Inhibitive Effect Of Acanthopanax Senticosus Injection On Gentamicin-induced Ototoxicity In Guinea Pigs

Aminoglycoside antibiotics ( AmAn) are widely used drugs for they have many advantages, such as stable nature, wide anti - bacterial spectrum, cheap and convenient. Although AmAn is suitable for clinical use, their severe side -effects nephrotoxicity and ototoxicity cant be ignored. AmAn ototoxicity has high incidence, once it happens and cant be reversible and even cause deafness. So it is urgent to know the mechanism of the AmAn ototoxicity and to supply theory evidences to prevent and cure the disease. There are many hypothesis about AmAn ototoxicity, for example, drugs storing up in inner ear, free radical damage, apoptosis, second messengers, mitochondrial DNA, metal ions, and so on.The traditional Chinese medicine Acanthopanax senticosus (ASS) injection is distilled from the root and stem and has the function of invigorating the blood and removing extravasated blood. By a great deal of experiments, evidences show that ASS helps to enhance non - special immune , fight cancer and aging, anti - fatigue , anti - radiation, and so on . ASS is widely used clinically in China as an effective remedy for cerebrovascular disorders, diabetes, neurasthenic and chronic bronchi et al with minimal side effects. But there is no report that ASS could be used for treat AmAn ototoxicity. According to ASS injection could dilate coronary artery, eliminate free radical and its other pharmacological effects, we speculated ASS could also dilate cochlea blood vessels, improve blood stream, ameliorate the disorders of free radicals, promote hair cells energy metabolism and improve the ability to deal with the situation of be short of bloodand oxygen. All these effects may decrease aminoglycoside ototoxicity. The aim of our experiment is to know if ASS could antagonism the AmAn ototoxicity and by which way it plays roles and supply theory evidences for clinical use.Methods1. Experimental groups and preparation of AmAn ototoxicity model Albino guinea pigs of either sex (200 ~250g) were obtained from the second clinical collage breeding laboratory of China medical university. Animals were divided into four groups at random. GM group: GM was injected intraper-itoneally in a volume of lOOmg/kg;ASS + GM group: injected GM lOOmg/kg+ ASS 200mg/kg in the two sides of abdominal separately;ASS group: injected ASS injection 2oomg/kg;the control group. Drugs were administrated 10 days and adjusted the drug dose by weighting.2. ABR testAuditory thresholds were measured by evoked ABR. Thresholds were taken for each animal at the beginning of the study, 10 days after the start of drug treatment. Animals were anesthetized with intraperitoneal injection of 1% sodium pentobarbital 40mg/kg. The positive needle electrode was inserted at the vertex, the midline of the scalp between the external auditory canals. The negative electrode was placed below the pinna of the left ear, and the ground electrode was inserted contralaterally. The sound stimuli consisted of 20ms tone bursts at the frequency of 2 Hz and were generated and delivered to the external auditory metus in a closed acoustic system through an ear bar connected to a DT -100 thansducer. The observe duration is 20ms and the intensity begin from 95dBnHl. The threshold is judged according to the wave P3.3. Preparation and detection of cochlea specimens3.1 SABC immunohistochemistry and TUNEL;animals from each group were headed, the temporal bones were immediately removed, two sides of otocyst were taken out. Open the otocyst, exposed the cochlea, put the cochlea into 4%' paraformaldehyde. After perfusion with the same fixative through the hole at the apical and immersed rinsed at 4t for 2 h, decalcification was done in neutral-ized 10% EDTA solution at 4% for 7 days, and then normal HE staining, investigate the expression of iNOS and Glu, the apoptosis with TUNEL method.3. 2 Cochlea samples for TEM and SEM: After recorded ABR and drug administration, 3 guinea pigs from each group were selected at random. We beheaded the guinea pigs rapidly, took out two sides of otocysts and opened them, exposed the cochleal, put the cochlea into 2. 5% glutaric dialdehyde. TTie bone near the apex, round window and oval window were opened, followed by perfu-sion from the apex with 2. 