Effect Of Ligustrazine On Cisplatin - Induced Apoptosis Of Rat Cochlea Hair Cells

the first part Experimental study on rat deafness model induced by intraperitoneal injection of cisplatinObjective: To explore the optimal dose and experimental condition of rat deafness model induced by intraperitoneal injection of cisplatin.Materials and methods: There are 50 healthy Wister rats. male and female are the same number. The weight is 200±20g.They are 6-8 weeks of age. All are purchased from experimental animal center of China Medical University. 50 rats were random- ly divided into 5 groups: blank control group; model group 1; model group2; model g- roup3; model group 4 and 10 rats in each group. Except for the blank control group, the following Cisplatin Injection the rest of the four groups of rats were given different doses of intraperitoneal injection. methods: blank control group: each rat received saline injections, a dose of 1ml / 100g·d-1,continued adm- inistration for 5 days. Model 1: each rats were given 0.4mg / ml concentration intra- peritonealed injection Cisplatin. The dose is 0.5ml / 100 g · d-1, continuous admin- istration for 5 days(equivalent to 2mg /Kg). Model 2: eachgroup rats were given 0.8mg/ml concentration Cisplatin Injection intraperitoneal injection, the dose of 0.5ml /100g·d-1, continuous administration for 5 days(equivalent to 4mg/Kg). Model 3: e- ach rats was given 1.6 mg/ml concentrate on Cisplatin by intraperitoneal injecti- on. the dose is 0.5ml/100g·d-1, continuous adm- inistration for 5 days(equivalent to 8mg/Kg). Model 4: each rats was given 3.2mg/ml concentration intraperitoneal injection Cisplatin. the dose is of 0.5ml/100 g ? d-1, continuous administration for 5 days(equivalent to 16mg/Kg). After the experiment, we d- etect the hearing threshold of ABR of rats, and observe the pathological changes in cochlear hair cells.Results: 1.The general condition and mortality of each groups of rats.The rats in control group were in good spirits. They have normal activity, normal amount of food. Givien high fives noise stimulation, sensitive reaction of auricle is well. Model group 1 rats have good mental state, activity decreased slightly. fresh wa- ter is normal.Given high fives noise stimulation, the Auricle response is slightly du- ll compair with the blank group reaction. Until the end of the experiment, there is no death rats. Rats mental state of model 2, 3, 4 groups is poor, especially 3, 4 group. They cur- led up in a cage corner, very little activity. Fresh water is also significantly reduced. Model group 4 rats in the experimental 4-5 days are almost no food. Giving high fiv- es noise stimulation, pinna response is very slow. The auricle response of 4 groups rats model is negative. No model group 2 rats death appeared, while the model 3, 4 r- ats died. One rat died at the third day and 2 rats died in the fifth day in Model group 3. The total mortality rate 30%; One died in the second day and 1 rats died in the third days, 1 rats died in the fourth days, 3 rats died in the fifth days in model group 4. The total mortality is 50%. 2. The results of ABR test in each group of rats The results of threshold detection in each group of rats shows that: Compared with blank control group rats, the ABR threshold increased significantly in model 1, 2, 3, 4 groups of rats(p < 0.01). There is significant difference. Comparing the result in model 1, 2, 3, 4 groups shows that ABR threshold increased significantly with the dose of cisplatin injection. 3.The morphological observation in cochlear basement membrane cells(1) The surface preparation of cochlear basilar membrane The results of the surface preparation of cochlear basilar membrane in each grou- p of rats shows that: It is clearly visible of three rows of hair cells. They are uniform, neat, no missing and dead cells with the surface preparation of cochlear basilar memb- rane in blank control group rats. The hair cells missing, but the arrangement in most of them is still relatively uniform neat, only occasional absence with the surface pre- paration of cochlear basilar membrane in Model 1rats.The fuzzy hair cells can be seen in most area of the surface preparation of cochlear basilar membrane in Model 2, but its general form is still relatively regular.The form of hair cells lost, and it is impossi- ble to identify the hair cell morphology. Occasionally we can see contours of 3-5 hair cells in Model 3 and 4 rats.