Purging Fire Blood: Method To Improve The Guinea Pig Cochlea Microcirculation Of Experimental Study

Objective：Sudden deafness is a common disease in otolaryngology, attributed to "acutedeaf" category in Traditional Chinese Medicine (TCM). At present, it isincreasing in incidence. There in TCM clinical practice a rich experience hasbeen explored and accumulated through a long-term diagnosis and treatment ofdeafness. Many compound agents have the action of multi-targeted regulation ofcochlear microcirculation; and emotional and psychological factors have becomethe important factors in onset and conversion of deafness and tinnitus. Modernmedical research shows that activating blood to resolve the stasis, or improvingmicrocirculation therapy, has a definite effect. So a therapy of “reducing fireand resolving stasis to dredge the orifice”(RRDO) might have a better effect,which is derived from an organic integration of activating blood to resolve thestasis plus clearing away liver fire, and has been confirmed by a large numberof clinical studies. The representative prescription for the therapy is XiehuoZhilong Granules (Granules for Reducing Fire to Cure Deafness), which has a goodeffect in a large number of clinical observations. This study was designed forobserving the impacts of RRDO on:(1) disturbance of cochlear microcirculationin guinea pig;(2) VEGF and bFGFmRNA expression in cochlear microcirculationdisorder of guinea pigs induced by photochemical method;(3) expressions ofiNOSmRNA and MMP-2mRNA in cochlear microcirculation disorder of guinea pigsinduced by photochemical method;(4) expressions of BCL-2mRNA, BAX mRNA and theirprotein in cochlear microcirculation disorder of guinea pigs induced byphotochemical method. Through the above investigations we study the improvementand regulation of cochlear microcirculation, and molecular mechanism of RRDOestablished on the basis of pathogenesis by "fire" and "stasis". Materials and methods:Animal model and grouping: Photochemical induction method was used inmodeling, then the guinea pig models were divided into five groups, namely normalgroup, model group, qi-promoting group, blood-activating group, andfire-reducing group, with10guinea pigs in each group.Experimental method:2ml of0.9%normal saline twice a day for the normalgroup;2ml of0.9%normal saline twice a day for the model group12-h after theoperation; and different treatments for the rest three groups. The specimensof the guinea pigs were sampled after quick decapitation on the2ndday afteradministration of anesthesia.(1) for fire-reducing group:12-h after theoperation, administration of the RRDO decoction,2ml, twice a day (amountingto1g/day for the raw medicine);(2) for the blood-activating group:administration of the ingredients activating blood to resolve stasis in the RRDOdecoction,2ml, twice a day (amounting to2g/day for the raw medicine);(3) forthe qi-promoting group: administration of the ingredients the qi-promotingsimply in the RRDO decoction,2ml, twice a day (amounting to4g/day for the rawmedicine). The course of treatment consisted of10days.Experimental indicators:(1) ABR threshold detection was conducted for allthe guinea pigs after modeling, before and1day after medication;(2) the striavascularis and hair cells were observed by using microscope-body anatomy andcochlea spiral ligament and basilar membrane stretched;(3) detections of MDAand CAT were done with cochlear TBA method and Visible light method;(4)expressions of cochlear VEGFmRNA and bFGFmRNA were determined by using Real-TimePCR;(5)) cochlear iNOSmRNA, MMP-2mRNA expressions were detected with Real-TimePCR;(6) cochlear BAX mRNA and BCL-2mRNA expressions were determined withReal-Time PCR;(7) VEGF, BCL-2and BAX protein expressions were determined byusing western blot.Statistical analysis: SPSS11.5package for statistical analysis was used.The measurement data of experimental data were expressed as (x±S); single-factor analysis of variance (One-Way ANOVA), and comparison betweengroups were conducted by using least significant difference method (LSD); andP﹤0.05, P﹤0.01were taken as significant and highly significant differencesin standards.Results:1. After the treatment, the ABR threshold was significantly lower in thefire-reducing group than in the blood-activating group and the qi-promotinggroup (P﹤0.05), and the ABR threshold was significantly lower in theblood-activating group than in the qi-promoting group (P﹤0.05). Compared withthe normal group and model group, the ABR thresholds in the three groups weresignificantly different (P﹤0.01).2. In the normal group spiral ligament showed normal vascular stria; in themodel group a large number of micro-vascular thrombi could be seen in the vesselsof spiral ligament stria, with vascular thickening to different degree andvascular disruption of parts of the vessels; in the qi-promoting group a largenumber of micro-vascular thrombi could be seen in the vessels of spiral ligamentstria, with vascular thickening to different degree and vascular disruption ofparts of the vessels; in the activating blood group the micro-vascular thrombicould were observed in spiral ligament stria, but with no significant vasculardisruption, and more micro-vascular thrombi compared to the fire-reducing group;in the fire-reducing group only a small amount of micro-vascular thrombi couldbe seen in spiral ligament stria, with no vascular disruption.3. The microstructure of the basilar membrane stretched: In the normal group,outer hair cells was distributed in three rows; clearly