Repair Mechanism Of TMP On Cochlea Hair Cell Damaged By Drug Ototoxicity

Objective:The drug toxicity of Amino-glycosides antibiotics (aminoglycoside antibiotics, AmAn) to cochlea and the vestibular has become a major problem for mankind. Therefore, many scholars have devoted to drug toxicity damage to the inner ear,ear hair cells regeneration research.It is generally believed that mammals cochlear hair cells do not have damage repair capacity. Recent studies show that vestibular hair cells in mammals maintain(ing)a certain level of regeneration and repair capacity after being damaged.In the normal machine, there is an effective antioxidant defense system to prevent the body from the injury of oxygen and its metabolites.TMP is an active ingredients of traditional Chinese medicine Chuanxiong,it can inhibit platelet aggregation, expansion of blood vessels, improve microcirculation and other role, it can direct the elimination of oxygen free radicals to prevent lipid peroxidation, and the protection of the role of hair cells.This study is intended to use guinea pig cochlea and blood for the study to TMP, gentamicin (GM) for experimental drug studies,observation gentamicin hearing on the impact of guinea pigs by testing blood and super-oxide Toki dismutase (SOD) activity and malondialdehyde (MDA) in mtDNA,as well as mitochondrial (mtDNA) deletions and 5 - Bromodeoxyuridine (BrdU) incorporation in cells in the cochlea of the expression Drug ototoxicity cell damage and repair mechanisms.Method:1 Experimental animals and administration division Ear reflex sensitive health-white guinea pig 4 groups, each with 10.Saline group:daily intraperitoneal injection of saline 2.5 ml / kg for 10 d;gentamicin group: daily intraperitoneal injection of GM100 mg / kg for 10 d;ligustrazine + gentamicin group:the left daily intraperitoneal injection GM100mg/kg right TMP140mg/kg intraperitoneal injection for 10 d;TMPgroup:TMP140mg/kg daily intraperitoneal injection for 10 d.Four groups of animals mixed farming,measuring body weight to adjusted daily dose.Stop injecting drugs guinea pigs were killed after six hours before the intraperitoneal injection BrdU100mg/kg weight2 Test the following: listening to auditory brainstem response (ABR) threshold detection;cochlear frozen sections of BrdU incorporation detection; SOD activity in the blood, MDA Determination;using nested polymerase chain (PCR) reaction for the detection of blood in the mtDNA deletion.Result:1. ABR threshold tests showed: before treatment ABR threshold in each group showed no significant difference (P> 0.05),after treatment,GM group ABR threshold is significantly higher than before treatment, and with normal control Comparing the difference was significant (P <0.01).GM+TMP group ABR threshold was also increased after treatment, but significantly lower than the GM (P <0.05).TMP group and the normal control group in the ABR threshold before and after treatment did not change significantly (P> 0.05).2. BrdU immunohistochemical results show that:optical microscopy,the normal control group and the most superficial coloring TMP group,the middle group GM,the GM+TMP deepest group showed strong expression (Fig 1).Image analysis showed that the normal control group and gray TMP group was significantly higher than the other two groups (P <0.05),Gray GM+TMP group was significantly lower than the GM group (P <0.05).3. SOD activity and MDA Determination of Value: after administration, GM group SOD activity in the cochlear tissue decreased significantly,MDA significantly increased, compared with control group (P <0.01).GM+TMP group cochlea SOD activity was significantly higher than that in the GM group, and the content of MDA were lower than GM,and the difference was significant (P <0.01,P <0.05).TMP group and the normal control group showed no significant difference (P> 0.05).4. Guinea pigs mtDNA deletion of the tests showed: The results showed that GM group and GM+TMP group electrophoresis images clear that the 404 bp band (Figure 2), consistent with the theory, the GM group and GM +TMP group mitochondria are missing;and compared with the incidence of mtDNA deletion,The results showed that GM group mtDNA deletion higher than the incidence of GM+TMP group, difference between the two groups is statistically significant (χ2 test,P <0.05).Discussion:Gentamicin-glycosides of antibiotics, it can lead to sensorineural hearing loss.Based on the experimental guinea pig given gentamicin ABR threshold tests verified the ototoxicity gentamicin caused by hearing impairment.recent years,the study found that many diseases