hepatoprotective (Tammam et al., 2023), and noteworthy antioxidant properties (Tanod et al., 2019a). Previous studies have reported the anti-inflammatory, antiviral, anticancer, and hepatoprotective properties of Sarcophyton crude extract (Riyadi et al., 2019; Zidan et al., 2016). These characters prompt the investigation into their potential to counteract the toxic effects of gentamicin.

The study will involve particular experimentation, including extract phytochemical analysis, biochemical assays, and histopathological examinations, seeking to unravel the intricate mechanisms underpinning the putative ameliorative effects of Sarcophyton extract on antibiotic-induced renal and testicular toxicity.

Methods

Collection and identification of Sarcophyton specimens

Soft coral specimens of the genus Sarcophyton were obtained in front of the National Institute of Oceanography and Fisheries at Hurghada province on the Egyptian Red Sea Coast. Soft coral Sarcophyton spp. was collected in January 2022 at 3–5 m depth using SCUBA, transported to the lab in ice bags, and stored at − 20 °C until analysis. Taxonomic experts carried out proper identification, ensuring the samples' authenticity. The collected samples were classified as Sarcophyton acutum, and the coral was stored in a freezer ( − 20 °C) until extraction time.

Preparation of Sarcophyton acutum methanol extract (SAME) by maceration

The frozen coral (50 g) was divided into smaller segments and ground into a fine powder using a mortar and pestle. Methanol extraction was performed by macerating the powdered samples in methanol (1:2 w/v) for five days at room temperature with occasional shaking (Zidan et al., 2016). The SAME extract was filtered, and the solvent was vaporized using a rotary evaporator to yield 3.14 ± 0.12 g of concentrated extract. The resulting methanol extract was stored at  − 20 °C until further analysis.

Analysis of the chemical composition of Sarcophyton acutum

Estimation of carbohydrate content

Anthrone method (Hedge et al., 1962) was applied to determine carbohydrate contents. As the standard, Glucose (Glc) was used to calculate the carbohydrate content in mg GlcE/g extract.

Estimation of protein content

The folin phenol reagent measured extract protein (Kingsley, 1939). As the standard, bovine serum albumin (BSA) was used to calculate the protein content in mg BSAE/g extract.

Estimation of total lipids content

The lipid content was determined with sulphuric acid-phosphoric acid-vanillin reagent following the Zollner and Kirsch (1962) method. As the standard, triglyceride (TG) was used to calculate the lipid content in mg TGE/g extract.

Estimation of total phenolic and flavonoid content

The total phenolic was determined using the Folin-Ciocalteu reagent, and the flavonoid content was determined using the aluminum chloride colorimetric method (Zouhri et al., 2023). Gallic acid (GA) and quercetin (Q) were used as standards, and the results were expressed as gallic acid equivalents (mg GAE/g extract) and quercetin equivalents (mg QE/g extract), respectively.

Estimation of total tannin contents

The Folin Ciocalteu assay determined the total tannin content in the extract (Mohammed & Manan, 2015). As the standard, gallic acid (GA) was used to calculate the tannin content of the extract in mg GAE /g extract.

Estimation of total alkaloid contents

The alkaline precipitation gravimetric method was used to determine alkaloid content, and data was calculated and expressed as mg/g extract (Harbone, 1973).

Estimation of saponin content

Saponin content was determined using vanillin and sulfuric acid reagents (Ali et al., 2023). As the standard, sapogenin (SE) was used to calculate the saponin content of the extract in mg SE/g extract.

Estimation of total antioxidant capacity

Total antioxidant capacity was determined using the phosphomolybdate method (Mabini & Barbosa, 2018). As the standard, Ascorbic acid (AA) was used to calculate the total antioxidant capacity of the extract in mg AAE/g extract.

Animals and treatment

Twenty-four adult male albino rats weighing 115.4 ± 4.2 g were housed in clean polypropylene cages (6 rats /cage). They were kept under ordinary husbandry circumstances with a 12-h light/dark cycle and a 22–25 °C temperature. The rats had unrestricted access to food and water throughout the experiment duration.

