OBJECTIVETo investigate the effects of salvia miltiorrhiza injection (SM) on gentamicin (GM)-induced expression of nitric oxide synthase (NOS) isoforms in guinea pig cochlea, and to explore the protective mechanism of SM on GM-induced ototoxicity.

METHODS40 guinea pigs were randomly assigned to 4 groups: control group, GM group, SM group and GM plus SM group. Expression of NOS isoforms in the guinea pig cochlea was detected by the SABC method of immunohistochemistry and microscope image analysis technique. Auditory threshold was tested by auditory brainstem response (ABR) measurement.

RESULTSInducible NOS (iNOS/NOS II) expression and ABR threshold in GM plus SM group were both significantly declined as compared with those in GM group (P < 0.01). Moreover, change of iNOS expression was in high correlation with that of ABR threshold ([r] > 0.7, P < 0.01). While expression of neuronal NOS (nNOS/NOS I) and endothelial NOS (eNOS / NOS III) showed no significant differences in all groups.

CONCLUSIONSM had no effect on the expression of nNOS and eNOS, but could inhibit iNOS high-expression induced by GM to reduce excessive generation of NO, therefore SM could protect against GM ototoxicity.

Animals ; Cochlea ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gentamicins ; toxicity ; Guinea Pigs ; Male ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Protective Agents ; pharmacology ; Salvia miltiorrhiza ; chemistry

Aminoglycoside antibiotics, due to their strong antibacterial effects and broad antimicrobial spectra, have been very commonly used in clinical practice in the past half century. However, aminoglycoside antibiotics manifest severe ototoxicity and nephrotoxicity, and are one of top factors in hearing loss. In this study, three members of the aminoglycoside antibiotics family, gentamycin, neomycin and streptomycin, were chosen as the representatives to be investigated for their toxicity to the embryonic development and the larva hair cells in zebrafish, and also to their target genes associated with hearing-related genes. The results showed that: (1) the lethal effect of all three drugs demonstrated a significant dependence on concentration, and the severity order of the lethal effect was streptomycin > neomycin > gentamycin; (2) all the three drugs caused the larva trunk bending in resting state at 5 dpf (day past fertilization), probably due to their ototoxicity in the physical imbalance and postural abnormalities; (3) impairment and reducing of the hair cells were observed in all three cases of drug treatment; (4) four genes, eya1, val, otx2 and dlx6a, which play an important role in the development of hearing organs, showed differential and significant decrease of gene expression in a drug concentration-dependent manner. This study for the first time reports the relevance between the expression of hearing genes and the three ototoxic antibiotics and also proved the feasibility of establishing a simple, accurate, intuitive and fast model with zebrafish for the detection of drug ototoxicity.
Aminoglycosides ; toxicity ; Animals ; Anti-Bacterial Agents ; toxicity ; Embryonic Development ; drug effects ; Gene Expression Regulation ; Gentamicins ; toxicity ; Hair Cells, Auditory ; cytology ; drug effects ; Hearing Disorders ; chemically induced ; genetics ; metabolism ; Homeodomain Proteins ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Larva ; drug effects ; Lateral Line System ; drug effects ; MafB Transcription Factor ; metabolism ; Models, Animal ; Neomycin ; toxicity ; Nerve Tissue Proteins ; metabolism ; Nuclear Proteins ; metabolism ; Otx Transcription Factors ; metabolism ; Protein Synthesis Inhibitors ; toxicity ; Protein Tyrosine Phosphatases ; metabolism ; Streptomycin ; toxicity ; Zebrafish ; embryology ; Zebrafish Proteins ; metabolism

OBJECTIVETo study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro.

METHODSAfter the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software.

RESULTSGM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05).

CONCLUSIONSELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.

Animals ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Gentamicins ; toxicity ; Hair Cells, Auditory, Inner ; drug effects ; pathology ; Hair Cells, Auditory, Outer ; drug effects ; pathology ; Mice ; Organ of Corti ; drug effects ; pathology ; Prescriptions ; Tablets

OBJECTIVETo explore an effective method for treating Gentamicin-induced deafness and the mechanism.

METHODSGuinea pigs were randomly divided into 5 groups: normal control group (group A), model group (group B), Ligustrazine group (group C), acupuncture group (group D) and Ligustrazine plus acupuncture group (group E). The group C, D and E were treated respectively by simple Ligustrazine, simple acupuncture at "Tinggong" (SI 19), "Yifeng" (TE 17), and "Waiguan" (TE 5), and Ligustrazine plus acupuncture. Ten days later, the auditory brainstem response (ABR) thresholds for the wave III , apoptosis and expressions of Bcl-2 and Bax proteins in the organ of Corti of the guinea pig were detected.

