Abstract
Objective
The clinical use of aminoglycoside antibiotics is limited in most countries because of auditory toxicity side effects. However, their use is common in developing countries because they are inexpensive and convenient. Salvia miltiorrhiza extracts are used clinically in China for their antioxidant properties. We investigated the effect of a clinically approved injectable S. miltiorrhiza solution on inducible nitric oxide synthase (iNOS) generation induced by the aminoglycoside antibiotic gentamicin and an ototoxicity protective mechanism.
Methods
Sixty adult guinea pigs were used in this study and divided into four groups. Auditory brainstem response (ABR) testing was performed before and after treatments and animals were sacrificed for morphological and immunostaining assays after determining threshold shifts in ABR. The cochleae were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to observe ultrastructural changes. In addition, hair cell loss, iNOS and caspase-3 expression, and apoptosis were measured.
Results
The result showed that hearing loss, iNOS overexpression accompanied with disorganization in the cochlea, and terminal deoxynucleotidyl transferase- mediated dUTP- biotin nick end labeling (TUNEL)-stained positive cells in animals treated with gentamicin. However, pretreatment with S. miltiorrhiza (3 g/kg/day for 10 days) decreased gentamicin-induced hearing loss, attenuated iNOS and caspase-3 expression, and decreased the number of apoptotic cells. Furthermore, it also reduced the ultrastructural damage due to ototoxicity as observed by SEM and TEM.
Conclusions
These findings indicate that S. miltiorrhiza protects against gentamicin-induced ototoxicity and could apply to the protection of ototoxicity.
1
Introduction
Aminoglycoside antibiotics such as gentamicin were discovered more than half a century ago and are the most commonly used antibiotics worldwide. Gentamicin has a high efficacy for serious Gram-negative bacterial infections and a low price. In developed nations, aminoglycosides tend to be reserved for severe bacterial or multidrug-resistant tubercular infections. The relatively inexpensive aminoglycosides are often prescribed as a first-line therapy for less severe conditions such as otitis media and bronchitis . However, the clinical use of aminoglycoside antibiotics has been limited in most countries because of side effects in auditory toxicity and impairing hearing ability during 1970s and 1980s . The use of aminoglycosides has declined in the past decades thanks to the development of new antibiotics with fewer side effects . Nevertheless, today aminoglycoside antibiotics are still the most commonly used antibiotics worldwide because of their efficacy and low cost. Streptomycin, an aminoglycoside antibiotic, has drawn considerable attention because it is the most effective drug for tuberculosis treatment . However, concern for the development of ototoxicity has often been eclipsed by the need for lifesaving treatment with streptomycin.
Studying harmful effects of these medicines may lead to solving their severe problems. The formation of free radicals is related to ototoxicity ; and glutathione, a primary cellular antioxidant that scavenges free radicals, can protect against ototoxicity . Nitric oxide (NO) is a free radical that is synthesized from an arginine substrate via nitric oxide synthase (NOS) catalysis. NOS exists in three isotypes: neuronal, endothelial, and inducible (nNOS, eNOS, and iNOS, respectively). Among them, nNOS and eNOS are expressed under physiological conditions; while iNOS is not usually expressed but can be stimulated by some stimulus such as lipopolysaccharides , noise exposure , and cochlear hydrops , leading to excessive NO synthesis. At physiological concentrations, NO is involved in the regulation of cochlear blood flow, auditory transduction, as well as neurotransmission in the cochlea and the vestibular system . But excess NO can exert cytotoxic effects in both the central nervous system (CNS) and the inner ear , leading to ototoxicity .
In China, many reports have discussed possible prevention methods for ototoxicity induced by gentamicin. Rhizome Salvia miltiorrhiza , a type of traditional Chinese medicine derived from the root of S. miltiorrhizae Bunge, is an example of such a standardized medication. S. miltiorrhiza extracts contain diterpene quinone and phenolic acid derivatives, including tanshinone, cryptotanshinone, isocryptotanshinone, miltirone, and tanshinol. These compounds have antioxidant properties and protect against lipid peroxidation, making them potential antidotes for free radical-based disorders . They have been shown to significantly attenuate the activity of superoxide and the increased concentration of malondialdehyde induced by kanamycin or cisplatin, consequently improving impaired hearing .
Previously, we reported the effect of S. miltiorrhiza on reducing iNOS expression of streptomycin-induced ototoxicity . In this study, we investigated the protective mechanism of S. miltiorrhiza on gentamicin-induced hearing loss.