After one week of adaptation, the rats were alienated into four groups six rats/group:

• 1. Control group (G1): Rats were administered 0.5 ml of water orally and injected with 0.5 ml of saline daily.

• 2. SAME group (G2): Rats received Sarcophyton acutum methanol extract (SAME) orally at a dose of 200 mg/kg/day dissolved in water for seven days (Zidan et al., 2016).

• 3. GEN group (G3): Rats were treated with gentamicin (80 mg ampoules, Memphis Pharm. & Chemical Ind., Cairo, Egypt) intraperitoneally (i.p) at a dose of 100 mg/kg/day diluted in saline for seven consecutive days (Abdelrahman & Abdelmageed, 2020).

• 4. GEN + SAME group (G4): Rats received gentamicin (100 mg/kg/day, i.p) along with SAME (200 mg/kg/day, orally) for seven days.

Animal sacrifice and tissue collection

On day 8 of the experiment, rats were anesthetized (60 mg sodium pentobarbital/kg, i.p.). The blood samples were collected using thin capillary glass tubes from the retro-orbital plexus into EDTA test tubes. Following blood collection, rats were sacrificed, and kidneys and testes were harvested. The organs were washed with 0.9% NaCl solution and weighed to calculate the renal realtine and testicular realtine weights. The organ weight-to-body weight ratio was determined using the formula: (Right organ weight + Left organ weight) / Bodyweight × 100. The harvested organs were either homogenized in buffer solution (weight-to-volume ratio of 10%) for further biochemical analysis or fixed in 10% formalin for histopathological analysis.

Determination of differential white blood cell

A drop of blood from collected blood samples was smeared onto a glass slide and stained with Wright-Giemsa. Differential counts were conducted by counting 200 cells/slide under a light microscope (Saadat et al., 2019).

Determination of kidney function markers

Blood urea levels were assessed using the urease colorimetric method, and serum creatinine levels were assayed using the Buffered Kinetic Jaffé reaction. Commercial kits (Spectrum Diagnostics, Egyptian Company for Biotechnology Cat. no. 318 001 and Cat. no. 234 001 respectively) were utilized.

Determination of testes function markers

Following kit protocols, the competitive inhibition enzyme immunoassay technique was applied to determine testosterone and luteinizing hormone (LH) levels. ELISA kits supplied by CUSABIO Biotech, (Cat. No. CSB-E05100r) and (Cat. No. CSB-E12654r), respectively, were utilized.

Determination of total antioxidant capacity and myeloperoxidase in tissue homogenate

Total antioxidant capacity (TAC) in renal and testicular tissues was determined using kits from Biodiagnostic (Giza, Egypt; CAT. no. TA 25 13). Myeloperoxidase (MPO) activities in tissue homogenates were also assessed using an ELISA kit from MyBioSource (San Diego, CA, USA, Cat.No: MBS046496) following the manufacturer's instructions.

Histopathological evaluation

Kidney and testes tissues were fixed for 24 h in 10% neutral formalin, ethanol dehydrated, and cleared in xylene, followed by Paraplast^® impregnation and embedding (Bancroft & Layton, 2019). Those embedded tissues were sectioned and stained with hematoxylin and eosin (H&E). Sections were examined and photographed using an Olympus CX41 microscope (Olympus, Tokyo, Japan).

Histo-morphometric analysis

The following morphometric parameters were measured in all groups using Fuji ImageJ software. Set the measuring unit using the scale tool to specify the unit of measurement (µm) and distance.

• 1. Freehand tool was used to determine areas of renal corpuscles, glomeruli, proximal and distal renal tubules, collecting tubules, seminiferous tubules, and data expressed in µm².

• 2. A straight line tool was used to determine the diameter of seminiferous tubules and seminiferous epithelial lining thickness and data expressed in µm.

• 3. Area of bowman’s space (µm²) = $\text{Renal corpuscles area }-\text{ Glomeruli area}$

• 4. Total interstitial tissue area (µm²) = $\text{Total testis tissue area }-Total\text{ seminiferous tubule area}$

• 5. Bergmann-Kliesch score marker for spermatogenesis (%) = no seminiferous tubules with sperms no longer attached to spermatogenic epithelium divided by the total number of seminiferous tubules(Sziva et al., 2022).