RESULTSIn the group E, the ABR threshold was significantly lower than that in the group C (P<0.05), and apoptotic cells, the expression of Bax protein and the ratio of Bax/Bcl-2 were lower than those in the group C and D in the organ of Corti, and Bcl-2 protein expression was increased.

CONCLUSIONAcupuncture at "Tinggong" (SI 19), "Yifeng" (TE 17), and "Waiguan" (TE 5) has a certain target-synergistic action on Ligustrazine and can increase therapeutic effect of Ligustrazine on Gentamicin-induced deafness, which are possible related with the inhibition of apoptosis, down-regulation of Bax expression and up-regulation of Bcl-2 expression.

Acupuncture Therapy ; Animals ; Anti-Bacterial Agents ; toxicity ; Deafness ; chemically induced ; therapy ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; Female ; Gentamicins ; toxicity ; Guinea Pigs ; Male ; Pyrazines ; therapeutic use

OBJECTIVETo explore the effect of acupuncture at "Neitinggong" drug-induced deafness.

METHODSGuinea pig deafness model was prepared by injection of gentamicin (GM). Acupuncture was respectively given at the points "Neitinggong" "Tinggong" (SI 19) and non-acupoints on the auricle in the experimental animals in different groups and the effects of different points on the auditory brainstem response and cochlear hair cells were observed.

RESULTSThere was a significant difference between GM group and Neitinggong group, and between GM group and Tinggong group. There was no significant difference between GM group and the auricle group, and between Neitinggong group and Tinggong group.

CONCLUSIONAcupuncture at "Neitinggong" can strength the function of the internal ear, and relieve the injury of cochlear hair cells caused by gentamicin, which is an effective acupoint for treatment of drug-induced deafness.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Anti-Bacterial Agents ; toxicity ; Cochlea ; drug effects ; pathology ; Deafness ; chemically induced ; prevention & control ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; Female ; Gentamicins ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; drug effects ; pathology ; Male

AIMTo study the antagonistic action of acanthopanax senticosus injection (ASS) on gentamicin ototoxicity.

METHODSGuinea pigs were randomly divided into control group, GM group, ASS group, and ASS + GM group. The changes of hearing threshold, cochlear morphology, expression of caspases-3 were determined by ABR, TEM, and Western blot, respectively.

RESULTSThe ABR threshold in GM group increased markedly. There was no significant difference in ABR threshold between ASS group and control group, but the ABR threshold in ASS group was much lower than that both in GM group and ASS + GM group. Severely injured hair cells with morphological characteristics of apoptosis were found under TEM in GM group, and the hair cells were less injured in ASS + GM group. The results of Western blot showed that the expression of caspase-3 increased markedly in GM group, but it increased slightly in ASS + GM group.

CONCLUSIONASS may antagonize the GM ototoxicity by inhibiting the expression of caspases-3.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Eleutherococcus ; Gentamicins ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; cytology ; drug effects ; Plant Extracts ; pharmacology

The nephrotoxicity of gentamicin (GM) has been widely recognized. Heat shock protein 72 (HSP72) has been reported to be a cytoprotectant. However, its cytoprotective effect against GM induced kidney injury has not yet been studied. In this study, we investigated the cytoprotective effect of HSP72 on GM-induced nephrotoxicity in vitro. Human Kidney tubular cell line, HK-2 cells were divided into four groups: control group, GM group (cells incubated with GM only), heat shock (HS) group (cells incubated at 43 degrees C for 30 min), and GM plus HS group, respectively. Lactate dehydrogenanse (LDH) release increased time-dependently from 24 hr to 96 hr compared to the data of cells treated with GM only. Results of NAG activities, superoxide dismutase (SOD) activities and malondialdehyde (MDA) content were similar to that of the LDH release. The amount of HSP72 positive cells increased significartly at 72 hr after cells were treated with GM only. Both HSP72 protein and gene expression increased significantly at 72 hr when cells were treated with GM. On the other hand, HS induced HSP72 expression markedly. Pretreatment of HS inhibited HK-2 cells from GM-induced injury. It could reduce LDH release and NAG activity. HS also increased SOD activity, and decreased MDA content when cells were damaged by GM. These findings suggested that HS may protect kidney cells from GM-induced injury. Pre-induction of HSP72 may provide therapeutic strategies for nephrotoxicity induced by GM.
Reactive Oxygen Species/metabolism ; RNA, Messenger/analysis ; Oxidation-Reduction ; L-Lactate Dehydrogenase/secretion ; Kidney Tubules, Proximal/chemistry/*drug effects ; Humans ; Heat ; HSP72 Heat-Shock Proteins/analysis/genetics/*physiology ; Gentamicins/*toxicity ; Cytoprotection ; Cells, Cultured