2
Materials and methods
Salvia miltiorrhiza injection solution (Injectio Salvia miltiorrhiza ) was obtained from Shanghai First Pharmaceutical Company (Shanghai, China). One milliliter of Injectio S. miltiorrhiza contains 1.5 g of S. miltiorrhiza root water extract. This is a standardized injectable preparation approved for clinical use (batch number: 030701; Department of Drug Administration, Shanghai, China) and is widely available over the counter. Gentamicin was purchased from Dalian Pharmaceutical Company (Dalian, China, purity ≥ 98%).
This study was performed in accordance with the Use of Laboratory Animals of China, and the animal experiments were approved by the Institutional Animal Care and Use Committee of China Medical University.
2.1
Animals and grouping
Sixty adult guinea pigs (~ 2 months, 200–260 g) with a sensitive Preyer reflex were used for this study. The animals were randomly divided into four groups (15 per group). The control group was injected intramuscularly (IM) with normal saline (2.5 mL/kg body weight/day); the gentamicin group received gentamicin only (100 mg/kg/day, IM); the treatment group received S. miltiorrhiza (3 g/kg/day, IM), followed by gentamicin (100 mg/kg/day, IM) 10 min later; and the S. miltiorrhiza group received S. miltiorrhiza only (3 g/kg/day, IM). The animals were properly labeled and given daily injection of the above drugs for 10 days. The animals were given free access to water and a regular diet, and they were allowed one week to acclimate before treatment.
2.2
ABR testing in the animal model of ototoxicity
Experimental guinea pigs were anesthetized with an intraperitoneal injection of 2% sodium phenobarbital. The animals were placed in an electrical shielding room for auditory brainstem response (ABR) recording. Briefly, a recording needle was placed subcutaneously in the scalp, a ground needle was placed subcutaneously in the mastoid, and a control needle was placed subcutaneously in the other mastoid. The click (duration of 0.1 ms) stimulation of a 12 kHz primary frequency was triggered by a sound stimulator (Danac-7) via a TDH-39 earphone to the ear. The signal was collected by a biological recording system (ATAC-450) with an analysis duration of 20 ms, 200 times on average. The stimulus interval was 90 ms, and the filter band-pass frequency was 100–3000 Hz. The hearing threshold was calculated from a 95 dB sound pressure level, with a 10 dB step decrease after the clear ABR wave was observed. The stimulation wave intensity was decreased by 5 dB each step when it was close to the hearing threshold until the P 3 wave diminished. Then, the sound intensity was regarded as the hearing threshold. The animals were determined to suffer from ototoxicity when auditory threshold shift was more than 20 dB.
2.3
Cochlea sample
The animals were decapitated after the last ABR recording, and the cochlea was swiftly separated. For all groups, the left side of the cochlea was designated for ultrastructural investigation (five from each group were analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively) and Western blot analysis (five cochleae for caspase-3 expression); the right side of the cochlea was designated for iNOS and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. To ensure efficient fixative perfusion, the round window and the cochlear apex were opened after the stapes was removed.
2.4
Preparation of frozen sections of cochlea
After ABR testing, 10 guinea pigs from each group were sacrificed by decapitation, and the cochleae were swiftly separated. After the otocyst was opened, the cochlea was exposed completely. The round window membrane was pricked under an anatomical microscope, the oval window was exposed through dislocation of the base of stapes, a small hole was drilled on the top of the cochlea, and then phosphate buffer containing 40 g/L paraformaldehyde was put into the fixation fluid at 4 °C for 2 h. After washing with phosphate buffer, the sample was put into 100 g/L ethylenediaminetetraacetic acid (EDTA) solution at 4 °C for 5 to 7 days for decalcification, then transferred into phosphate buffer containing 250 g/L sucrose, and left at 4 °C overnight. The next day, the sample was embedded in optimal cutting temperature (OCT) compound for frozen sectioning with a cryostat.
2.5
Preparation of SEM
The cochlea was fixed with 2.5% glutaraldehyde for approximately 4–6 h. The bony capsule was removed, and the basilar membrane was dehydrated, dried, gilded, and then observed under SEM (JEOL JSM-T300 SEM, Japan).
2.6
Preparation of TEM
The cochlea was fixed with 2.5% glutaraldehyde for approximately 4–6 h and decalcified in 10% EDTA for 10 days. The basilar membrane and spiral ligament were rinsed, postfixed with osmium tetroxide, dehydrated with a graded series of ethanol concentrations, and embedded in resin. Serial midmodiolar sections were cut at 50 nm thickness, stained, and observed under TEM (JEOL JSM-T300 electron microscope, Japan).
