Experimental Study On Effects Of Xiehuo Huayu Tongqiao Method On Caspase - 9/6 And Bax / Bcl - 2 In Cochlear IRI Rats

Objective:Currently, there are 360 million people suffer from hearing impairment, and there is a rising trend. Chinese medicine believes that ‘Huo’ and ‘Yu’ are the major causes of sudden deafness, Chinese medicine has accumulated rich experience in the diagnosis and treatment of deafness through long-term clinical practice. the use of RRDO method achieved good effect in the clinical treatment of sudden deafness, it is an indirect proof of the good effect of the RRDO method to IRI in cochlear, and the multi-target regulation effects of RRDO, however the mechanisms need to be clarified.most of them are sensorineural deafness. Blood circulation disorder of the inner ear is the most important factor to cause sensorineural deafness, which is mainly caused by ischemia and reperfusion injury. The pathological mechanisms of structure and function of inner ear damaged induced by IRI are complex, the mechanism may be associated with the overproduction of oxygen free radicals and apoptosis. Cochlea of the inner ear is extremely sensitive to reducing or interrupting blood and oxygen supply, the mechanism of IRI leading the changes of structural and functional in the inner ear are very complex, the mechanism may be related to the release of inflammatory mediators, the main cause of increasing production of oxygen free radicals in the cochlea, the result is damage of changes in cell membrane function, the function of cells and organelles. Oxygen free radicals and lipid peroxides degradation of malondialdehyde(MDA) can interfere with the metabolism of proteins, sugars and nucleic acids, and decrease activity of the enzyme, the template dysfunction and tissue damage cell structure of nucleic acids. At the same time, oxygen free radicals is also related with the body’s antioxidant enzyme activity, Such as catalase(CAT), etc. When peroxidase is not sufficient to remove the free radicals in body, the damage of tissue will appear. Thus, CAT, MDA is an important index to evaluate the cochlea IRI. Many experiments show that apoptosis is a major form of cell damage cochlear IRI. Apoptosis promotes a kind of internal mechanisms after cells are stimulated, cell death is mainly caused by endogenous endonuclease activation. B-cell lymphoma / leukemia-2 proto-oncogene(Bcl-2) is gene family to regulate cell proliferation and apoptosis status. Bcl-2 is the main anti-apoptotic factor, Bax、Bad、Bak、Bid are the pro-apoptotic gene, which may be the Bcl-2 form heterodimers of Bcl-2 inhibitory effect produced. The high expression of Bcl-2 can promote the forming the heterodimer of Bcl-2/Bax, which can help to inhibit the cell apoptosis. The high expression of Bax can promote the homodimers forming of Bax/Bax and the cell apoptosis. The Bcl-2/Bax imbalance exists, and apoptosis and proliferation may increase apoptosis. Thus, the Bcl-2 、Bax imbalance are related with cochlea IRI. Many experiments show that apoptosis is a major form of cell cochlear injury IRI. Apoptotic process can occur through exogenous and endogenous pathway, the Caspases activation of these two pathways can degrade the sub-structure of cells. Caspases are not only an important molecule in apoptosis phenomena, but also a key protease. The Caspase-8 of extrinsic apoptosis pathway involved in apoptosis implementation, and induced Caspase-3 activation, then started the Caspase cascade reaction and execution classes Caspase cleavage of a specific substrate resulting in apoptosis. Caspase-6 of extrinsic apoptosis pathway belongs to the performing protein that can react with the nucleus, cut sheet fibroin, cause disintegration of the nuclear lamina, lead to chromosome condensation, and promote apoptosis. In intrinsic apoptosis pathway apoptosis, Caspase-9 belongs to the starting protein, which can shear a series sub-structure of cell to lead to cell death. Two apoptotic pathways are independent, while are closely linked. Therefore, in cochlea IRI Caspase-6 and 9 are important indicators to evaluate the apoptotic damage.Sudden deafness is attributed to ‘acute deaf’ category in Traditional Chinese Medicine(TCM). In TCM clinical practice, the rich experience has been explored and accumulated through a long-term diagnosis and treatment of deafness. Therefore, a therapy of ‘reducing fire and resolving stasis to dredge the orifice’(RRDO) might have a better effect. In this experiment, a rat cochlea IRI model was adopted to observe the effect of RRDO to the Catalase(CAT), malondialdehyde(MDA), the apoptosis associated protein(e.g. Bcl-2、Bax、Bad、Bak、Bid). and apoptosis regulatory protein(e.g. Caspase-6 and Caspase-9), by these methods we study the protective effect of RRDO to the cochlea of the inner ear in IRI from the molecular level, through above investigations, we will clarify the improvement and regulation the molecular mechanism of RRDO established on the basis of pathogenesis by "fire" and "stasis", and provide experimental basis for clinical application and mechanism of action. Materials and methods:Animal model and grouping: Photochemical induction method was used in modeling, the rat models were divided into five groups, namely normal group, model group, clearing liver-fire group, blood-activating group, and fire-reducing group, with 20 rats in each group.Experimental method: For the clearing liver-fire group: 12-h after the operation, administration of the ingredients activating blood to resolve stasis in the RRDO decoction, 1ml/100 g, twice a day for 7 days.normal saline was given corresponding for 7 days. For fire-reducing group: 12-h after the operation, administration of the RRDO decoction, 1ml/100 g, twice a day for 7 days. For the blood-activating group: 12-h after the operation, administration of the ingredients activating blood to resolve stasis in the RRDO decoction, 1ml/100 g, twice a day for 7 days. The course of treatment consisted of 