OBJECTIVETo investigate uptake and accumulation of gentamicin by cells in the guinea pig inner ear after intratympanic injection using a fluorescent probe--gentamicin-Texas-red conjunction (GTTR).

METHODSAdult guinea pigs (n = 80) were administered a single dose of GTrR to the middle ear cavity through the intact membrane and survived for 12 h, 24 h, 48 h, 3 d, 4 d, 7 d, 14 d and 28 d. The distribution of GTTR in the cochlear and vestibular cells was observed after staining with phalloidin-alexa-488. Texas Red and DMSO were injected into the tympanum as control.

RESULTSDiffuse staining of gentamicin in the labyrinth was observed initially after local drug administration. At later time point the outer hair cells and sensory cells of vestibular organ were staining more densely than the support cells in the inner ear. The peak level of fluorescent density was reached 3 days after local injection. The GTTR was observed in the infracuticular zone.

CONCLUSIONSGTTR was a potential fluorescent probe to investigate the pharmacokinetics and mechanisms of gentamicin accumulation in local application.

Animals ; Anti-Bacterial Agents ; administration & dosage ; pharmacokinetics ; toxicity ; Ear, Inner ; metabolism ; Fluorescent Dyes ; Gentamicins ; administration & dosage ; pharmacokinetics ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; metabolism

To examine the significance of heat shock protein 70 mRNA in ototoxicity resulted from gentamicin (GM), twenty healthy albino guinea pigs (200-250 g) of either sex with a positive Prier reflex were divided into two groups randomly. In GM group the animals received 100 mg/kg GM daily by intraperitoneal injection for 10 d. In saline control group the animals received 2.5 ml/kg saline daily by intraperitoneal injection for 10 d. Auditory brainstem response (ABR) thresholds were recorded in each animal before and 1 d after GM or saline administration. After the second ABR measurement, the expression of HSP70 mRNA in guinea pig cochlea was observed with in situ hybridization and image quantitative analysis system. The results showed that the threshold of ABR in the GM group was significantly higher than that of the saline control (P< 0.001). The expression of HSP70 mRNA was more intensive in stria vascularis, spiral ligament and spiral ganglion cells in the GM group than that of the saline control group. These results suggest that administration of gentamicin can induce the expression of HSP 70 mRNA in guinea pig cochlea, and that this effect may protect hearing function from ototoxicity.
Animals ; Cochlea ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; physiology ; Female ; Gentamicins ; toxicity ; Guinea Pigs ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation

AIMTo investigate the effects of injectio Salvia Miltiorrhiza (SM) on gentamicin (GM)-induced free radical formation in guinea pig cochlea, and to explore possible mechanisms on GM-induced ototoxicity.

METHODSBiochemical assays of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in guinea pig cochlea, combined with auditory brainstem response (ABR) measurement and transmission electron microscopic observation were used in this investigation.

RESULTSSOD activity was significantly declined while MDA content was distinctly increased in cochlear tissues after GM injection (P < 0.01). Moreover, they were well correlated with auditory function damage (|r| > 0.7). Co-treatment with SM evidently enhanced SOD activity and decreased MDA content (P < 0.01, P < 0.05). Furthermore, auditory function was markedly ameliorated. Morphological changes of cochlea were consistent with those of hearing function.

CONCLUSIONLipid peroxidation elicited by free radical was involved in GM-induced cochleotoxicity. SM might enhance SOD activity and prevent lipid peroxidation. As the result it might alleviate GM ototoxicity, and improve auditory function.

Animals ; Cochlea ; drug effects ; metabolism ; Free Radicals ; metabolism ; Gentamicins ; toxicity ; Guinea Pigs ; Lipid Peroxidation ; Malondialdehyde ; analysis ; Salvia miltiorrhiza ; Superoxide Dismutase ; metabolism