Experimental Study On Effect Of Xiehuo Huayu Tongqiao Method On Caspase - 8/3 And Fas System In Rat Cochlea Ischemia Reperfusion Injury

Purposes:When the hair cells of the central auditory, auditory nerve or spiral organ occurs lesions the barriers of the feeling about the voices and nerve impulse conduction will happen. This is what we called sensorineural hearing loss. It is a clinical comman and frequen disease in the department of ENT(ear, nose and throat). The disorder of blood flow in the inner ear mainly contributes to the sensorineural hearing loss. More and more researches confirm that the ischemia alone won’t cause the disorder of blood flow in the inner ear. And it is mainly induced by the reperfusion injury after the ischemia. Ischemia-reperfusion injury is a complex physiological and pathological process and multiple factors and targets are involved in the reaction. In the recent reports of Chinese medicine treatment about inner ear diseases, activating blood to resolve the stasis medicines are the most wide and effective. The second effective therapy is clearing liver-heat and reducing fire. In our study, the activating blood to resolve the stasis and clearing liver-heat and reducing fire were combined to form “ reducing fire and resolving stasis to dredge the orifice”(RRDO) which might have a better effect.The study was designed to explore(1) the application of vascular occlusion in the rat model of cochlear ischemia-reperfusion injury;(2) the influences of RRDO on the rat model of of cochlear ischemia-reperfusion injury caused by the vascular occlusion;(3) molecular mechanisms of how RRDO improves ischemia-reperfusion injury in the cochlear rat model. Materials and methods:Animal model and grouping: Vascular occlusion method was used in modeling, then the rat models were divided into five groups, namely normal group, fire-reducing group, model group, qi-promoting and blood-activating group, and fire-reducing and stasis resolving group, with 24 rats in each group. Experimental method: treated with normal diet without any administration for the normal group; after modeling, treated with normal diet without any administration for model group; 2ml of the decoction twice a day for the fire-reducing group one day after the operation, last for 7 days; and different treatments for the rest two groups.(1) for qi-promoting and blood-activating group: one day after the operation, administration of the decoction, 2ml, twice a day(amounting to 2g/day for the raw medicine);(2) for the fire-reducing and stasis resolving group: administration of the ingredients in the RRDO decoction, 2ml, twice a day(amounting to 5g/day for the raw medicine); The course of treatment consisted of 7 days. Experimental indicators:(1) ABR threshold detection was conducted for all the rats after modeling, 1 day before and 7 days after medication;(2) the stria vascularis and hair cells were observed by using microscope-body anatomy and cochlea spiral ligament and basilar membrane stretched;(3) detections of MDA and CAT were done with cochlear TBA method and Visible light method;(4) expressions of cochlear Caspase-8 and Caspase-3 mRNA were determined by using Real-Time PCR, then the protein expression of Caspase-8 and Caspase-3 were detected by western blot;(5) the expressions of cochlear Fas and FasL mRNA were determined by using Real-Time PCR, then the protein expression of Fas and FasL were detected by western blot;(6) the expressions of cochlear XIAP and XAF1 mRNA were determined by using Gradient PCR, then the protein expression of XIAP and XAF1 were detected by western blot. Statistical analysis: SPSS18.0 package for statistical analysis was used. The measurement data of experimental data were expressed as sx)( ±; single-factor analysis of variance(One-Way ANOVA), and comparison between groups were conducted by using least significant difference method(LSD); and P ﹤0.05, P ﹤0.01 were taken as significant and highly significant differences in standards. Results: 1. After the treatment, the ABR threshold was significantly lower in the fire-reducing and stasis resolving group than in the qi-promoting and group blood-activating, fire-reducing group and model group(P<0.05), and the ABR threshold was significantly lower in normal group than in other groups(P<0.05). 2. The microstructure of the basilar membrane stretched: In the normal group, outer hair cells was distributed in three rows; clearly displayed, orderly arranged, evenly distributed, and with no obvious missing; in the model group and fire-reducing group, most of the outer hair cells were deformed, arranged in serious disorder, disintegrated and destructed was lodged seriously, connections of cell disappeared, the cilia of cell was lodged seriously; in the qi-promoting and blood-activating group the outer hair cells arranged relatively orderly, partly deformed, and with partial necrosis; in the fire-reducing and stasis resolving group the outer hair cells arranged more orderly, missing sparsely, and with unclear contour occasionally. The damage of hair cells in the model groups and fire-reducing group was serious, the damage of out hair cell in the fire-reducing and the qi-promoting and blood-activating groups the damage of outer hair cells was relatively mild, and the damage was more evident in the qi-promoting and blood-activating group than in the fire-reducing and stasis resolving group. 