2.7
iNOS expression
The reagent kit to assay iNOS was provided by Wuhan Boshide Biotechnology Co., Ltd. (Lot no. BA0362, Wuhan, China). Following the manufacturer’s instructions, the primary antibody was used at a 1:200 dilution. In the negative control group, the primary antibody was replaced with 0.01 M phosphate buffer, while the remaining procedure was the same. The intensity of iNOS staining was determined by the mean gray value from an image analysis system (see below).
2.8
TUNEL staining
In order to visualize the apoptotic cells, we used an in situ cell death detection kit (Wuhan Boshide Biotech., MK1020). The tissue sections were treated with proteinase K (20 μg/mL) for 20 min at room temperature and washed twice with phosphate-buffered saline (PBS). They were incubated with the TUNEL mixture solution for 1 h at 37 °C and washed three times with PBS. The sections were incubated for 30 min with horseradish peroxidase at 37 °C and then washed with PBS. Staining was conducted using 3,3′-diaminobenzidine (DAB), and counterstaining was carried out using hematoxylin.
2.9
Western blot for caspase-3
After ABR measurement, cochlear tissues from guinea pigs were dissected and homogenized in ice-cold lysis buffer [25 mM N -2-hydroxyl piperazine- N ’-2-ethane sulfonic acid, pH 8.0, 5 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors]. Cell debris and the nuclear fraction were removed by centrifugation at 10,000 rpm for 20 min at 4 °C. The supernatant was extracted and stored at − 80 °C until ready for use. Samples were incubated in Laemmli sample buffer (62.5 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol) at 95 °C for 10 min. The supernatants were separated by 8% SDS-polyacrylamide gel electrophoresis and electrically transferred to a polyvinylidene difluoride membrane. Nonspecific binding sites were blocked with Tris-buffered saline (100 mM Tris, 0.9% NaCl, pH 7.5), 0.1% Tween 20 (TBS-T), and 5% nonfat dry milk at room temperature for 4 h. Membranes were then incubated in a 1:500 dilution of rabbit polyclonal anti-caspase-3 in TBS-T for 24 h at 4 °C. After three washes in TBS-T, membranes were incubated in a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG anitbody. Immunoreactive caspase-3 protein was detected by enhanced chemiluminescence. The membranes were then stripped and restained with anti-glyceraldehyde-3-phosphate dehydrogenase at a concentration of 1:10,000 for the protein loading control.
2.10
MicroImage analysis
The MetaMorph/Coolsnapfx/AX70 MicroImage analysis system (jointly produced by the USA and Japan) was used, and 20 sections were randomly collected for each group. The slice was put under a microscope, the image was visualized, and the picture was taken with a digital camera and transferred to a computer. After every part of the cochlea to be analyzed was sketched, the gray values of cellular iNOS-positive staining in Corti’s organ, stria vascularis, and spiral ganglion were respectively measured under the same conditions with image analysis software, and then the mean gray value of cellular iNOS-positive staining in each part was calculated. Image color shade is divided into 256 scales according to a computer image analyzer. The binary representations of gray value assume that 0 is black and the maximum value is 255. The higher the color value, the lighter the shade of gray is and the lower the color value, the darker the shade of gray is.
2.11
Analysis of hair cell loss
After ABR measurement, the other five animals of each group were decapitated. The otocyst was quickly removed, and the round and oval windows were pricked under an anatomical microscope. For succinate dehydrogenase (SDH) activity assay, a solution containing 0.05 M sodium succinate, 0.05 M phosphate buffer, and 0.05% tetranitro blue tetrazolium was slowly perfused through the round window for about 1 min, the cochlea was immersed in the solution for 1 h at 37 °C, and then the cochlea was immersed in 4% paraformaldehyde for 24 h. The reduction of SDH activity is commonly related to the damage to the hair cells. After that, the Corti’s organ was dissected into three pieces consisting of the basal, middle, and apical turns, and each part was mounted with glycerin on a glass slide. Specimens were examined under a light microscope to determine the number of missing hair cells. Cytocochleograms were made by plotting the percentage of hair cells remaining in each part of the four individual rows (1 row of IHC and 3 rows of OHCs) measured in millimeters. Summary of cytocochleograms was made by averaging the percentage of remaining hair cells from IHCs and OHCs of each group of animals, which represented the measurement of the treatment response.
2.12
Statistical analysis
Data were presented as mean ± standard deviation (SD). Significant differences between groups were analyzed with SPSS V.11.5 (SPSS Inc., Chicago, IL, USA) using the Student’s t -test and the f -test. A p- value < 0.05 was considered statistically significant.
