Male Wistar albino rats were sourced from the animal house at the National Organization for Drug Control and Research (NODCAR).

Cefotaxime (dissolved in normal saline, 200 mg/kg/day). It was given by intramuscular injection for 14 days (Al Sammak et al., 2021). The Geranium oil was obtained from the National Research Center (NRC), Dokki, Egypt, and was extracted from the P. graveolens plant. The main components and their concentrations of this oil have been detailed in (Fahmy et al., 2023).

The current experimental study was conducted on male Wistar albino rats, aged 3–4 months and weighing between 170–200 g. The handling of the rats followed the protocols established by the NODCAR. They were selected from a pure strain to ensure a consistent and uniform genetic background. The animals always had free access to food and water and were kept in an environment maintained at 21°C–24°C with 40%–60% relative humidity and a 12-h light-dark cycle. To minimize stress, the animals were handled gently, avoiding squeezing, pressure, or rough maneuvers. All animal experimentation protocols were approved and supervised by the ethical committee of the National Organization of Drug Control and Research (NODCAR), in accordance with the organization’s guidelines. The approval reference number is NODCAR/II/39/2022. The study methodology is illustrated in Figure 1, which provides a schematic overview of the key steps involved in the research process.

Experimental Animals (30) Wistar albino male rats were randomly assigned into 5 groups of 6 rats in each group:

I. Control group (Negative group): Animals were received normal saline by oral gavage 14 days.

At the end of the experiment, 24 h after the last manipulation, the rats were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg body weight) and xylazine (10 mg/kg body weight), both warmed to 37°C. Following anesthetization, the rats were euthanized, and blood samples were collected for serum separation. The liver and kidneys were immediately harvested and preserved at −80°C for biochemical analysis. A portion of the tissue was homogenized in protease/phosphatase-complemented lysis buffer (10% glycerol; 200 mM NaCl, 5 mM EDTA, 10 mM Tris, pH 7.4) for ELISA assays. The homogenate was centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was stored at −80°C for further use. The organs from each group were preserved in 10% formalin-buffered saline for histology and immunohistochemistry. The animals that were used were then frozen until they were incineration.

For RT-PCR gene expression analysis, about 30 mg of tissue was preserved in RNA lysis solution at −80°C until further processing. The expression of MAPK and Nrf2 genes was evaluated via real-time quantitative reverse transcription PCR (RT-PCR). Total RNA was extracted from the frozen samples using TRIzol^® reagent (Invitrogen, Sigma–Aldrich, St. Louis, MO) following standard procedures. This RNA was then converted into complementary DNA (cDNA) using SMARTScribe™ Reverse Transcriptase (Clontech Laboratories, Inc., a Takara Bio Company). RT-PCR was conducted with a Real-Time PCR v 7.9 System (DTlite, DNA Technology, LLC, Moscow, Russia) and SYBR^® Green PCR Master Mix (QIAGEN) in a 25 µL reaction volume. The cycling conditions included an initial step at 95°C for 15 s, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. The primer sequences used are detailed in Table 1. Data were analyzed using ABI Prism sequence detection system software and quantified with PE Biosystems v17 Sequence Detection Software. Gene expression levels were determined using the comparative threshold cycle method, with all data normalized against the GAPDH gene as a reference control (Table 1).

The levels of reduced glutathione (GSH) and malondialdehyde (MDA) in the tissue were quantified spectrophotometrically according to the method outlined by Mihara and Uchiyama 1978. Catalase Activity (CAT) was determined spectrophotometrically according to the method of Aebi 1984, using the reagent kits purchased from Biodiagnostic company (Egypt). Nitric oxide (NO) levels were measured spectrophotometrically using the method described by Montgomery and Dymock 1961, with reagent kits obtained from Biodiagnostic Company (Egypt).

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in serum were measured using reagent kits (Cat. nos. AL 1031, AS 1061) obtained from Bio diagnostics. For analyzing Aspartate (ALP), biochemical kits Spin React (Barcelona, Spain) were utilized (Salem et al., 2023). Colorimetric assay kits from Biomed Diagnostics (Cairo, Egypt) were utilized to measure blood urea, serum creatinine, serum albumin, and total protein levels (Khames, et al., 2019).

Rat TNF-α and IL-1β levels were measured in serum using enzyme-linked immunosorbent assay ELISA kits, following the manufacturer’s instructions provided by RayBiotech Inc. (Parkway Lane Suite, Norcross, GA) (Khames, et al., 2019). Beclin 1 levels were measured using an ELISA employing the sandwich technique, as provided by Sino Geneclon Biotech Co. Ltd. (Hangzhou, China) (Mohammed et al., 2020).

