Wistar rats (n=30; 4 days old), were obtained from Animal Center of China Medical University (Shenyang, China). The animals were housed in stainless steel wire-mesh cages (5 rats per cage) under standard laboratory condition (25°C, relative humidity 60%, and 12 h dark-and-light cycle). The animals were allowed free access to water and food.

The rats were randomly divided into the control and GM groups. For the GM group, each rat received a dose of 80 mg/kg GM via intramuscular injection. For the control group, each animal received an equal volume of normal saline. The injections were administered once a day for 7 days consecutively.

On the 7th day, 300 µl blood was collected by cardiac puncture. Blood samples were immediately placed in 1.5 ml centrifuge tubes containing heparin. After centrifugation at 2,000 × g for 10 min, plasma samples were collected and stored at 4°C. Plasma analysis was conducted within 2 days of collection. A total of 10 rats (randomly 5 per group) were sacrificed by overdose of anesthetic. The kidneys, cochlear tissue and liver were collected. The samples were processed soon after. The study was approved by the ethics committee of Sichuan University (Chengdu, China).

Plasma serum creatinine (SCr) and blood urea nitrogen (BUN) levels were determined with Serum Creatinine kit (Beijing Leadman Biochemistry Co., Ltd., Beijing, China) and Blood Urea Nitrogen kit (Beijing Leadman Biochemistry Co., Ltd.), respectively, with an AU480 Chemistry system (Beckman Coulter, Inc., Brea, CA, USA) according to manufacturer's instructions.

Total RNA was isolated from tissue samples with TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturers' instructions. Total RNAs were quantified with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Samples of total RNA from ears, kidney and liver of rats from the same group were pooled for subsequent GeneChip analysis. Prior to the analysis, pooled total RNA samples were purified using an RNeasy Total RNA Mini Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions.

The GeneChip scan was performed with an Affymetrix GeneChip Rat 230 2.0 array (Affymetrix; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Gene expression changes were represented as ratios between the GM and control groups. Gene expression profiles of liver tissue were used for normalization. The difference value of expression ratio between cochlear and liver, or kidney and liver was calculated to determine the change tendency. A ratio >1 indicated that the gene expression was upregulated, and vice versa. Selected genes were classified into four categories for further analysis, as follows: Upregulated in cochlear; downregulated in cochlear; upregulated in kidney; and downregulated in kidney. Genes exhibiting similar tendencies were selected.

Cochlear, kidney and liver tissues from the rats were preserved in 10% phosphate-buffered formalin. Tissues fixed with neutral formalin were embedded in paraffin and sectioned at 3 µm. Hematoxylin and eosin (H&E) staining was performed to observe GM-induced ototoxicity, indicated by loss of cochlear hair cells and inflammation.

To identify Ifi44 protein in the cochlear and kidney, sectioned paraffin-embedded tissue samples were deparaffinized for immunohistochemistry. Slides were incubated with 3% H₂O₂ at room temperature for 10 min to eliminate endogenous peroxidases, and washed with distilled water and PBS. The slides were then incubated with 5% goat serum (ZsBio, Beijing, China) at room temperature for 10 min. Primary antibody (rabbit anti-rat-IFI44 primary antibody; GTX32667; 1:100; GeneTex, Inc., Irvine, CA, USA.) incubation was performed at 37°C for 2 h. PBS was used as blank control for primary antibody incubation. After washing with PBS, biotinylated goat anti-rabbit IgG secondary antibody (ZB-2010; 1:200; ZsBio) incubation was performed at 37°C for 30 min. The slides were washed with PBS and incubated with HRP-streptavidin (ZB-2404; 1:500; ZsBio) working buffer at 37°C for 30 min. The slides were washed with PBS and incubated with diaminobenzidine at room temperature for 10 min, followed by washing with water and H&E staining. The sections were imaged with an Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan).

