Specific Blocker Of Inos Eit On Bacterial Endotoxin-induced In Vitro Cultured Rat Organ Of Corti Damage Protection Role,

IntroductionLipopolysaccharide( LPS) is component of Gram - negative bacterium s cellular wall . It can stimulate variety of cells to produce cellular factors , free radical and nitric oxide ( NO). Small amounts of the things above can activiate the immune system to kill the pathogen. At present ,some scholars considered that NO plays an essential role in certain inner disorder. Pathogenesis of inner ear disorder is not certain and there's no effective treatment. We choose LPS to construct a model of ototoxicity with emphasis on NO/NOS (isform of nitric oxide syn-thase ) , and use the special inhibitor of iNOS to protect organ of corti of cell culture from the damage. The finding reported here will hopefully enable further progress towards successful ototoxicity treatment.Materials and MethodsMaterial: 30 healthy 3 - day - old Wistar rat were randomly divided into five groups . The first group : normal control group, cultivated only ;The second group:be given 100nM EIT only;The third group:be given 50 ug/ml LPS; The fourth group : be given 200ug/ml LPS; The fifth group: be given 200ug/ml LPS + 100nM EIT. Method: Wistar rat were killed on the third born day, cochlaes were dissected with the aid of watchmaker forceps and a stereomicroscope . Organs of Corti were freed from surrounding tissues and placed on a 30mm culture dish with culture supplement and treatment . After 48 hours , speci-mens were fixed for 4 hours in 4% paraformal - dehyde in 0. IMphosphate buffer(PH7. 4). Specimens were rinsed in 0. 1M PBS, incubated in 0. 25% TritonX - 100 for 5min and immersed in TRITC - labled PHALLOIDIN in PBS for 30min specimens were examined under a fluorescence microscope with appropriate filters for FITC ,the data were analyzed by Students Test.ResultsHair cell incubated in nomal culture supplement for 48 hours remained normal, specimens incubated in lOOnM EIT, show undamaged, the ones incubated in 50ug/ml LPS is almost normal, the specimens incubated in 200ug/ml LPS show great damage in both inner hair cell and outer hair cell, thes pecimens incubated in 200ug/ml LPS plus lOOnM EIT decreased the damage both in inner hair cell and outer hair cell. The data from the last two groups show statistic difference.DiscussionLPS is component of Gram - negative bacterium's cell wall and routinely used for experiments of NO induction by most of investigators. We found hair cells incubated in basal culture medium at a final concentration of 50ug/ml LPS for 48 hours show undamage , while hair cells incubated at a final concentration of 200ug/ml LPS show serious damage. It can suggest the ototoxicity of LPS is dose - depended. EIT can only inhibit iNOS which induces a large amount of ototoxic NO. It cant inhibit cNOS which induce small amount of NO related to the normal phsiological activity. So it hardly damage hair cells. SoEIT,a special inhibitor of iNOS can prevent the damage of LPS to the hair cells, it may be a very promising medicine to the inner disorder.ConclusionEIT,a special inhibitor of iNOS can effectivelly protect LPS -treated Cortis Organ explanted in culture.