Statistical analysis

Differences between obtained values (mean ± standard deviation) were analyzed by a one-way variance test followed by the Tukey–Kramer multiple comparison test using Graph Pad Prism version 5.01 (Graph pad software, INC, La Jolla, CA, United States).

Results

Quantitative phytochemical analysis of SAME

The quantitative study of primary and secondary metabolites reveals various chemical constituents in the SAME (Table 1). Regarding primary metabolites, protein has the highest concentration, followed by carbohydrates and lipids. However, the alkaloids represent the highest secondary metabolites in the SAME, followed by saponins, phenolics, flavonoids, and tannins. The obtained data revealed that SAME displayed a remarkable antioxidant capacity with an average total antioxidant capability of 10.503 ± 0.632 mg AAE/g extract.

Table 1 Primary and secondary metabolites and total antioxidant capacity

Effect of GEN and SAME treatment on body weight and kidney and testes weight

Table 2 depicts alterations in body weight and relative organ weight. The SAME-treated (G2) group showed body and organ weights comparable to the control (G1) rats. GEN injection (G3) led to reduced body weight, increased kidney weight, and decreased testes weight compared to both control (G1) and SAME-treated (G2) rats. Conversely, administering SAME with GEN (G4) resulted in increased body weight, reduced kidney weight, and increased testes weight compared to GEN-treated (G3) rats. This group (G4) also exhibited slight changes in body weight and relative organ weight compared to controls (G1) and SAME-treated (G2) rats.

Table 2 Initial and final body weight, Kidney weight, and testis weight in different experimental groups

Effect of GEN and SAME treatment on differential white blood cell count

Table 3 displays the differential white blood cell percentages. SAME (G2) treatment caused minimal changes compared to the control rats (G1) in differential white blood cell count. GEN injection (G3) significantly increased lymphocyte percentage and decreased neutrophil percentage in rats compared to controls (G1) and SAME-treated (G2) rats while showing no significant increase in monocytes and eosinophils. Administering SAME with GEN (G4) resulted in insignificant changes in lymphocytes, monocytes, and eosinophils percentages, with a notable rise in neutrophil percentage compared to GEN-treated rats (G3). However, G4 showed no significant changes compared to controls (G1) and SAME-treated (G2) rats.

Table 3 White blood cells % in different experimental groups

Effects of GEN and SAME treatment on renal and testicular function markers in the blood of rats

Urea and creatinine levels, indicators of renal function, were assessed, as shown in Table 4. SAME treatment (G2) had no significant impact on urea and creatinine levels compared to controls (G1). GEN injection (G3) significantly increased both markers compared to controls (G1) and SAME-treated (G2) rats. Administering SAME with GEN (G4) significantly reduced urea and creatinine levels compared to GEN-treated rats (G3), although these levels remained higher than controls (G1) and SAME-treated rats (G2).

Table 4 Urea, creatinine, luteinizing hormone and testosterone levels in different experimental groups

Plasma luteinizing hormone (LH) and testosterone levels, indicators of testicular function, were examined (Table 4). SAME-treated rats (G2) exhibited no significant changes compared to controls (G1). GEN injection (G3) significantly decreased both markers compared to controls (G1) and SAME-treated rats (G2). Administering SAME with GEN (G4) significantly increased LH and slightly increased testosterone compared to GEN-treated (G3) rats. However, G4 also showed decreased significantly LH levels and slightly reduced testosterone levels compared to controls (G1) and SAME-treated rats (G2).

Effects of GEN and SAME treatment on renal and testicular total antioxidant capacity and myeloperoxidase levels

Table 5 presents the analysis of total antioxidant capacity (TAC) and myeloperoxidase (MPO) levels. The administration of SAME (G2) exhibited no significant impact on TAC and MPO levels compared to the control of rats (G1) in renal and testicular tissue. In contrast, GEN injection (G3) significantly reduced TAC levels and increased MPO levels compared to controls (G1) and SAME-treated rats (G2) in both tissues. Administering SAME with GEN (G4) significantly elevated TAC levels and reduced MPO levels compared to GEN-treated rats (G3). However, this group demonstrated significantly lower TAC levels and insignificantly higher MPO levels than controls (G1) and SAME-treated rats (G2).