3. The three fluorescent signal of the outer hair cells of the model group and drug intervention group on the basement membrane markedly increased and enhanced. Compared with normal group difference was statistically significant(P<0.01). And compared with model group, the activing blood to resolve the stasis group and RRDO hair cells significantly reduce the fluorescence signal. the performance of the RRDO was more obvious( P<0.05)。Hair cells signals of clearing liver-heat and reducing fire group compared with the two groups was significantly enhanced.(P<0.01) 4.The MDA level of the fire-reducing and stasis resolving group and the qi-promoting and blood-activating group was significantly lower than those of the model group and fire-reducing group(P<0.05), while CAT level of the fire-reducing and stasis resolving group was significantly higher than those of the qi-promoting and blood-activating group(P<0.05). The level of CAT in both the fire-reducing and stasis resolving group and the qi-promoting and blood-activating group were significantly higher than those of model group and fire-reducing group(P<0.01). 5. When compared with normal group, the expressions of Caspase-8 and Caspase-3 mRNA in other 4 groups were significantly higher than in the model group(P<0.01~0.05); while it was lower in the fire-reducing and stasis resolving group than in the qi-promoting and blood-activating group than that in fire-reducing group(P<0.05). For the expression of Caspase-8 and Caspase-3 protein, the results were similar to those of mRNA detection. The expressions of Caspase-8 and Caspase-3 protein in other 4 groups were significantly higher than in the model group(P<0.05); while it was lower in the fire-reducing and stasis resolving group than in the qi-promoting and blood-activating group than that in fire-reducing group(P<0.05). 6. When compared with model group and fire-reducing group, the expressions of Fas and FasL mRNA in normal group were significantly lower(P<0.01~0.05), while those in the fire-reducing and stasis resolving group than in the qi-promoting and blood-activating group were significantly decreased(P<0.05). For the expression of Fas and FasL protein, the results were similar to those of mRNA detection. When compared with model group and fire-reducing group, the expressions of Fas and FasL protein in normal group were significantly lower(P<0.01), while those in the fire-reducing and stasis resolving group than in the qi-promoting and blood-activating group were significantly decreased(P<0.05). 7.This experiment observed XIAP gene of cochlear ischemia-reperfusion injury caused by vascular blocking method may have a protective effect by gradient PCR and Western methods. Show that cell apoptosis is one of the important mechanisms of cochlear ischemia-reperfusion injury.RRDO can raise XIAP gene expression and inhibit the Caspase to block programmed cell death. At the same time the ischemia-reperfusion injury of the cochlea increases XAF1 expression. Its antagonism to XIAP enhance and accelerate damage tissue cell apoptosis. By traditional Chinese medicine intervention study on the control of the activing blood to resolve the stasis medicines and RRDO can reduce XAF1 expression. While the latter change were more significant(P<0.05). Conclusion: 1. Vessel occlusion method can be used to establish the rat cochlear ischemia-reperfusion injury model. This method is simple, reliable, high success rate and less harm to animal, so it worth to expand the application in the future. 2. Cochlear ischemia-reperfusion injury could remarkably increase the hearing threshold. 3. RRDO therapy can effectively improve cochlear microcirculation disturbance, reduce the level of ABR and improve hearing of model animal. 4. RRDO therapy can significantly deceases the MDA level, while increase CAT activity, which is better than the effect of qi-promoting and blood-activating therapy. 5. RRDO therapy can significantly reduce the expression of Caspase 3and Caspase 8 for rats with microcirculation disturbance of the cochlea, and the effect is better than those of promoting qi and activating blood to resolve stasis. 6. RRDO therapy may improve the appotosis of hair cell through decreasing the expression of Caspase 3and Caspase 8. 7. RRDO therapy can significantly reduce the expression of Fas and FasL for rats with microcirculation disturbance of the cochlea, and the effect is better than those of promoting qi and activating blood to resolve stasis. 8.RRDO can raise XIAP gene expression and inhibit the Caspase to block programmed cell death. At the same time the ischemia-reperfusion injury of the cochlea increases XAF1 expression. Its antagonism to XIAP enhance and accelerate damage tissue cell apoptosis. By traditional Chinese medicine intervention study on the control of the activing blood to resolve the stasis medicines and RRDO can reduce XAF1 expression. 9. The effect of RRDO therapy to improve cell apoptosis is better than that of promoting qi and activating blood therapy.