(2) the pathology of cochlearThe results of the cochlea pathological HE staining in each group of rats shows that: It could be clearly distinguish the cochlea hair cells form and arrangement with the cochlea pathological HE staining. The cochlea hair cells are uniform and neat. the hairy structure attached to the basement membrane in blank control group rats.The cochlea hair cells are uniform and neat, occasional absence, but the capillary structure is sparsed in Model 1. The cochlea hair cells are absent obviously,but the capillary structure is melt in Model 2.The cochlea hair cells are absent obviously, disordered arrangement. the c attached capillary structure is almost invisible in Model 3 and 4 group rats.Conclusion: 1. The method that Cisplatin injected into rats abdominal cavity could lead to the rise of ABR thresholds of rats and damaged the cochlear hair cell. 2.The dose of Cisplatin 2 mg/kg per day consecutive five days can cause rat cochlea damaged, but it is not enough to as a deaf animal model which can be used in scien- tific experiments. 3.The dose of Cisplatin 8 mg/kg and 16 mg/kg per day consecutive five days can lead to excessive damage in the rat cochlea hair cells. They are not suitable as a deaf ani- mal model which can be used in scientific experiments. 4.The dose of Cisplatin 4 mg/kg per day consecutive five days cause rat cochlea da- mage. The degree of damage is moderate. The model is stable and can be used as animal model of deafness for scientific experiments.The second parts The experimental research of the mechanism of Ligustrazine intervene internal apoptosis way in cisplatin induced model rats cochlear deafnessObjective :To study the mechanism of Ligustrazine intervene internal apoptosis way of cisplatin induced model rats cochlear deafnessMaterials and methods: 62 rats were divided into two groups respectively with random number average: normal group(N group, 11), model replication group(group M, 51). Do nothing with normal group, breeding normally; Model replication group rats were made of the drug-induced deafness model which were induced by the method intraperitoneal injectionof cisplatin. After the model replication were made successfully, using the method of random numbers, normal group rats were randomly divided into two groups: normal control group(N1), ligustrazine control group(N2); Model replication groups were randomly divided into two groups: model control group(M1), TMP treatment group(M2). 1-5 days in the experiment: normal group was given saline by intraperitoneal injection 5 ml/kg, continuous injection for 5 days. Model replication group was given cisplatin intraperitoneal injection 4 mg/kg, continuous injection for 5 days.6-12 days in the experiment: normal group was given saline by intraperitoneal injection 5 ml/kg, continuous injection for 7 days. The ligustrazine compared group was given ligustrazine by intraperitoneal injection 140 mg/kg, continuous injection for 7 days. In the model compared group was given saline by intraperitoneal injection 5 ml/kg, continuous injection for 7 days. The ligustrazine therapy group was given ligustrazine by intraperitoneal injection 140 mg/kg, continuous injection for 7 days. After the treatment1 h, put the rat into death, strip the cochlea, used in p53, bax, bcl-2, caspase-9, caspase-3 m RNA and protein expression level detection in- 80 ℃ refrigerator.Results: 1. The evaluation results of model:The result of N and M group rats ABR test after model replicated showed: compared with normal group, ABR threshold of model group rats increased significantly(p < 0.01), with statistical significance. The cochlear basilar membrane cells in N group rats are neat, uniform. there is no obvious lack of cells; most of the cochlear basilar membrane cells in M group rats are deformation, arranged disorderly and numerous dissolved the damaged hair cells. cell connection is incomplete. 2.The experiment results of p53, bax, BCL- 2, caspase- 9, caspase 3 protein expression level in each group rats cochlea tissues show that: Compared with blank control group, model compare group and model group with ligustrazine in the rat cochlea tissues bax, p53, caspase3, 9 protein expression level increased significantly, while the BCL 2 protein expression level decreased significantly(p < 0.01); Compared with model control group, model with ligustrazine group bax, p53 protein expression in rat cochlea tissues were significantly reduced, and the BCL- 2 protein expression level was significantly increased(p < 0.01).Compared with blank control group and the ligustrazine blank group, all the indicators are no significant difference(p > 0.05). 