2
Materials and methods
Salvia miltiorrhiza injection solution (Injectio Salvia miltiorrhiza ) was obtained from Shanghai First Pharmaceutical Company (Shanghai, China). One milliliter of Injectio S. miltiorrhiza contains 1.5 g of S. miltiorrhiza root water extract. This is a standardized injectable preparation approved for clinical use (batch number: 030701; Department of Drug Administration, Shanghai, China) and is widely available over the counter. Gentamicin was purchased from Dalian Pharmaceutical Company (Dalian, China, purity ≥ 98%).
This study was performed in accordance with the Use of Laboratory Animals of China, and the animal experiments were approved by the Institutional Animal Care and Use Committee of China Medical University.
2.1
Animals and grouping
Sixty adult guinea pigs (~ 2 months, 200–260 g) with a sensitive Preyer reflex were used for this study. The animals were randomly divided into four groups (15 per group). The control group was injected intramuscularly (IM) with normal saline (2.5 mL/kg body weight/day); the gentamicin group received gentamicin only (100 mg/kg/day, IM); the treatment group received S. miltiorrhiza (3 g/kg/day, IM), followed by gentamicin (100 mg/kg/day, IM) 10 min later; and the S. miltiorrhiza group received S. miltiorrhiza only (3 g/kg/day, IM). The animals were properly labeled and given daily injection of the above drugs for 10 days. The animals were given free access to water and a regular diet, and they were allowed one week to acclimate before treatment.
2.2
ABR testing in the animal model of ototoxicity
Experimental guinea pigs were anesthetized with an intraperitoneal injection of 2% sodium phenobarbital. The animals were placed in an electrical shielding room for auditory brainstem response (ABR) recording. Briefly, a recording needle was placed subcutaneously in the scalp, a ground needle was placed subcutaneously in the mastoid, and a control needle was placed subcutaneously in the other mastoid. The click (duration of 0.1 ms) stimulation of a 12 kHz primary frequency was triggered by a sound stimulator (Danac-7) via a TDH-39 earphone to the ear. The signal was collected by a biological recording system (ATAC-450) with an analysis duration of 20 ms, 200 times on average. The stimulus interval was 90 ms, and the filter band-pass frequency was 100–3000 Hz. The hearing threshold was calculated from a 95 dB sound pressure level, with a 10 dB step decrease after the clear ABR wave was observed. The stimulation wave intensity was decreased by 5 dB each step when it was close to the hearing threshold until the P 3 wave diminished. Then, the sound intensity was regarded as the hearing threshold. The animals were determined to suffer from ototoxicity when auditory threshold shift was more than 20 dB.
2.3
Cochlea sample
The animals were decapitated after the last ABR recording, and the cochlea was swiftly separated. For all groups, the left side of the cochlea was designated for ultrastructural investigation (five from each group were analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively) and Western blot analysis (five cochleae for caspase-3 expression); the right side of the cochlea was designated for iNOS and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. To ensure efficient fixative perfusion, the round window and the cochlear apex were opened after the stapes was removed.
2.4
Preparation of frozen sections of cochlea
After ABR testing, 10 guinea pigs from each group were sacrificed by decapitation, and the cochleae were swiftly separated. After the otocyst was opened, the cochlea was exposed completely. The round window membrane was pricked under an anatomical microscope, the oval window was exposed through dislocation of the base of stapes, a small hole was drilled on the top of the cochlea, and then phosphate buffer containing 40 g/L paraformaldehyde was put into the fixation fluid at 4 °C for 2 h. After washing with phosphate buffer, the sample was put into 100 g/L ethylenediaminetetraacetic acid (EDTA) solution at 4 °C for 5 to 7 days for decalcification, then transferred into phosphate buffer containing 250 g/L sucrose, and left at 4 °C overnight. The next day, the sample was embedded in optimal cutting temperature (OCT) compound for frozen sectioning with a cryostat.
2.5
Preparation of SEM
The cochlea was fixed with 2.5% glutaraldehyde for approximately 4–6 h. The bony capsule was removed, and the basilar membrane was dehydrated, dried, gilded, and then observed under SEM (JEOL JSM-T300 SEM, Japan).
2.6
Preparation of TEM
The cochlea was fixed with 2.5% glutaraldehyde for approximately 4–6 h and decalcified in 10% EDTA for 10 days. The basilar membrane and spiral ligament were rinsed, postfixed with osmium tetroxide, dehydrated with a graded series of ethanol concentrations, and embedded in resin. Serial midmodiolar sections were cut at 50 nm thickness, stained, and observed under TEM (JEOL JSM-T300 electron microscope, Japan).