exist in oxygen free radical injury.Oxygen free radicals is currently poisoning,such as poisoning the basis of the pathologic process.Gentamicin and iron chelate iron and the formation of gentamicin complex,it can induce the formation of oxygen free radicals,oxygen free radicals in the cochlea increase in the number of these free radicals and hair cell membrane inositol phosphate ester closely integrated The combination provides a kind of free radicals formed by the electron donor needs, resulting in oxidation-reduction reaction between lipid peroxidation,and SOD, glutathione peroxidase (GPX),catalase ( CAT) was significantly consumption, resulting in tissue and cell damage. Based on the experimental guinea pigs given gentamicin in the blood after MDA levels were significantly increased activity of SOD and the decreased significantly, the results confirmed the cochlea gentamicin can promote the excessive oxygen free radicals and lead to a hearing ototoxicity injury.Many gene mutations and the pathogenesis of the deaf. Mitochondrial oxidative phosphorylation through the process of cell ATP is the essential source of energy, this process is a process of oxygen free radicals.The present study considers that reactive oxygen species (ROS) that can damage DNA and mutant DNA strand breaks. MtDNA in particular, because of its structural proteins or other non-DNA-binding protein protected, no correction in the replication and DNA repair function itself, the ROS susceptible to injury. Molecular biology studies have shown that amino-glycosides antibiotics through 125 rRNA and mitochondrial function and inhibit oxidative phosphorylation of the respiratory chain in the process of protein synthesis, and further the cause respiratory chain defects and oxidative phosphorylation efficiency, a lack of ATP, which cochlea and vestibule leading to the injury or death. Through this experiment by giving gentamicin after deletion of mitochondrial DNA in the blood the result was significant it shows the determination of gentamicin can induce mitochondrial DNA deletion or mutation caused ototoxicity lead to hearing damageTMP of Chinese medicine has increased the activity of SOD, scavenging oxygen free radicals, improve blood circulation, such as anti-lipid peroxidation pharmacological activity. In this study give gentamicin at the same time give TMP, in accordance with the determination of ABR threshold, SOD activity in the blood, MDA and mtDNA deletion results show TMP could antagonize the ototoxicity gentamicin, improve IP Adriamycin poisoning after a hearing impairmentThe research shows that BrdU is ideal target to reflect cell proliferation. In the body tissues and cells there is no endogenous BrdU exist. The introduction of in vivo BrdU, to replace the thymidine by the proliferation of the cells in S phase cells specific to the nucleus of its intake by immunohistochemical methods can be specific markers to show BrdU-positive cells. In this experiment in the introduction of exogenous BrdU, the immunohistochemical staining, the digital camera to take photographs, after the image morphology software analysis results showed that the normal control group and gray TMP group was significantly higher than the other two groups (P <0.05). , GM + TMP group was significantly lower than the gray GM group (P <0.05). smaller gray value, the stronger the positive reaction that TMP can promote the formation of new hair cells, and repair fuction of the toxicity of gentamicin ear.Conclusion:Gentamicin can induce ototoxicity, hearing impairment, and outer hair cells by interfering with the energy metabolism, and promote the formation of oxygen free radicals, the body mtDNA deletion or mutation, resulting in the cochlear and vestibular cell injury or death hearing loss. And through its TMP improve microcirculation, the protection of the role of vascular endothelial cells, effectively reduce the toxicity of gentamicin ear, and the body by reducing the formation of oxygen free radicals and increase the activity of SOD and Reduce blood mtDNA deletion speculate but also reduce the cochlea of mtDNA deletion and promote the cochlea Freshmen such as the role of hair cells, effective antagonist of the toxicity of gentamicin ear and improve hearing.The results of this experimental study to clarify and improve the amino-glycosides antibiotic TMP ototoxicity mechanism and drug intervention ototoxicity cell repair mechanisms provide a theoretical basis for the prevention and treatment of deafness poisoning provide a theoretical basis for the development of new drugs and to find new ways.