Table 5 Total antioxidant capacity (TAC) and myeloperoxidase (MPO) levels kidney and testes tissues in different experimental groups

Histological and morphometric evaluation of renal tissue

The examination of renal tissue sections from control (G1) and SAME-treated rats (G2) revealed intact nephron structures characterized by regular in shape and size renal corpuscles, proximal and distal convoluted tubules, and collecting tubules (Fig. 1). The renal corpuscle has a central glomerulus tuft surrounded by Bowman's capsule, while renal tubules include proximal convoluted tubules, distal convoluted tubules, and collecting tubules. One epithelial cell layer surrounds a central lumen in these tubules (Fig. 2A and B).

Fig. 1

Morphometric analysis of renal tissue in different experimental groups. (A) A low magnification light micrograph of renal tissue of rats showing renal corpuscle (RC) and collecting tubules (CT) (H&E – X100). Histograms (B-G) showing the area of renal corpuscle (B), glomerulus tuft area (C), bowman’s space area (D), proximal convoluted tubule area (E), distal convoluted tubule area (F) and collecting tubules area (G) in different experimental groups. Notice a significant increase in the renal corpuscle area, glomeruli tufts, bowman’s space, and renal tubules in the GEN-treated group compared to the control and SAME-treated groups. SAME, Sarcophyton acutum methanol extract; GEN, Gentamicin. Anova results are represented as means ± SD, *p < 0.05, **P < 0.01 and ***P < 0.001. while # represents significant difference compared to GEN + SAME-treated groups (^#p < 0.05, ^##p < 0.01, ^###p < 0.001)

Fig. 2

Ameliorative Effect of SAME on GEN-induced kidney injury in rats (H&E X400). Control (A) and SAME-treated (B) kidney sections showing normal kidney structure of glomerulus (G), surrounded by Bowman’s space (BS) and Bowman's capsule (BC) and renal tubules (PCT: Proximal convoluted tubules (PCT), distal convoluted tubule (DCT) and Collecting tubules (CT). (C–D) GEN-treated kidney sections showing glomerulus collapse and Bowman's space dilation (star), renal tubules hypertrophic degeneration (head arrow), severe tubular necrosis (TN), formation of hyaline cast (HC), interstitial lymphocytes infiltration (LI). (E) GEN + SAME treated renal section showing normal glomerulus (G) surrounded by Bowman’s space (BS), tubular hypertrophic degeneration (Head arrows), cytoplasmic vacuolization (V), regular Proximal convoluted tubules (PCT), and distal convoluted tubule (DCT). SAME, Sarcophyton acutum methanol extract; GEN, Gentamicin

In contrast, renal tissue from GEN-treated rats (G3) exhibited notable tissue damage, with significant increases in the size of renal corpuscles, hypertrophy of glomeruli tufts, dilation of Bowman's space, and expansion of tubules compared to control and SAME-treated rats (Fig. 1). Additionally, renal corpuscles showed collapsed glomeruli tufts, while renal tubules displayed severe epithelial lining hypertrophy and necrosis (Fig. 2C and D). GEN injection (G3) also led to a significant increase in interstitial leucocyte infiltration and the formation of tubular hyaline casts. Due to tubular rupture, some hyaline casts were also identified as intermixed with inflammatory cells in the interstitium (Fig. 2D).

Administering SAME with GEN (G4) ameliorates the damaging effects of GEN, as evidenced by mild swelling of renal corpuscles and insignificant expansion of renal tubules (Fig. 1). Furthermore, there was a reduction in tubular epithelial vacuolization and hypertrophy, along with the prevention of tubular hyaline deposition and leucocyte infiltration (Fig. 2E).

Histological and morphometric evaluation of testes tissue

Testicular tissue sections from both control (G1) and SAME-treated rats (G2) exhibited predominantly seminiferous tubules surrounded by relatively thin interstitial tissue (Fig. 3A). These tubules were lined by spermatogenic epithelia, with scattered basal Sertoli cells among spermatogonia. The presence of spermatocytes, spermatids, and spermatozoa verified the normal spermatogenesis. Leydig cells were observed scattered within the interstitial tissue (Fig. 4A and B).

