| BackgroundIt has been known that around a third of the inner ear mutations in fish are associated with human syndrome and non syndrome deafness. Zebrafish has emerged as an important vertebrate animal model. Although there is no outer and middle ear in zebrafish, it has typical inner ear structures for vertebrate. The molecules and the morphology of the inner ear hair cells in zebrafish are very similar to that of the mammal. In addition, zebrafish has another sensory organs—the lateral line, which is comprised of several neuromasts, each neuromast containing a cluster of hair cells. What’s more, the zebrafish possesses a lot of advantages such as small body, simple cultivation, in vitro fertilization, in vitro growth, transparent embryos, strong fertility, short reproductive cycle, varies breeds and so on. Thus zebrafish has obvous advantages to study the inner ear development and the ototoxic drugs.As one of the clinical commonly used chemotherapy durgs, cisplatin plays an important role in the treatment of head and neck malignant tumor such as nasopharyngeal carcinoma (NPC). While in the progress of treatment of tumor, cisplatin has different degrees of side effects on the body. It has been reported that about 93% of the patients with malignant tumors who accept cisplatin chemotherapy developed progressive or irreversible nerve deafness sexy voice, seriously affecting their life quality. For the further study of the features generation mechanism, drug prevention and regeneration after injury of cisplatin ototoxicity, many scholars have adopted different animal models and in vitro experiments and obtained some research results, but the damage mechanism of cisplatin in inner ear hair cells are still not very clear. Many studies have shown that some important mechanisms participate in cisplatin ototoxicity, such as DNA damage, oxidative stress and inflammation factor (IL-6, IL-8 and TNF-a, etc.) production, etc.Reactive oxygen species (ROS) is the chemical activity molecules produced in the process of metabolism, including oxygen ions and oxygen free radicals and hydrogen peroxide, etc. Under normal circumstances, the body produces a certain amount of ROS, and ROS will be involved in cell signal transduction process, plays an important role in maintaining the body. But when the body is stimulated excessive reactive oxygen species, high levels of reactive oxygen species can damage cells and genetic structure.Neutrophils capture mechanism (NETs) is a newly discovered in recent years as a neutrophil anti-infection mechanism. Not only that, but in autoimmune diseases, tumors, thrombotic disease, the NETs have also been found participation, producing adverse effect to the body.STAT3 is one of the important part of signal transduction and transcriptional activation factor (STATs) family, it is a kind of DNA binding protein, with functions of signal transduction and transcriptional regulation, and is an important part of the JAK-STAT signal pathway. In normal tissue cells, the STAT3 is mainly involved in cell proliferation, differentiation and apoptosis, etc. Many peptide ligands such as growth factors, cytokines and so on on the cell surface combined with the corresponding receptors, which is combined with receptor tyrosine protein kinase in the cytoplasm and lead to the phosphorylation of receptor tyrosine residues and JAKs activation; The activation of JAKs and receptors raised within the cytoplasm of STAT3, and activate STAT3. SH2 domain structure on STAT3 is combined with tyrosine phosphorylation sites on the receptor to homologous or heterologous dimmers in intracytoplasmic; Dimer transfers into the nucleus, combining with the specific sequences of DNA in target gene promoter, induced the corresponding gene expression. In addition, the activation erea of some STAT3 protein are serine residues. Through this process, STAT3 ligand excitation of transient signal can be converted to the change of gene expression and regulate a variety of physiological functions of the cell. Under normal circumstances, the activation of STAT3 is short and very quickly, but when the STAT3 protein expression, it may lead to a wide variety of tumor. Studies have shown that in a lot of head and neck cancer cells the quantity of STAT3 expression is obviously higher than normal tissue cells. JAK kinase inhibitors, STAT3 decoy oligonucleotide, STAT3 signal pathway inhibitors and epidermal growth factor receptor, and some natural biological agents can be applied to STAT3 signal transduction pathway and reduce some downstream of STAT3 gene expression so as to induce the head and neck tumor cell apoptosis.Now it has been proved that STATs family of STAT1 and STAT6 are activated in the hair cells death caused by cisplatin, and inhibit STAT1 and STAT6 respectively, cisplatin on hair cell damage will be reduced. There has been a number of studies that research the STAT3 in promoting cell proliferation, but the report in the process of apoptosis research is still rare. Research has shown that STAT3 expression increased in the ischemia reperfusion brain injury. This paper mainly discusses the STAT3 