3. The experiment results of p53, bax, BCL- 2, caspase- 9, caspase 3 RNA expression level in each group rats cochlea tissues show that: Compared with blank control group, model compare group and model group with ligustrazine in the rat cochlea tissues bax, p53, caspase3, 9 RNA expression level increased significantly, while the BCL 2 protein expression level decreased significantly(p < 0.01); Compared with model control group, model with ligustrazine group bax, p53 protein expression in rat cochlea tissues were significantly reduced, and the BCL- 2 RNA expression level was significantly increased(p < 0.01), Compared with blank control group and the ligustrazine blank group, all the indicators are no significant difference(p > 0.05).Conclusion:1. Give the rats by intraperitoneal injection of cisplatin dosage(4 mg/kg), once a day, five days, drug-induced deafness rat model can be successfully induced. 2. Ligustrazine can significantly decrease the p53, bax, caspase- 9, caspase 3 m RNA and protein expression level, increase the BCL- 2 m RNA and protein expression level in cochlear tissue of rat models. 3. Ligustrazine on drug resistance model rats cochlear deafness antiapoptotic effect may be realized by blocking or inhibit apoptosis internal way of beginning.The third part The experimental research of the mechanism of Ligustrazine intervene external apoptosis way in cisplatin induced model rats cochlear deafnessObjective: To study the mechanism of Ligustrazine intervene external apoptosis way of cisplatin induced model rats cochlear deafnessMaterials and methods: 62 rats were divided into two groups respectively with random number average: normal group(N group, 11), model replication group(group M, 51).Do nothing with normal group, breeding normally; Model replication group rats were made of the drug-induced deafness model which were induced by the method intraperitoneal injection of cisplatin. After the model replication were made successfully, using the method of random numbers, normal group rats were randomly divided into two groups: normal control group(N1), ligustrazine control group(N2); Model replication groups were randomly divided into two groups: model control group(M1), TMP treatment group(M2).1-5 days in the experiment: normal group was given saline by intraperitoneal injection 5 ml/kg, continuous injection for 5 days. Model replication group was given cisplatin intraperitoneal injection 4 mg/kg, continuous injection for 5 days. 6-12 days in the experiment: normal group was given saline by intraperitoneal injection 5 ml/kg, continuous injection for 7 days. The ligustrazine compared group was given ligustrazine by intraperitoneal injection 140 mg/kg, continuous injection for 7 days. In the model compared group was given saline by intraperitoneal injection 5 ml/kg, continuous injection for 7 days. The ligustrazine therapy group was given ligustrazine by intraperitoneal injection 140 mg/kg, continuous injection for 7 days. After 1h the last treatment, put the rat into death, strip the cochlea, used in fas/fas L、caspase-8 m RNA and protein expression level detection in- 80 ℃ refrigerator.Results: 1.The fas/fas L, caspase- 8 protein expression levels in each group of the rat cochlea tissues 2.The experiment results shows that : Compared with blank control group, model compare group and model group with ligustrazine in the rat cochlea tissues fas/fas L、caspase-8 protein expression level increased significantly(p<0.01);Compared with model control group, model with ligustrazine group, fas/fas L、caspase-8 protein expression in rat cochlea tissues were significantly reduced(p<0.01); Compared with blank control group and the ligustrazine blank group, all the indicators are no significant difference(p > 0.05). 3.The fas/fas L, caspase-8 RNA expression levels in each group of the rat cochlea tissues The experiment results shows that : Compared with blank control group, fas/fas L、caspase-8 m RNA expression level of model compare group and model group with ligustrazine in the rat cochlea tissues increased significantly(p<0.01);Compared with model control group, fas/fas L、caspase-8 m RNA expression of model with ligustrazine group in ratcochlea tissues were significantly reduced(p<0.01).Compared with blank control group and the ligustrazine blank group, all the indicators are no significant difference(p > 0.05).Conclusion:1. Ligustrazine can significantly decrease the fas/fas L 、 caspase-8 m RNA and protein expression level. 2.The antiapoptotic effect of Ligustrazine on drug resistance model rats cochlear deafness may be realized by blocking or inhibit apoptosis external way of beginning.