Fig. 3

Morphometric analysis of testicular tissue in different experimental groups. (A) A low magnification light micrograph of testicular tissue of rats showing seminiferous tubules (ST), interstitial tissue (IS), and seminiferous epithelial layer (Double head arrows) (H&E – X100). Histograms (B-G) showing seminiferous tubular diameter (B), seminiferous epithelial layer thickness (C), Bergmann − Kliesch score percentage (D), and interstitial tissue area (E). Notice a significant reduction in seminiferous tubular diameter, seminiferous epithelial layer thickness, Bergmann − Kliesch score percentage, and a significant increase in interstitial tissue area compared to the control and SAME-treated groups.Abbreviations: SAME, Sarcophyton acutum methanol extract; GEN, Gentamicin. Anova results are represented as means ± SD, *p < 0.05, **P < 0.01 & ***P < 0.001. while ^# represents significant difference compared to GEN + SAME-treated groups (^###p < 0.001)

Fig. 4

Ameliorative effect of SAME on GEN-induced testicular injury in rats (H&E - X400). Control (A) and SAME-treated (B) testicular sections showing the normal appearance of seminiferous tubules surrounded by basal lamina (BL) and containing spermatogonia (Sg), primary spermatocytes (Ps), spermatids (St), spermatozoa (Sz), Sertoli cells (Se), Leydig cells (LC). (C) GEN-treated testicular section showing seminiferous tubule shrinkage, irregular outline (Head arrow), disturbed spermatogenic layers and giant vacuoles (arrow), with necrotic eosinophilic debris in the lumen (star). (D) GEN + SAME treated testicular section showing irregular outline (Head arrow), complete spermatogenic layers, sperms in the lumen and few necrotic cells (star). SAME, Sarcophyton acutum methanol extract; GEN, Gentamicin

In contrast, GEN injection (G3) induced significant testicular damage, characterized by seminiferous tubule atrophy with a notable reduction in tubule size, decreased spermatogenic layer thickness, and low Bergmann − Kliesch score percentage (Fig. 3). Severe distortion of the spermatogenic lining with a corrugated basal lamina and spermatogenic epithelial vacuolization degeneration were observed. This alteration, associated with incomplete spermatogenesis, absence of spermatozoa in degenerated tubular lumens, and accumulation of necrotic eosinophilic debris, was observed (Fig. 4C). Interstitial expansion and degeneration of interstitial cells were also evident (Fig. 4).

Administering SAME with GEN (G4) ameliorates the GEN testicular damage. This group (G4) showed moderate damage to seminiferous tubules, characterized by normal tubular size and spermatogenic layer, with a significantly higher Bergmann − Kliesch score percentage (Fig. 3). Co-administration of SAME with GEN (G4) prevented inhibition of spermatogenesis, evidenced less spermatogenic lining vacuolization, reduced spermatogenic epithelial necrosis, and presence of luminal spermatozoa (Fig. 4D).

Discussion

Gentamicin-induced nephrotoxicity and testicular damage are well-documented, with oxidative stress, inflammation, and cellular dysfunction playing key roles (Aly, 2019; El-Sayed et al., 2022; Mahmoud et al., 2021). This research focuses on the potential of Sarcophyton acutum methanol extract (SAME) in mitigating these toxic effects, leveraging the extract's antioxidant and anti-inflammatory properties(Fouad et al., 2021; Tanod et al., 2019b). Gentamicin's nephrotoxic effects are well-documented, resulting from its accumulation in renal tubules, leading to cellular injury and apoptosis. This accumulation can lead to acute tubular necrosis and inflammation, a serious condition that can result in kidney failure if not promptly treated (Erseckin et al., 2022; Laaroussi et al., 2021; Nadeem et al., 2023). In rats treated with gentamicin (G3), severe kidney destruction, tubule dilation, cellular swelling, and necrosis were observed, accompanied by a decline in total antioxidant capacity (TAC) renal activity. These changes are linked to gentamicin-induced oxidative damage, which decreases renal TAC levels and increases membrane peroxidation, triggering hypertrophy and necrosis of tubular epithelial cells (Gomaa et al., 2018).