Male rats were euthanized at the end of the trial, and tiny, fresh liver and kidney specimens were gathered and quickly preserved in 10% formalin solution for at least 24 h. The specimens underwent the standard paraffin embedding procedure, which involves dehydration in progressively higher grades of ethyl alcohol, clearing in various xylene changes, and embedding in various melted paraffin wax changes at 60°C. A microtome was used to cut paraffin blocks into 5-micron pieces. All tissue sections were examined under an Olympus BH-2 microscope for characterization of histopathological changes at scale bar 20 µm. The tissue sections were thick sections that were stained with Haematoxylin and Eosin (H.and E.) for histological evaluation of hepato-renal injury. Following an injection of cefotaxime and oil, the following parameters were selected to represent the degree of morphological damage to the liver: Congestion, Lymphatic infiltration, Necrosis, Hydropic degeneration and Pyknosis. And kidney: Lymphatic infiltration, Tubular degeneration, Fragmented glomeruli, and vacuolated glomeruli. These parameters were assessed on a scale ranging from absent or normal (−), mild (+), to moderate (++). Additionally, liver fibrosis was evaluated in Masson’s trichrome-stained liver sections. Coded samples were examined and scored under a light microscope in a blinded manner.

Sections of 4 μm thick were cut from various animal species, and immunohistochemistry was carried out. After deparaffinizing, rehydrating, and using H₂O₂ in methanol to inhibit endogenous peroxidase activity, sections were prepared. In a microwave, sections were pre-treated in citrate buffer (pH 6.0). Sections were treated with anti-caspase-3 antibodies (1:200) at room temperature. Sections were treated first with streptavidin peroxidase, then with biotinylated goat anti-polyvalent, and lastly with DAB plus chromogen. Hematoxylin was used as a counterstain on the slides. Under a light microscope, the slides were examined to determine the degree of cell immunopositivity.

The data were analyzed statistically using GraphPad Prism Software Inc., version 6.0. Results are presented as mean ± standard error of the mean (SEM). Statistical significance for biochemical parameters was determined with a threshold of p < 0.05. Analysis was conducted using one-way ANOVA, followed by a Tukey test for multiple comparisons.

In the present study, results showed that intramuscular injection of cefotaxime during the course of the experimental induced significant (p < 0.05) increase in AST, ALT and ALP serum activities as shown in Table 2. The table presents the effects of different treatments on serum activites of hepatic enzymes, specifically AST, ALT and ALP, which are markers of liver function and damage. In the control group, the activities of AST, ALT, and ALP were found to be 42.19 ± 0.89 IU/L, 29.88 ± 0.59 IU/L, and 59.25 ± 1.18 IU/L, respectively. These values represent normal liver enzyme activities in serum. For the DMSO group, which served as a vehicle control, enzyme activitites were similar to the control, with AST at 42.03 ± 0.66 IU/L, ALT at 31.57 ± 0.29 IU/L, and ALP at 64 ± 0.43 IU/L. In contrast, the group treated with cefotaxime exhibited a marked increase in liver enzyme activities: AST levels rose significantly to 201 ± 4.7 IU/L, ALT levels to 157.7 ± 6.64 IU/L, and ALP levels to 235.2 ± 5.75 IU/L. These elevations indicate substantial hepatotoxicity and liver damage caused by cefotaxime.

Comparing with cefotaxime group, liver enzyme activities decreased with the combination of cefotaxime and geranium oil. The AST, ALT, and ALP activities dropped to 83.1 ± 1.5 IU/L, 69.58 ± 1.23 IU/L, and 99.34 ± 0.42 IU/L, respectively. These decreases imply that geranium oil offers some defense against cefotaxime-induced liver damage. Enzyme activities for AST, ALT, and ALP were 42.69 ± 0.99 IU/L, 34.21 ± 0.33 IU/L, and 62.31 ± 1.4 IU/L for the geranium oil group alone. These results were in line with those of the control group, suggesting that geranium oil alone has no deleterious effects on liver function. Overall, these findings show that although cefotaxime causes severe liver damage, geranium oil successfully reduces hepatic damage and returns enzyme levels to normal.

As showed in Table 3, Creatinine, albumin, and urea serum levels were normal in the control and DMSO groups. Renal impairment was evidenced by a considerable increase in serum creatinine (2.02 ± 0.06 mg/dL) and urea (87.25 ± 1.2 mg/dL) levels during cefotaxime treatment, and a decrease in serum albumin levels (2.8 ± 0.03 g/dL). After receiving cefotaxime + geranium oil treatment, serum levels of albumin (3.45 ± 0.02 g/dL) and urea (49.88 ± 0.59 mg/dL) were restored, indicating a potential protective effect against kidney impairment. Creatinine, albumin, or urea levels were not signficantly different from the control group while using geranium oil alone. This research suggests that kidney damage is induced by cefotaxime, but that these effects can be prevented by geranium oil.