expression in zebrafish hair cells death caused by cisplatin, and interfered with STAT3 inhibitors, we proved that STAT3 in zebrafish hair cells death caused by cisplatin is activated, and with inhibition of STAT3, cisplatin on zebrafish hair cell damage reduced. Using specific markers of neutrophils transgenic zebrafish Tg (lyz: DsRed) we observed the phenomenon that in the process of cisplatin injury, neutrophils move to neuromasts, we measure that neutrophils may participate in the damage process of cisplatin on the hair cells.ObjectiveEstablish cisplatin on zebrafish youth hair cell damage model; Research the role of STAT3 in zebrafish lateral line caused by cisplatin youth hair cells damage.Methods1. Zebrafish cisplatin injury hair cells in zebrafish lateral line1.1 The treatment of 5dpf zebrafish with cisplatinCisplatin powder was dissolved with system water to 3333um, stored- 20℃. 5dpf zebrafish were randomly divided into the system of water control group, different concentrations (125 um,250um,500um,1000um)cisplatin groups, treated respectively 1 h,2 h,3 h,4 h,6 h,with7 fish of each group. Repeat 3 times.1.2 Examination of neuromasts in zebrafishThe zebrafish in the control group and cisplatin treatment group were respectively dealed with 1 h,2 h,3 h,4 h,6 h, discard processing liquid, liquid with PBS,5 min at a time, a total of 3 times. Then put 2uM fluorescent reactive dye solution Yo-Pro-1, avoid light staining 1 h, discard Yo-Pro-1, cleaning with PBS,5 min at a time, a total of 3 times. Using 0.02% MESS anesthesia, slides, zebrafish from left side, fluorescence microscope, taking pictures, choose SOI, SO2,01, OC1 four fixed neural mound hair cells count.1.3 Apoptosis detection of hair cells in zebrafish lateral line5dpf AB zebrafish were randomly divided into the system of water control group and 250um cisplatin treatment group, every group were treated for 3 h, respectively. Discard processing liquid, with PBS. cleaning,5 min at a time, a total of 3 times. Then zebrafish were treated for 1h in TUNEL dyeing stain in a dark environment at 37℃, then discard TUNEL staining fluid, clean with PBS,5 min at a time, a total of 3 times. Puts zebrafish on slides, take the left side, fluorescence microscope, taking pictures. Count the number of dead hair cells.10 fish were used in each group. Repeat 3 times.2. Neutrophils may participate in the damage process of hair cells in zebrafish lateral line by cisplatinTake 5dpf transgenic zebrafish Tg (1yz:DsRed), and randomly divided them into control group and experimental group. The experimental group were treated with 250 um cisplatin for 3 h, the control group join the amount of system water. Afer processed, clean with system water. Stained in 2uM Yo-Pro-1 for 30 min in dark, rinse three times in system water, each time 5 min. Observation under the fluorescence microscope. Each group of ten fish, repeat 3 times.3. STAT3 is actived in the process of hair cell death in zebrafish lateral line caused by cisplatin.3.1 RT-qPCR method to detect the expression of STAT3 in zebrafish neuromast (see detailed steps method)3.2 in situ hybridization method to detect expression of STAT3 in zebrafish neuromast (see detailed steps method).3.3 STAT3 inhibitors S3I-201 can reduce hair cell death in zebrafish lateral line caused by cisplatin.Take 5dpf transgenic zebrafish (dsred), and randomly divided them into control group, cisplatin,. S3I-201+cisplatin treatment group(each group of 10 fish). S3I-201+cisplatin treatment group were treated with 50 um S3I-201 for 2 h, then add 250 um cisplatin, cisplatin group to join 250 um cisplatin, control group to join the amount of water system, deal with 3 h. Discard processing liquid, with PBS cleaning,5 min at a time, a total of 3 times. Stained in 2 uM Yo-Pro-1 for 30 min in dark, rinse three times in system water, each time 5 min. Using 0.02%MESS anesthesia, slides, zebrafish from left side, fluorescence microscope, taking pictures, choose SO1, SO2,01, OC1 four fixed neural mound hair cells count.4. N-acetyl cysteine (NAC) decreases the damage of hair cells on zebrafish lateral line by cisplatin4.1 N-acetyl cysteine and cisplatin co-treate 5dpf zebrafish5dpf zebrafish were randomly divided into control group, cisplatin group and NAC+cisplatin group, the NAC+cisplatin group were pretreated with different concentrations of n-acetyl cysteine (NAC) (10100 um um, um,50,200 um) for 2h, then add 250 um cisplatin solution to co-treat for 3 h, the cisplatin group was only treated with 250 um cisplatin for 3h, the control group to join the amount of system water.Control group and experimental group are respectively treated for 3h, discard processing liquid, and liquid with PBS,5 min at a time, a total of 3 times. Then in 2uM fluorescent reactive dye solution Yo-Pro-1, avoid light staining for 1 h, discard Yo-Pro-1, clean with PBS liquid,5 min at a time, a total of 3 times. Then zebrafish were taken into 0.02% MESS anesthesia, slides, letting it sleep on left side, fluorescence microscope, taking pictures, choose SO1, SO2,01, OC1 four fixed neural mound hair cells to count.5. The statistical analysisUsing