Renal tubule dilation can occur due to various factors, including toxic injury, urinary tract obstruction, inflammation, fibrosis, and nephropathy (Hanif et al., 2024; Mishra et al., 2021). In this study, gentamicin-induced oxidative damage caused hypertrophy, necrosis, and tubular dilation, similar to findings by Mishra et al. (2021). This tubular dilation promotes hyaline cast deposition in renal tubules. Protein precipitation and cast formation in dilated tubules lead to obstructing tubules, and tubular rupture further impairs renal function (Ali et al., 2021; Hanif et al., 2024).

This study recorded a significant elevation in urea and creatinine levels in the GEN-treated group (G3), marking the retention of nitrogenous wastes. Two mechanisms may contribute to this retention: Reduced glomerular filtration and ductal obstruction. Gentamicin oxidative damage can reduce antioxidant defense, activating inflammatory mechanisms and leading to mesenchymal cell contraction, thus reducing the glomerular infiltration rate (Monfared et al., 2023).

Damage to glomeruli and renal tubules during gentamicin-induced tubular necrosis causes ductal obstruction by necrotic cells, impaired tubular reabsorption activity leading to retention of the nitrogenous substances and subsequently increased urea and creatinine in the blood (Abdeen et al., 2021; Mirazi et al., 2022). In agreement with previous studies, gentamicin injection caused reduced glomerular filtration rate, tubular hydropic degeneration and necrosis, and increased serum urea and creatinine levels (Bai et al., 2023; Erseckin et al., 2022; Mahmoud et al., 2021).

The tubular obstruction and rupture caused by cast formation can elicit an interstitial inflammatory response (Dvanajscak et al., 2020). Damaged renal cells release cytokines and chemokines, causing macrophage and lymphocyte infiltration, which contribute to renal damage by releasing pro-inflammatory and pro-fibrotic cytokines (Araújo et al., 2020; Black et al., 2019).

This study found hyaline casts and leucocyte infiltration in GEN-treated rats, indicating interstitial nephritis and ongoing kidney injury, characterized by decreased neutrophils and increased eosinophils and lymphocytes in the blood. Neutrophil migration to inflamed tissues temporarily decreases blood neutrophil counts (Rosales, 2018). In acute interstitial nephritis, eosinophils and inflammatory cell infiltration are observed (Black et al., 2019).

Leucocyte migration can increase MPO levels, marking inflammatory tissue (Abdel-Hamid et al., 2018). In this study, leucocyte infiltration further exacerbates renal inflammation and damage. Infiltrated inflammatory cells release myeloperoxidase (MPO), contributing to further damage to renal tissue by producing more reactive oxygen species (ROS). Inflammatory responses triggered by GEN-induced renal tissue damage exacerbate renal injury, with increased MPO activity and leukocyte infiltration (Bai et al., 2023), including neutrophils, eosinophils, basophils, and monocytes in the interstitium (Gupta et al., 2022).

Moreover, this study found that gentamicin-induced nephrotoxicity was associated with decreased antioxidant activity and increased renal inflammatory marker MPO, leading to glomeruli dysfunction, tubular necrosis, obstruction, and leucocyte infiltration. Previously, several studies reported that overproduced ROS caused cellular injury and dysfunction, reduced antioxidant defense (↑oxidation markers,↓ antioxidant enzymes, and ↓TAC), and activated inflammatory markers (↑MPO), leading to tubular necrosis and leukocyte infiltration in gentamicin–treated rats (Monfared et al., 2023; Taher et al., 2021).

Additionally, gentamicin injection caused a drop in body weight and increased kidney weight, consistent with previous studies (Aurori et al., 2023; Nadeem et al., 2023). Mishra et al. (2021) reported that the decline in body weight due to gentamicin injection might be linked to renal tubule damage, causing dehydration and weight loss, or improved catabolism and reduced food intake, while an increase in kidney weight might be a result of tubular cells inflammation and edema.

Interestingly, the detrimental effects of gentamicin extend beyond the kidneys to affect the testes. Testicular cells, under oxidative stress, secrete inflammatory cytokines, which trigger an inflammatory response leading to spermatogenic cells apoptosis and damage to the structure of the blood–testicle barrier (Ok et al., 2020).