5% glutaric dialdehyde. The samples were prepared according to routine procedures for TEM and SEM.3. 3 Western blot methods;All the animals were headed and took off oto-custs. Put them into the 0.9% physiological saline and separate the bony - cartilaginous cochlea capsule. After ultrasonic smashed and 4t centrifuged 17500rpm for 2 hours, the samples were made standard. The processes to dispose the protein are electrophoresis and transduct membrane, use 5% calf serum protein act 1 hour, incubate with the rabbit polyclonal antibody overnight, PBS flush, add special labeled antibody 2 hours. The results were analyzed with GIS -700D gel image system.3. 4 OHCs preparation: Animals were killed and the temporal bones were rapidly removed from the skull. After the bulla was opened, the bony capsule of the cochlea was picked away to expose the spiral ligament. The organs of Corti were incubated for 15min in balance salt containing 0. 5mg/mL trypsin. After terminate the digest, hair cells were mechanically dissociated by gently blowing with a micro syringe and were then transferred into the 24 - well.3. 5 Calcium imaging with fluo - 3;Add 0. 5ul fluo - 3/ AM to each well, incubate 30 min then terminate, observe the change of OHCs [ Ca2 + ] i.4. Image analysis: MetaMorph - Coolsnapfx - AX70 micro image analysis system was used to test average gray value of iNOS and Glu positive reaction of each group. Apoptosis: Apoptosis positive cell nucleus is expressed by yellowish brown pellet, and the normal cell nucleus is blue. Western blot results were analyzed with GIS -700D gel image system.5. Statistical analysis: The data were represented byx ± s, t test was used with SPSS10.0 for statistical analysis.Results1. ABR thresholdsNo significant differences in the ABR thresholds were found between experimental groups at the beginning of the study;GM alone caused thresholds elevation. Combination groups could significantly decrease the thresholds. No differences in the ABR thresholds also were found between ASS group and control group.2. Immunohistochemistry for iNOS and GluThere were no iNOS expression in control group and ASS group. Localization of iNOS in the cochlea of guinea pigs from GM group and ASS + GM group was significantly deeper than that in normal group. Whereas iNOS staining in ASS + GM group were significantly lower than that in GM group. Glu were expressed in GM group much stronger than those of control group.3. Observation under TEM and SEMThe structure of hair cells in control group and ASS group is normal and has no much change. But the hair cells of GM group showed the typical morphologic character-, their nuclear chromatin condensation and concentrated cytoplasm. The organells kept intact and budding into several membranes - encapsulate bodies. The number of the mitochondrion decreased and the structure were not clear. The ASS + GM group hair cells membrane were smooth and there were some balloons and no formation of encapsulate bodies. Under SEM, IHCs and OHCs of control group were clear;the sensory hair losses to varying degrees in hair cells of GM group while the combination group had less hair cell lose.4. The expression of caspase - 3The expression of caspase - 3 in control group and ASS group were a little, but in GM group were much more. The western blot picture also showed the expression of caspase - 3 in ASS + GM group were much lower than that of GM group and much higher than that of control group and ASS group.5. The results of apoptosisThere were no apoptosis cells in control and ASS group cochlea. But in GMgroup, there were lots of apoptosis cells. The number of apoptosis cell in ASS + GM group was much lower than that of GM group.6. The effect of Glu and ASS to OHC [Ca2+ ]iGlu can enhance the OHCs [ Ca2 + ] i while ASS can decrease the OHCs [ Ca2 + ] i caused by Glu.Conclusion1. During GM ototoxicity, iNOS expression strengthens, so we speculated ASS could alleviate GM cochlea injure by inhibiting the expression of iNOS.2. Apoptosis is a kind of way of GM ototoxicity, while ASS could antagonism it by decreasing the caspase - 3 expression.3. There is Glu immune histochemistry reactions increasing which supply more Glu may be involved in GM ototoxicity.4. Glu can enhance the OHC [Ca2+ ]i while ASS can decrease the OHC [ Ca2 + ] i caused by Glu.