| Objective:Aminoglycoside antibiotics (aminoglycoside antibiotics, AGs) or by micro-organisms taken by the semi-synthetic system for a class of neutral sugars and amino sugar or amino alcohol glycoside key ring combination of antibiotics alkaline soluble in water.The mechanism is mainly inhibition of bacterial protein synthesis, mostly through the kidney after intramuscular injection to the prototype discharge, cause reversible or irreversible vestibular, cochlear and kidney damage, the ear has become the drug of renal toxicity, a major problem for human.Through the application of this study was to observe the effect of immunohistochemistry TMP (TMP) interfere with gentamicin (GM) poisoning, and kidney in guinea pigs 5-bromodeoxyuridine (BrdU) labeled cells positive positioning in the organization and distribution, and evoked response and auditory brainstem (ABR) test verified from the morphology and function in traditional Chinese medicine poisoning GM TMP can promote hair cells in guinea pigs and kidney tissue proliferation (ie regeneration), and the proliferation of cells in the cochlea by electron microscopy source; application PCR reaction, observe the effect of TMP intervention guinea pig renal mitochondrial poisoning GM DNA (mtDNA) deletion and to explore TMP promote regeneration of damaged cells and its mechanism.Method:(1) Experimental animals and administration divisionThe pinna reflection sensitive health guinea pig (BaiSeGong mesh) 40, only weigh 200 330g, male and female, not limited, random divided into four groups (each group of 10 only) gentamycin group (GM):continuous 10 daily GM100 mg/kg intraperitoneal injection; Traditional Chinese medicine ligustrazine+gentamycin group (TMP+GM):continuous 10 GM 100mg intraperitoneal injection daily left and right/kg intraperitoneal injection TMP140mg/kg; Traditional Chinese medicine ligustrazine group (TMP):continuous 10 daily abdomen.(2) Experiment testing the followingListen brainstem response (ABR) threshold detection; cochlear frozen sections and paraffin-embedded sections of renal BrdU detection; transmission electron microscopy of cochlear hair cells of the situation; by nested polymerase chain (PCR) reaction for the detection of mtDNA in renal tissues missing.Result:(1) Chinese intervention in GM ototoxicity TMP ABR threshold changes of cochlear determination resultsBefore treatment ABR thresholds in each group showed no significant (P>0.05), GM group after treatment was significantly higher ABR thresholds, and the difference was significant compared with control (P<0.01). GM+TMP group of ABR threshold, although there are increased after treatment, but significantly lower than the GM (P<0.05). TMP group and control group before and after treatment ABR thresholds did not change significantly (P>0.05).(2) Brdu incorporation of the results of immunohistochemistry1) Brdu cochlear hair cells mixed results of immunohistochemistryCochlear hair cells observed under light microscope, Brdu the normal control group and TMP group were the most light colored, GM group centered, GM+TMP group most, was strongly positive expression (Fig3.1). Image analysis showed that the normal control group and TMP group Brdu gray value were significantly higher than the other two groups (P<0.05), GM+TMP group of gray value lower than the GM group (P<0.05, Tab.3.2).2) Brdu incorporation of the kidney tissue immunohistochemical resultsLight microscope the kidney tissue, Brdu the normal control group and TMP group were the most light colored, GM group centered, GM+TMP group most, was strongly positive expression (Fig.3.2). Image analysis showed that the normal control group and TMP group Brdu gray value were significantly higher than the other two groups (P<0.05), GM+TMP group of gray value lower than the GM group (P<0.05, Tab3.3).(3) TEM morphological changes of cochlear hair cells1 d GM group after stopping nuclear membrane of outer hair cell shrinkage, depression, nuclear chromatin condensation, condensation, chromatin margination, vacuolar degeneration of mitochondria (Fig 3.4-B); TMP+GM Group:withdrawal 1d cochlear hair cells after the damage was lighter than the GM group, hair cells in the cell body and nuclear morphology were normal, vacuolar degeneration less (Fig 3.4-C). Therefore, TMP can reduce GM ototoxicity and improve the role of auditory function. We listen to functional recovery after treatment shows that the new guinea pigs found that the cells from the basement membrane of the basal surface and the damaged sensory epithelium of the middle layer of transitional cell layer to the cavity surface. Bubble has engulfed the cytoplasm; nucleus oval or irregular shape. The feeling in the damaged surface layer of cells on the bellows of the new free surface of cells can be seen that microvilli, tight junctions with surrounding cells exist (Tab3.4).(4) MtDNA4568 deletion in guinea pig bloodThe results showed that GM group and the GM+TMP group, gel electrophoresis can be seen clearly 404bp band (Tab3.7), consistent with the theory, indicating that GM group and the GM+TMP group were mitochondrial DNA deletion; and relatively mtDNA4568 the incidence of missing, The results showed that GM group mtDNA4568 deletion was higher than GM+TMP group, a statistically significant difference between the two groups (χ2 test, P<0.05, Tab3.4).Conclusion:GM is AGs, is a broad-spectrum antibiotics against gram-negative bacteria. It is an ear nephrotoxic drugs, can lead to sensorineural hearing loss and kidney damage. TMP is a "blood gas medicine," said traditional Chinese medicine extracted Chuanxiong effective monomer, now widely used in clinical treatment of blood stasis syndrome of both diseases. Chinese TMP significantly on the hydroxyl radical scavenging reduce the kanamycin in the cochlea of guinea pigs caused by free radicals, improve hearing.The first experimental observation of the TMP on GM ototoxicity hearing of guinea pigs, the results show that the TMP to GM ototoxicity in guinea pigs was significantly lower ABR thresholds, suggesting that TMP can significantly reduce the damage leaving GM ototoxicity hearing can be improved. Our group found by scanning electron microscopy:GM ototoxicity in guinea pig cochlea after stopping the second transfer 30d inner and outer hair cell stereocilia and the normal control group showed no significant difference. While the third switch to a newborn cochlea stereocilia appears that gentamicin after injury can regenerate hair cells.In this study, the introduction of exogenous BrdU, after immunohistochemical staining, the digital camera to take pictures, images processed by the morphological analysis software showed that TMP can promote cochlear hair cells and kidney cells (regeneration). The TMP can reduce transmission electron microscopy proved GM ototoxicity and improve the role of auditory function.We listen to functional recovery after treatment shows that the new guinea pigs found that the cells from the basement membrane of the basal surface and the damaged sensory epithelium of the middle layer of transitional cell layer to the cavity surface. Bubble has engulfed the cytoplasm; nucleus oval or irregular shape. The feeling in the damaged surface layer of cells on the bellows of the new free surface can be seen that cell microvilli in close connection with the surrounding cells (Fig 3.5).In summary, this study shows that the absence of GM by making mtDNA renal toxicity resulting from the ear, so that hair cells and kidney damage, resulting in renal toxicity of the ear; TMP activity through its reduction of the role of oxygen free radicals, etc., effectively reduce GM ototoxicity in guinea pigs of the hearing threshold, effectively reduce the toxicity of GM-oto-renal damage, and to promote the proliferation of damaged cochlear hair cells, in order to clarify and improve the mechanism of renal toxicity, and Ear AGs intervention TMP cell injury and repair mechanisms of drug toxicity to provide a theoretical basis Ear drugs for prevention and treatment provide the theoretical basis of renal toxicity, and to find new ways to develop new drugs.
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