Extracted RNA was converted to cDNA by reverse transcription of 1 µg RNA with random primers and AMV reverse transcriptase (Applied Biosystems, Thermo Fisher Scientific, Inc.). The reverse transcription conditions were 42°C for 1 h and 99°C for 5 min. Primers (Table I) were designed using Primer 3 software (http://primer3.ut.ee) and synthesized by Genscript Biotech Corporation (Nanjing, China). The reverse transcription and qPCR were performed out on an ABI PRISM 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). qPCR was performed in a total volume of 20 µl, with each well containing 10 µl SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 2 µl cDNA, and 0.4 µM Ifi44 or Gapdh primers. The PCR condition consisted of initial denaturation step at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. The relative level of gene expression was calculated using the 2^−ΔΔCt method (15).

To prepare protein samples for western blotting, prepared tissues (cochlear, kidneys, and livers) were cut into pieces and washed with PBS three times. Tissue pieces were homogenized in RIPA lysis buffer (P0013B; Beyotime Institute of Biotechnology, Shanghai, China) containing PMSF, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was collected and protein concentration was determined using BCA Protein Assay reagents (Pierce, Thermo Fisher Scientific, Inc.). Protein loading buffer (5X; P0015; Beyotime Institute of Biotechnology) was added into the supernatant, and then boiled for 10 min. The protein samples were stored at −70°C until use. Protein samples were loaded, 100 µg for each well, onto a SDS-PAGE gel and transferred to a PVDF membrane (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The membrane was then incubated with 5% nonfat dry milk for 3 h at room temperature, and then with rabbit anti-rat-IFI44 primary antibody (GTX32667; 1:1,000; GeneTex) and rabbit anti-rat-GAPDH primary antibody (GTX100118; 1:1,000; GeneTex) for 2 h at room temperature. The membrane was washed with 0.1% TBST for 5 times and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (ZB-2301; 1:2,500; ZsBio) as the secondary antibody for 1 h at room temperature. After final washing with 0.1% TBST for 5 times, protein bands were detected with an enhance chemiluminescence assay kit (Pierce, Thermo Fisher Scientific, Inc.).

All experiments were conducted in triplicate and repeated at least twice. The group mean ± standard deviations were calculated for each measured parameter. Statistical differences between the groups were evaluated using the Student's t test with SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

H&E staining of cochlear tissue from the rats demonstrated distinct inflammatory invasion in the GM group (Fig. 1A and B). Increased blood SCr and BUN levels are indicators of kidney dysfunction. In the GM group, there were significant increases in the levels of both indicators compared with the levels in the control group (P<0.05; Fig. 1C).

Morphological changes in the cochlear tissue, as revealed by H&E staining, along with the biochemical changes in blood, indicated that GM induced ototoxicity and nephrotoxicity in the experimental group rats.

The Affymetrix Rat Genome 230 2.0 microarray contains 31,000 probe sets corresponding to ~24,000 annotated rat genes and 6,693 expressed sequence tags. As GM predominantly causes damage in the kidney and ear, to explore the similarities in gene expression changes between the kidney and ear, genes significantly altered in the liver tissue from both groups were excluded from further analysis. The genes that were unchanged in the liver tissue between the two groups were classified into four categories: Upregulated in the kidneys; downregulated in the kidneys; upregulated in the cochlear; and downregulated in the cochlear (Tables II–V). Compared with the control group, nine genes exhibited similar expression changes in the kidneys and ears in the GM group (Table VI).

Ifi44 was one of the significantly upregulated genes in cochlear and kidney tissue, but not changed in the liver tissue of the GM group with a ratio of 2.45184 (GM cochlear vs. control cochlear) and 3.2915 (GM kidney vs. control kidney), respectively. As Ifi44 is associated with inflammation processes, further analysis was conducted to verify its expression in the cochlear and kidney tissues.

To verify the changes of Ifi44 expression in GM group rats compared with control rats, RT-qPCR, western blotting and immunohistochemistry were performed. As demonstrated in Figs. 2 and 3, the expression of Ifi44, at the transcriptional and translational levels, was increased in ear and kidney tissue in the GM group rats compared with control group rats. These results were consistent with the results of the GeneChip microarray.