As showed in Table 4, catalase activity was significantly lower (8.64 ± 0.11 units) after receiving cefotaxime therapy than in the DMSO (14.34 ± 0.18) and control groups (14.38 ± 0.38 units) groups. Catalase activity was higher with geranium oil (10.96 ± 0.4) as compared to the cefotaxime group. Nitric oxide (NO) levels were significantly higher after cefotaxime treatment (127.7 ± 4.3) than in the control (35.86 ± 6.05) and DMSO groups (44.52 ± 0.54) groups. When compared to the cefotaxime group, geranium oil treatment resulted in a considerable reduction of nitric oxide levels (76.46 ± 1.77). The cefotaxime group had significantly higher MDA levels (74.33 ± 0.59 units) than the DMSO group (31.63 ± 0.5) and control group (28.81 ± 1.1). Compared to the cefotaxime group, MDA levels were significantly lower (52.86 ± 0.85) after geranium oil treatment. GSH levels were significantly lower in the cefotaxime group (64.34 ± 0.27 units) than in the DMSO group (27.31 ± 0.59) and control group (25.86 ± 0.54). GSH levels were significantly higher (40.41 ± 0.56) after geranium oil treatment than in the cefotaxime group.

As showed in Table 5, cefotaxime therapy raised nitric oxide levels (78.04 ± 2.89 units) compared to the control (31.08 ± 1.35) and DMSO groups (32.64 ± 0.75), which was similar to liver tissue. When compared to the cefotaxime group, geranium oil administration resulted in a considerable reduction of nitric oxide levels (51.44 ± 0.3). Compared to the control (27.07 ± 0.56) and DMSO groups (28.44 ± 0.61) groups, the MDA levels were considerably higher in the cefotaxime group (69.93 ± 1.48 units). MDA levels were substantially lower (47.18 ± 0.46) in the geranium oil group compared to the cefotaxime group. Compared to the control group (14.5 ± 0.1) and DMSO group (14.26 ± 0.03), cefotaxime significantly reduced GSH levels (7.86 ± 0.104 units). GSH levels were higher in the geranium oil group (10.57 ± 0.2) than in the cefotaxime group. The cefotaxime group (8.64 ± 0.11) had significantly lower catalase activity than the DMSO (14.34 ± 0.18) and control groups (14.38 ± 0.38). Catalase activity was higher with geranium oil (10.96 ± 0.4) as compared to the cefotaxime group.

Exposure to cefotaxime resulted in a significant increase in TNF-α levels with an average of 127.65 compared to 41.84 in the control group, indicating an inflammatory response. Treatment with geranium oil in combination with cefotaxime decreased TNF-α levels to an average of 76.46, suggesting a potential protective or anti-inflammatory effect of geranium oil (Figure 2A). Similarly, IL-1 Beta levels in the Cefotaxime group increased to an average of about 145.53, while in the Cefotaxime + Geranium oil group they decreased to an average of 77.78, indicating that Geranium oil significantly reduced the cefotaxime-induced elevation of IL-1 Beta. This increase indicates a marked inflammatory response (Figure 2B). In the cefotaxime group, levels of the autophagy marker Beclin 1 were considerably reduced, falling to an average of 28.95 from the control average of 106.03. This suggests that cefotaxime may block autophagy. But Beclin 1 levels rose to an average of 64.57 when Geranium oil (Cefotaxime + Geranium) was present, suggesting that Geranium oil may help restore autophagic function to some extent (Figure 2C).

In the cefotaxime-treated group, there was a significant increase in pro-inflammatory markers compared to the control (p < 0.05, TNF-α; p < 0.05, for IL-1β), suggesting an inflammatory response. Co-administration with Geranium oil significantly reduced these elevated levels, as indicated by TNF-α values dropping from 127.65 ± 1.32 to 41.84 ± 4.353 (p < 0.05) and IL-1β levels from 145.53 ± 1.99 to 77.78 ± 6.241 (p < 0.05). Furthermore, Beclin one levels, which were reduced in the cefotaxime group (p < 0.05), were partially restored upon treatment with Geranium oil (64.57 ± 3.733, p < 0.05), suggesting a protective effect on autophagy pathways.