10 days.Experiment contents:(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) the m RNA expressions of BCl-2、Bax of cochlear were determined with Real-Time PCR method;(5) BCl-2、Bax and Bad、Bak、Bid protein expressions were determined with western blotting method;(6) the m RNA expressions of cochlear Caspase-6 and Caspase-9 were determined using Real-Time PCR method;(7) the m RNA expressions of cochlear Caspase-6,Caspase-9,Caspase-12 were determined with western blotting method.Statistical analysis: The SPSS18.0 software package was used to perform statistical and analysis. The measurement of experimental data was expressed as sx)( ?; Comparison between two groups was done with independent sample T-test, differences among groups were compared with analysis of variance; and Chi-squared test was used for data analysis. P<0.05, P<0.05 were separately taken as significant and highly significant differences in standards. Results: 1. After the treatment, the ABR threshold was significantly lower in the fire-reducing group than in the qi-promoting and group blood-activating, clearing liver-fire 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 clearing liver-fire 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 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 saline 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 group. 3.Cochlear IRI can reduce the activity of CAT in Cochlear organization(P <0.05), in the fire-reducing group and blood-activating group, the activity of CAT was significantly increased(P <0.05), furthermore the promoting activity of fire-reducing group to CAT is better than blood-activating group. 4. Cochlear IRI can reduce the activity of MDA in Cochlear organization(P<0.05), in the fire-reducing group and blood-activating group, the activity of MDA was significantly increased(P <0.05), furthermore the promoting activity of blood-activating group to CAT is better than fire-reducing group. 5. Cochlear IRI can reduce the expression of Bcl-2 m RNA in Cochlear organization(P <0.05), the m RNA expression of Bcl-2 in fire-reducing group was obviously higher than that in the model group(P <0.05). 6. Cochlear IRI can promote the expression of Bax m RNA in Cochlear organization(P <0.05), the m RNA expression of Bax in blood-activating group was lower than that in the model group(P < 0.05), the m RNA expression of Bax in fire-reducing group was obviously lower than that in the model group(P <0.05). 7. The m RNA expression of Bcl-2 in model group was obviously lower than that in the control group(P <0.05); however in the fire-reducing group and blood-activating group, the expression of Bcl-2 was significantly increased(P <0.05). 8. The m RNA expression of Bax in model group were obviously lower than that in the control group(P <0.05); however the fire-reducing group and blood-activating group compared with the model group, the expression of Bclax was significantly increased(P <0.05).The the expression of fire-reducing group and blood-activating was significant different(P <0.05). 9. Cochlear IRI can promote the expression of Caspase-6 m RNA in Cochlear organization(P <0.05); however the expression of Caspase-6 m RNA in fire-reducing group was significantly lower than that in model group(P < 0.05). 10. Cochlear IRI can promote the expression of Caspase-9 m RNA in Cochlear organization(P <0.05); however the expression of Caspase-9 m RNA in fire-reducing group was significantly lower than that in model group(P <0.05),The the expression of fire-reducing group and blood-activating was significant different(P <0.05). 11. Cochlear IRI can promote the expression of Caspase-12 m RNA in Cochlear organization(P <0.05); however the expression of Caspase-9 m RNA in fire-reducing group was significantly lower than that in model group(P <0.05),The the expression of fire-reducing group and blood-activating was significant different(P <0.05). 12. The m RNA expression of Caspase-6 in model group was obviously lower than that in the control group(P<0.05); however in the fire-reducing group and blood-activating group compared with the model group, the expression of Caspase-6 was significantly inhibited(P < 0.05). 13. The m RNA expression of Caspase-9 in model group was obviously higher than that in the control group(P<0.05); however in the fire-reducing group and blood-activating group compared with the model group, the expression of Caspase-9 was significantly decreased(P <0.05). 14. The m RNA expression of Caspase-12 in model group was obviously higher than that in the control group(P<0.05); however in the fire-reducing group and blood-activating group compared with the model group, the expression of Caspase-9 was significantly decreased(P <0.05). 15.The fire- reducing group and blood- activating group, GRP78 protein is lower than the model group and clearing liver- fire group(P < 0.05).The the expression of fire-reducing group and blood-activating was significant different(P <0.05). Conclusions: 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. Cochlear IRI can reduce the activity of CAT in cochlear organization, MDA contents were increased obviously, both CAT and MDA are the main cause of hearing impaired deaf. 5. In cochlear IRI, RRDO can significantly increase in CAT activity, and decrease MDA activity, meantime it can strengthen anti-lipid peroxidation, and reduce the role of lipid peroxidation. The imbalance axis of CAT/MDA oxidation is corrected, the free radical damage induced by membrane lipid peroxidation was improved in Cochlear IRI. 6. RRDO can obviously increase the expression of Bcl-2 in Cochlear tissue, and decrease the expression of Bax, RRDO also can corrected the imbalance axis of Bcl-2、Bax and inhibit the cells apoptosis. 7. RRDO can obviously decrease the expression of Caspase 6 /9/12 in Cochlear tissue, RRDO also can inhibit the cell apoptosis and protect the cochlear tissue in inner ear IRI, through inhibiting the activities of apoptotic protein and initial protein.