2.7
iNOS expression
The reagent kit to assay iNOS was provided by Wuhan Boshide Biotechnology Co., Ltd. (Lot no. BA0362, Wuhan, China). Following the manufacturer’s instructions, the primary antibody was used at a 1:200 dilution. In the negative control group, the primary antibody was replaced with 0.01 M phosphate buffer, while the remaining procedure was the same. The intensity of iNOS staining was determined by the mean gray value from an image analysis system (see below).
2.8
TUNEL staining
In order to visualize the apoptotic cells, we used an in situ cell death detection kit (Wuhan Boshide Biotech., MK1020). The tissue sections were treated with proteinase K (20 μg/mL) for 20 min at room temperature and washed twice with phosphate-buffered saline (PBS). They were incubated with the TUNEL mixture solution for 1 h at 37 °C and washed three times with PBS. The sections were incubated for 30 min with horseradish peroxidase at 37 °C and then washed with PBS. Staining was conducted using 3,3′-diaminobenzidine (DAB), and counterstaining was carried out using hematoxylin.
2.9
Western blot for caspase-3
After ABR measurement, cochlear tissues from guinea pigs were dissected and homogenized in ice-cold lysis buffer [25 mM N -2-hydroxyl piperazine- N ’-2-ethane sulfonic acid, pH 8.0, 5 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors]. Cell debris and the nuclear fraction were removed by centrifugation at 10,000 rpm for 20 min at 4 °C. The supernatant was extracted and stored at − 80 °C until ready for use. Samples were incubated in Laemmli sample buffer (62.5 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol) at 95 °C for 10 min. The supernatants were separated by 8% SDS-polyacrylamide gel electrophoresis and electrically transferred to a polyvinylidene difluoride membrane. Nonspecific binding sites were blocked with Tris-buffered saline (100 mM Tris, 0.9% NaCl, pH 7.5), 0.1% Tween 20 (TBS-T), and 5% nonfat dry milk at room temperature for 4 h. Membranes were then incubated in a 1:500 dilution of rabbit polyclonal anti-caspase-3 in TBS-T for 24 h at 4 °C. After three washes in TBS-T, membranes were incubated in a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG anitbody. Immunoreactive caspase-3 protein was detected by enhanced chemiluminescence. The membranes were then stripped and restained with anti-glyceraldehyde-3-phosphate dehydrogenase at a concentration of 1:10,000 for the protein loading control.
2.10
MicroImage analysis
The MetaMorph/Coolsnapfx/AX70 MicroImage analysis system (jointly produced by the USA and Japan) was used, and 20 sections were randomly collected for each group. The slice was put under a microscope, the image was visualized, and the picture was taken with a digital camera and transferred to a computer. After every part of the cochlea to be analyzed was sketched, the gray values of cellular iNOS-positive staining in Corti’s organ, stria vascularis, and spiral ganglion were respectively measured under the same conditions with image analysis software, and then the mean gray value of cellular iNOS-positive staining in each part was calculated. Image color shade is divided into 256 scales according to a computer image analyzer. The binary representations of gray value assume that 0 is black and the maximum value is 255. The higher the color value, the lighter the shade of gray is and the lower the color value, the darker the shade of gray is.
2.11
Analysis of hair cell loss
After ABR measurement, the other five animals of each group were decapitated. The otocyst was quickly removed, and the round and oval windows were pricked under an anatomical microscope. For succinate dehydrogenase (SDH) activity assay, a solution containing 0.05 M sodium succinate, 0.05 M phosphate buffer, and 0.05% tetranitro blue tetrazolium was slowly perfused through the round window for about 1 min, the cochlea was immersed in the solution for 1 h at 37 °C, and then the cochlea was immersed in 4% paraformaldehyde for 24 h. The reduction of SDH activity is commonly related to the damage to the hair cells. After that, the Corti’s organ was dissected into three pieces consisting of the basal, middle, and apical turns, and each part was mounted with glycerin on a glass slide. Specimens were examined under a light microscope to determine the number of missing hair cells. Cytocochleograms were made by plotting the percentage of hair cells remaining in each part of the four individual rows (1 row of IHC and 3 rows of OHCs) measured in millimeters. Summary of cytocochleograms was made by averaging the percentage of remaining hair cells from IHCs and OHCs of each group of animals, which represented the measurement of the treatment response.
2.12
Statistical analysis
Data were presented as mean ± standard deviation (SD). Significant differences between groups were analyzed with SPSS V.11.5 (SPSS Inc., Chicago, IL, USA) using the Student’s t -test and the f -test. A p- value < 0.05 was considered statistically significant.
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