SPSS 19.0 statistical analysis software, data analysis, data to(average± standard deviation), two factors multiple level compares the repeated measure analysis of variance, comparing the two groups using independent sample t test, multiple comparison using one-way analysis of variance (one-way ANOVA), f test taken Levene test.Set P< 0.05 difference was statistically significant.Result1. Cisplatin injury hair cells in Zebrafish lateral lineAfter cisplatin treatment, count SO1, SO2,01, OC1 four neural high number of hair cells. Normal zebrafish of these four neuromasts hair cell mean was 10.4167 ± 0.92983;Use 250 um cisplatin treatment for 3 h, counting cells mean was 5.2381 ± 0.81577;With 250 um cisplatin 6 h after processing, counting cells mean was 0.0357 ±0.08964;Use 1000 um cisplatin treatment after 3 h, counting cells mean was 0.5± 0.45415; With 1000 um cisplatin for 6 h, counting cells to mean of 0+0. By the statistics of the number of their hair cells, cisplatin treatment group were less than the normal control group (P< 0.05). You can see from the data, zebrafish lateral line hair cells were gradually reduced, increased concentration of platinum and cisplatin effect, the longer lateral line hair cells decreased, the more obvious. Indicate that cisplatin on zebrafish lateral line hair cells injury with concentration and time dependence.2. Hair cells apoptosis detection after cisplatin injuryBy TUNEL staining to observe the zebrafish lateral line hair cells apoptosis signal,250 um cisplatin treatment for 3 h, lateral line hair cells apoptosis signal number is:0.7333±0.14710. Hair cell apoptosis signal number in control group is: 0.0883±0.13786. The hair cell apoptosis signal number cisplatin treatment group is more than the control group (P< 0.01).3. Neutrophils may participate in the damage process of hair cells in zebrafish lateral line by cisplatinNeutrophils is developed by the bone marrow hematopoietic stem cells enter the blood,and organization. Under normal circumstances, the blood neutrophils, generally attached to the vascular wall, the other half with the circulation of the blood. Bone marrow reserves a large number of mature neutrophils, when needed they enter the blood circulation in the body immediately. In the process of zebrafish treatment with cisplatin, we observed the phenomenon of neutrophils to move to neuromasts; While the control group zebrafish without this phenomenon, which can be concluded that neutrophils may participate in the damage process of hair cells in zebrafish lateral line by cisplatin, but the specific role the neutrophils play in the process remains to be seen.4. STAT3 expression increase in hair cells lesions of zebrafish lateral line caused by cisplatinIn situ hybridization results showed:STAT3 signal in the lateral line neumasts is significantly higher than the control group, and cisplatin treatment 3h signal is higher than 6h, prompt STAT3 expression is highest when dealing with 3 h. Real-time fluorescent quantitative PCR result is consistent with the results of in situ hybridization. STAT3 inhibitors S3I-201+cisplatin group compared with cisplatin treatment group, the number of hair cells respectively is 3.6364±0.71906,2.25± 0.55572, differences being statistically significant (P< 0.05). While compared with the control group(9.3462± 0.99759), the number of hair cells significantly reduced(P < 0.01).5. N-acetyl cysteine (NAC) decreases the damage of hair cells by cisplatinThe hair cells number of treatment group of NAC+cisplatin respectively in zebrafish lateral line is:4.4±0.5525,5.4531±0.8814,6.0625±1.1893,7.4706± 0.4039. Cisplatin treatment group of zebrafish lateral line hair cell number is:3.5833 ±0.9885. Control group zebrafish side hair cells number is:9.25±0.9843. Statistics show that number of hair cells of zebrafish lateral line by NAC pretreatment significantly better than the hair cells in zebrafish that only with cisplatin treatment (P< 0.01), and with the increase of concentration of n-acetyl cysteine, the stronger , the protection, speculated that ROS plays an important role in hair cell damage caused by cisplatin.Conclusion1. Immersed in cisplatin dosage can result zebrafish lateral line nerve high decrease in the number of hair cells, and there is time and the concentration dependence; Cisplatin made hair cell apoptosis increases in zebrafish lateral line neuromasts.2. Neutrophils may participate in the damage process of cisplatin hair cells in zebrafish lateral line. Further observation way of neutrophils in the damage process, explore the neutrophils capture mechanism (NETs) whether to participate in the process.3. STAT3 in zebrafish side hair cells lesions caused by cisplatin expression; Inhibition of STAT3 can reduce the damage of hair cells on lateral line by cisplatin. It indicates STAT3 plays a promoting role in cisplatin injury of lateral line hair cells.4. ROS system inhibitors n-acetyl cysteine has certain protective effect on cisplatin injury on zebrafish lateral line hair cells, which may provide experimental basis for clinical application. |
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