MAPKs are activated through phosphorylation and subsequently stimulate the downstream transcription factor NF-κB. Our findings indicate that cefotaxime treatment in rats led to increased phosphorylation levels of p-38 MAPK. However, geranium oil treatment significantly decreased the phosphorylated protein levels of p-38 MAPKs, indicating the geranium oil’s protective effect in the cefotaxime-induced toxicity on rats as shown in Figures 3A, C. On the other hand, our findings showed that geranium oil treatment exert inhibitory effect on Nrf2 after its elevation by cefotaxime treatment as shown in Figures 3B, D.

As shown Table 6 and in A photomicrograph of a liver sample stained with Hematoxylin and Eosin Figure 4: The control rat’s anatomy (Figure 4A): was normal, with no histological abnormalities, as well as a typical central vein and hepatocyte arrangement. Hepatocyte nuclei were seen as dark red objects inside the cells, with the cytoplasm dyed red. The sinusoids and the typical portal region, along with comparable hepatic strands that go from the margin of the lobule to the central vein. In G2 DMSO group (Figure 4B): standard composition of hepatic tissue, but there were numerous congested blood vessels. Moreover, G3 (Cefotaxime group): In liver sections (Figure 4C): there was altered lobular form and nuclear deterioration in some locations, as well as disarray of normal hepatic cells, necrosis, and hydropic degeneration. Enlargement and moderate congestion of the hepatic central vein were discovered. In the portal area, moderate lymphocyte infiltration was seen. While in G4 (Geranium oil + cefotaxime group): Hepatocytes and portal components (Figure 4D): appeared to be in good health. The region is infiltrated by small mononuclear cells. Necrotic changes were not detected. It was possible to see a few clogged blood vessels. There was mild hydropic degeneration. Few numbers of hepatic cells revealed pyknotic feature. As expected in G5 (Geranium oil only group). Hepatic architecture appeared normal, with hepatic cords properly aligned around sinusoids. There was no evidence of mononuclear cell invasion. There were no alterations in fatty degeneration noted. The blood vessels appeared to be normal as in Figure 4E.

The H&E staining results represented in Table 7 and Figure 5. G1: Renal (Malpighian) corpuscles with glomerular capillaries, Bowman’s capsules, and urine space had a typical histological appearance in the renal cortex. Proximal convoluted tubules had a small lumen lined with cubical cells at the base with round nuclei. Distal convoluted tubules had a wide lumen, which was surrounded by simple cubical cells with sphere nuclei in the center or at the apex. The rats in the G2and5 had renal cortical structures that were similar to those in the control group. The group treated with cefotaxime exhibited some degree of degeneration; glomerular asymmetry in size and structure, with some glomeruli shrinking in size due to atrophied glomerular tuft and cystic appearance. Some vacuolated, fragmented and congested glomeruli were seen. Cell nuclei showed full or partial destruction in the proximal and distal convoluted tubules. Among the damaged tubules and renal corpuscles, there was mononuclear cell invasion. Renal tubules with disturbed epithelial linings and desquamation were seen. Some renal tubules showed luminal cast. G4 (Figure 5D) Geranium oil + cefotaxime group: some tubular epithelial cells in renal convoluted tubules appeared to have entire cytoplasm, whereas others had mild vacuolation. The glomeruli with normal and standard size inside Bowman’s capsule. There was no evidence of lymphatic invasion. The most of renal glomeruli and tubules appeared normal.

Histological examination of tissue samples from groups G1, G2, and G5 (Figures 6A, B, E) revealed delicate collagen fibers within the central vein walls of hepatic lobules. Minimal collagen deposition was also observed in the interlobular septa and surrounding the portal vein. In the cefotaxime group (Figure 6C), a marked increase in collagen fiber deposition was observed, leading to the formation of a fibrous septum within the hepatic lobule. Fiber accumulation intensified in the portal region, interacting with nearby septa, and wrapping around each other. Compared to the model group, the G4 (Figure 6D) group treated with both geranium oil and cefotaxime exhibited a significant reduction in collagenous fibrous tissue. Fibrosis was markedly decreased, with only a minimal amount of thin, blue-stained fibers observed around blood vessels.

IHC was used to examine the expression of the caspase −3 in all the groups' rat livers. While DMSO and Geranium oil had no effect on caspase −3 levels (Figures 7B, E), the cefotaxime group showed a significant rise in caspase −3 positive levels (Figure 7C), which was dramatically reduced by geranium oil administration (Figure 7D).

IHC was used to examine the levels of caspase −3 in all the groups' rat kidney. While Vehicle control DMSO and geranium oil had no effect on caspase −3 levels (Figures 8A, B, E), the cefotaxime injected group showed a significant rise in caspase −3 positive levels (Figure 8C), which was dramatically reduced by geranium oil treatment (Figure 8D).