In this study, total antioxidant capacity (TAC) levels were decreased, and MPO activity increased in testicular tissues following gentamicin exposure. These results indicate oxidative stress and inflammatory cells infiltration in gentamicin-treated rats, as reported by Mohamed et al. (2021). Increased testicular MPO activity promotes tissue injury, reduces antioxidant enzymes, and impairs testicular function (Abarikwu et al., 2022; Mega et al., 2022).

The study highlights that reduced total antioxidant capacity (TAC) due to gentamicin treatment leads to oxidative damage and impaired spermatogenesis, potentially resulting in testicular seminiferous tubule atrophy and widening of the interstitial space. Similarly, gentamycin-induced testicular cell damage may be due to oxidative stress, which peroxides membrane lipids, altering sperm motility and reducing the total antioxidant capacity of testicular tissue (Abdelhaffez et al., 2019; El-Sayed et al., 2022).

In this study, gentamicin-induced interstitial tissue damage, MPO upregulation, and TAC reduction deteriorate testicular injury, impair Leydig cell function, and reduce testosterone production. Previous studies show that low testosterone in gentamicin-treated rat’s results from Leydig cell damage, free radical generation, and antioxidant reduction, leading to germ cell detachment, apoptosis, spermatogenesis failure, and infertility (Abdelhaffez et al., 2019; Aly, 2019; Aly & Hassan, 2018).

This study confirmed that gentamicin impaired spermatogenesis by reducing seminiferous tubule epithelia and causing the absence of matured sperms in most seminiferous tubule lumens. This disruption is linked to reduced testosterone and gentamicin-induced oxidative damage to spermatogenic cells, leading to degeneration of seminiferous tubules and sperm apoptosis (El-Sayed et al., 2022; Hamad et al., 2023; Taha et al., 2022).

In this study, GEN-treated rats showed atrophic tubules with corrugated outlines, widened interstitial spaces, and reduced testis's weight. The enlarged interstitial space may result from myoid cell contraction, seminiferous tubule shrinkage, or reduced spermatogonia (Hashim et al., 2022). The decline in testosterone levels causes spermatogenesis disruption, seminiferous tubule atrophy, and premature sperm detachment, leading to a significant decrease in testes weight (Aly, 2019; El-Sayed et al., 2022).

Natural antioxidants like phenols, flavonoids, and tannins are crucial for human defense and disease prevention by preventing or delaying oxidation by free radicals (Mohammed & Manan, 2015). This study confirms SAME's antioxidant and anti-inflammatory activity by alkaloids, saponins, phenolics, flavonoids, and tannins. Previous studies reported that Sarcophyton extracts are rich in antioxidant and anti-inflammatory natural products, including phenols, saponins, tannins, steroids, triterpenoids, alkaloids, and flavonoids (Fouad et al., 2021; Tanod et al., 2019b).

The DPPH test assesses the antioxidant activity of plant extracts containing polyphenol components, which act as hydrogen atom donors and free radical capturing agents (Motto et al., 2021). This study confirmed SAME antioxidant capacity by reduction in DPPH molecules. Abdeen et al. (2021) reported that the high antioxidant activity of the date extract is related to the existence of total phenols, flavonoids, and scavenging of DPPH radicals' assay.

As evidenced in the present study, SAME extract can protect the cells against gentamicin oxidative damage by significantly increasing TAC levels in kidney and testes tissues compared to GEN-treated rats. These impacts might relate to the synergistic effect of steroids, phenolic, flavonoids, tannin, alkaloids and saponins compounds of SAME, which effectively act as hydrogen donors, rendering them potent antioxidants. Similarly, soft coral extract Sarcophyton glaucum has a powerful antioxidant effect by increasing antioxidant enzymes in acetaminophen–intoxicated rats (Tammam et al., 2023). The methanolic extract of Sarcophyton flexuosum showed significant antioxidant activity, attributed to its unique composition of bioactive compounds, including terpenoids, steroids, and alkaloids (Byju et al., 2015). Phenolic compounds possess scavenging power due to their ability to neutralize and stabilize free radicals by acting as hydrogen atom donors (Thouri et al., 2017), rendering them potent antioxidants and thus limiting gentamicin renal toxicity (Gomaa et al., 2018). Tannins can inhibit lipid peroxidation and lipoxygenases while scavenging hydroxyl, superoxide, and peroxyl radicals, promoting cellular prooxidant states (Iamkeng et al., 2022).

