Study design and animal model

This experimental animal study was conducted in June 2023 at the Medicine Faculty of Andalas University in Padang. The Research Ethics Committee of Medicine Faculty Andalas University in Padang deemed this study ethically viable (No. 178/UN.16.2/KEP-FK/2023), and it was in accordance with the international guidelines for the use and care of laboratory animals. Standard protocols are followed for treating, experimenting, and disposing of experimental animals.

Twenty-four healthy male Wistar rats (Rattus norvegicus) aged between 8 and 12 weeks and weighing 200 and 300 g were used in this investigation. Every rat had limitless amounts of food and liquids and was housed in a cage for 12 h of light and 12 h of darkness each day. The temperature in the cage was set to be between 22°C and 25°C. All rats' body weight and functional index values were measured one day before intervention to ensure they all met the inclusion and exclusion criteria.

Researchers have shown that a single intraperitoneal (i.p.) administration of cisplatin at 15 mg/kg induces toxicity in the cochlea of rats. On day 10, we used an immunohistochemistry test to examine the expression of superoxide dismutase (SOD) in rat cochlear tissue in order to assess the impact of thymoquinone on cisplatin-induced oxidative stress (Figure 2).

Experimental intervention

We divided all rats into four groups (n = 6 in each group) using simple random sampling, as shown in Table 1. The group that got cisplatin 15 mg/kg (i.p.) and did not get thymoquinone therapy is called the Cis group; the group that got cisplatin 15 mg/kg (i.p.) plus 25 mg/kg/day thymoquinone intragastric gavage (i.g.) for five days is called the Cis+TQ25 group; the group that got cisplatin 15 mg/kg (i.p.) plus 50 mg/kg/day thymoquinone (i.g.) for five days is called the Cis+TQ50 group; and the group that got a single dose of 0.5 mL normal saline (i.p.) as a placebo and did not get thymoquinone therapy is called the control group. On day 10, we will test the expression of superoxide dismutase (SOD) in rat cochlear tissue using an immunohistochemistry examination to observe the antioxidant effects of thymoquinone on cisplatin-induced oxidative stress.

The sample was divided into four groups (n = 6 in each group). Group Cis only received a single dose of cisplatin 15 mg/kg intraperitoneally (i.p.); Group Cis+TQ25 received a single dose of cisplatin 15 mg/kg (i.p.) plus 25 mg/kg/day thymoquinone intragastric gavage (i.g.) for five days; the Cis+TQ50 group received a single dose of 15 mg/kg cisplatin (i.p.) plus 50 mg/kg/day thymoquinone (i.g.) for five days; and the control group was given a single dose of normal saline 0.5 mL (i.p.). Cis, cisplatin; TQ, thymoquinone.

Thymoquinone was procured from Sigma-Aldrich Chemical Co. (Spruce Street, St. Louis, Missouri 63103, USA) with a purity level of 98% and dissolved in corn oil afterward. The administration of the drug was conducted via intragastric gavage. Cisplatin was procured from Kalbe Farma Co. (Jendral Suprapto Street, Cempaka Putih, Jakarta 10510, Indonesia) and acquired through a medical prescription.

Immunohistochemistry examination

The cochlea was dissected after the experimental animals had been given intraperitoneally injected doses of ketamine (100 mg/kg) and xylazine (7.5 mg/kg) to induce anesthesia. The cochlear tissue intended for histopathological analysis was immersed in a 10% formalin solution and allowed to be fixed for 48 hours. The specimens underwent decalcification and were then embedded in paraffin blocks. We partitioned the block into segments, each with a thickness of 4 mm.

We performed immunohistochemical (IHC) testing of trial animal cochlea tissue using primary antibodies against mouse Mn-SOD. The results were read using an Olympus light microscope with a wide field of view, obtained positive expression when a brown color was found in the cytoplasm of epithelial cells, and assessed the staining intensity of MnSOD expression in the cochlear tissue of experimental animals. Staining intensity was calculated using the ImageJ program (ImageJ 1.49v software, National Institute of Health, Bethesda, USA). By isolating the brown-stained area, the color intensity was reported as an intensity value.

Statistical analysis

We expressed the descriptive data using the mean and standard deviation (SD). We used the Shapiro-Wilk test to assess data normality, Levene's test to assess data homogeneity, and the one-way analysis of variance (ANOVA) test to assess differences among results. In response to identifying a statistically significant difference, we conducted a post hoc analysis. We set the statistical significance level at p < 0.05. We analyzed the data using SPSS version 22 (IBM Corporation, Armonk, NY, USA).

Results

Figures & Tables

Discussion

Cisplatin causes toxicity in the cochlea (ototoxicity) through intrinsic and extrinsic pathways [13]. This is also consistent with the findings of Kishimoto-Urata et al. [14], who demonstrated that cisplatin elicits oxidative stress within the mitochondria through the intrinsic pathway. Cisplatin not only makes more ROS but also stops the cochlea’s antioxidant enzymes from expressing their mRNA. Endogenous antioxidants (SOD, CAT, GPx, and GSH-ST) are made by the body to protect itself from oxidative stress [11].

The imbalance between ROS production and antioxidant functions can lead to oxidative stress, which further contributes to apoptosis in the cochlea [1]. The antioxidants reduce the formation of new free radicals by converting free radicals into more stable forms [15]. Cisplatin affects the activity of numerous enzymes, such as catalase, Zn-SOD, Mn-SODA, and Cu, that protect cells from oxidative damage. Research conducted by Kim et al. [16] reported that cisplatin (6 mg/kg/day) administration for ten days will cause nephrotoxicity and a reduction in SOD expression. A similar finding was reported by Kitada [17], who found that kidney Zn-SOD and Mn-SOD expression are decreased by cisplatin toxicity. Previous research has shown that a single intraperitoneal injection of cisplatin at 15 mg/kg can harm test animals’ cochleas [5]. Other studies also suggested that single-dose cisplatin administration will decrease SOD activity in the first week of treatment [18]. This study demonstrated that ten days following therapy, a single intraperitoneal injection of 15 mg/kg cisplatin reduced SOD expression in cochlear tissue.

This study shows that administration of thymoquinone 25 mg/kg or 50 mg/kg (i.g.) for five days may raise the expression of SOD in cochlear tissue. Pharmacological research has focused on thymoquinone in recent years [19]. Thymoquinone, the primary active ingredient of black cumin (Nigella sativa), is a useful antioxidant that can stop ototoxicity caused by cisplatin [7]. Thymoquinone is a potent antioxidant (IC50 = 80.43 µg/mL) that enhances the activity of antioxidant enzymes and eliminates reactive oxygen species, resulting in high total antioxidant status values in the blood [6].

Thymoquinone has a potent ability to scavenge free radicals, particularly hydroxyl and superoxide anions [20]. The administration of thymoquinone orally to adult male Wistar rats increased the levels of SOD, CAT, GPx, and GSH-ST, providing protection against the harm that cisplatin inflicted on their liver, hearts, and digestive tracts [21]. According to research by Shahid [22], intestinal SOD expression was elevated in response to cisplatin toxicity when thymoquinone was administered orally at a dosage of 1.5 mg/kg/day (i.g.) for 14 days.

In conclusion, thymoquinone decreases oxidative stress caused by cisplatin by upregulating SOD expression in Wistar rat cochlea.

The authors declare that there is no conflict of interest regarding the publication of this paper.

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