In this study, SAME administration with gentamicin ameliorated its damaging effect as kidney tissue showed mild tubular dilation and mild tubular epithelia hypertrophic degeneration, accompanied by a significant reduction in urea and creatinine levels compared to the GEN-treated group. Gomaa et al. (2018) suggest that flavonoid compounds may be crucial in mitigating oxidative damage in renal epithelial cells, offering a protective mechanism against gentamicin-induced injury. Laaroussi et al. (2021) extract has high phenolic and flavonoid content, strong DPPH radical scavenging capacity, and powerful antioxidant capacities, potentially improving gentamicin-oxidative stress and alleviating hepatorenal injuries. Soft coral Litophyton sp extract reduces hepato-nephrotoxicity either by lowering lipid peroxidation and changing the antioxidant defense system or by giving free radicals an electron to reduce their reactivity, thus reducing serum levels of creatinine and urea in 1,2-dimethylhydrazine intoxicated rats (Ashry et al., 2022).

SAME's antioxidant ability to shield cells from gentamicin-oxidative stress in testes was marked by increased TAC, which protects interstitial tissue Leydig cells, thus maintaining normal testosterone levels leading to preservation of spermatogenesis process as characterized by conservation of spermatogenic epithelial layer thickness, presence of sperms in seminiferous tubules lumens and preserved of testes weight. Abdelhaffez et al. (2019) reported that antioxidants suppressed gentamicin-induced lipid peroxidation, thus protecting spermatogenesis as marked by maintaining the germinal epithelium layer thickness. Taha et al. (2022) reported that drinking Zamzam water can considerably enhance testicular weight in gentamicin-treated rats through two mechanisms: modulating the pituitary–gonadal axis by upregulating testosterone levels and improving spermatogenesis and sperms maturation through its antioxidant properties.

This study has also suggested an anti-inflammatory activity of SAME extract in gentamicin-treated rats, which was marked by a reduction in the infiltration of inflammatory cells and reduced activity of MPO in kidneys and testes. Similarly, Abdeen et al. (2021) assumed that suppression of ROS-induced inflammatory reaction could be related to the scavenging activity of the antioxidant components of date extract. Sarcophyton components reduce inflammation by inhibiting nitric oxide and nuclear factor-kappa B, thus preventing chronic inflammation (Riyadi et al., 2019; Tanod et al., 2019b).

The anti-inflammatory activity of SAME might be attributed to its flavonoid content, which reduces renal oxidative stress and inflammation. Flavonoid-rich extracts significantly lowered serum urea and creatinine, increased TAC, and reduced MPO in renal tissue compared to gentamicin treatment (Taher et al., 2021). Flavonoid-rich extracts anti-inflammatory effects attenuated gentamicin-induced renal damage by reducing inflammatory cell infiltration and MPO levels (Babaeenezhad et al., 2021; Mahmoud et al., 2021). Flavonoid-rich plant extracts reduced MPO levels and testicular injury in oxidative damage (Ajiboye et al. (2022).

Conclusion

Sarcophyton acutum methanol extract (SAME) shows promise in mitigating gentamicin-induced renal and testicular toxicity through its antioxidant and anti-inflammatory properties. SAME's antioxidant composition includes phenols, flavonoids, tannins, alkaloids, and saponins. Their synergistic activity increased total antioxidant capacity (TAC) levels in kidney and testes tissues, counteracting gentamicin-oxidative stress. Moreover, SAME reduces inflammatory cell infiltration and myeloperoxidase (MPO) activity in renal and testicular tissues, potentially due to its flavonoid content. These findings highlight SAME's potential therapeutic role in combating antibiotic-induced organ damage, emphasizing the importance of exploring marine-derived compounds for medical applications. Further research is warranted to elucidate SAME's mechanisms of action and clinical utility in preventing and treating antibiotic-induced renal and testicular toxicity.