displayed, orderlyarranged, evenly distributed, and with no obvious missing; in the model group,most of the outer hair cells were deformed, arranged in serious disorder,disintegrated and destructed was lodged seriously, connections of celldisappeared, the cilia of cell was lodged seriously; in the qi-promoting group the part of outer hair cells was deformed, there were more flakes defect,connection of some cells disappeared; in the blood-activating group the outerhair cells arranged relatively orderly, partly deformed, and with partialnecrosis; in the fire-reducing group the outer hair cells arranged more orderly,missing sparsely, and with unclear contour occasionally. The damage of hair cellsin the model and the qi-promoting groups was serious, the damage of out haircell in the fire-reducing and the blood-activating groups the damage of outerhair cells was relatively mild, and the damage was more evident in theblood-activating group than in the fire-reducing group.4. The MDA level of the fire-reducing group was significantly lower thanthose of the blood-activating and the qi-promoting groups (P﹤0.05), while CATlevel was significantly higher than those of the blood-activating and theqi-promoting groups (P﹤0.05). Compared with the normal group and model group,the three groups were significantly different in the two indicators(P﹤0.01).5. The expressions of VEGFmRNA in all the3treatment groups weresignificantly higher than in the model group (p﹤0.01); it was higher in thefire-reducing group than in the blood-activating and the qi-promoting groups(P﹤0.05), and higher in the blood-activating group than in the qi-promotinggroup (P﹤0.05). The expression of VEGFmRNA in the fire-reducing group was about2.4times what it was in the blood-activating group, about6.8times what itwas in the qi-promoting group, and about8.8times what it was in the model group.For the expression of VEGF protein, in VEGF/β-actin there was no significantdifference between the qi-promoting group and the model group (P﹥0.05); therewas a significant difference between the blood-activating group and theqi-promoting group (P﹤0.05); and there was a significant difference betweenthe fire-reducing group and the blood-activating group (P﹤0.05). The expressionof bFGF mRNA of all the3treatment groups was significantly higher than thatof the model group (P﹤0.05); it was higher in the fire-reducing group than inthe blood-activating and the qi-promoting groups (P﹤0.05); and was higher inthe blood-activating group than in the qi-promoting group (P﹤0.05). The expression of bFGF mRNA in the fire-reducing group was about1.4times what itwas in the blood-activating group, about4.2times what it was in the qi-promotinggroup, and about6.3times what it was in the model group.6. In the normal group and all the3treatment groups, the expressions ofiNOSmRNA were significantly lower than that in the model group (P﹤0.05). Theexpression of iNOSmRNA in the fire-reducing group was similar with that in theblood-activating (P﹥0.05). The expression of iNOSmRNA in the model group wasabout1.3times what it was in the qi-promoting group, about4.7times what itwas in the blood-activating group, and about4.6times what it was in thefire-reducing group. In every treatment group, the expression of MMP-2mRNA wassignificantly lower than that in the model group (P﹤0.05). In the model group,the expression of MMP-2mRNA was1.5times what it was in the qi-promoting group;2.4times what it was in the blood-activating group; and7.3times what it wasin the fire-reducing group.7. In the normal group and all the3treatment groups, the expressions ofBAX mRNA were significantly lower than that in the model group (P﹤0.05). Theexpression of BAX mRNA in the fire-reducing group was lower than those in theblood-activating group and the qi-promoting group (P﹤0.05), the expression ofBAX mRNA in the blood-activating group was lower than that in the qi-promotinggroup (P﹤0.05). The expression of BAX mRNA in the model group was about2.2times what it was in the qi-promoting group o; about4.4times what it was inthe blood-activating group; about6.4times what it was in the fire-reducinggroup; and about14.8times what it was in the normal group. The expression ofBAX mRNA in the qi-promoting group was about2times what it was in theblood-activating group; about3times what it was in the fire-reducing group.The expression of BAX mRNA in the blood-activating group was about1.5timeswhat it was in the fire-reducing group. In the3treatment groups the expressionsof BCL-2mRNA were significantly higher than that the model group (P﹤0.05),and the expression of the fire-reducing group was higher than those in theblood-activating group and the qi-promoting group (P﹤0.05); the expression of the blood-activating group was higher than that in the qi-promoting group (P﹤0.05). In The fire-reducing group the expression of BCL-2mRNA was about2.5times what it was in the blood-activating group, about6times what it was inthe qi-promoting group, and about17times what it was in the model group.8. In protein expression the ratio of BCL-2/Bax, the fire-reducing group﹥the blood-activating group﹥the qi-promoting group﹥the model group. Andthere was significant difference among all the groups (P﹤0.05).Conclusion:1. RRDO therapy has the functions of improving microcirculation andenhancing anti-lipid peroxidation.2. RRDO therapy can effectively improve the photochemically induced cochlearmicrocirculation disturbance, and it is better than activating blood to resolvestasis.3. The simple qi-promoting therapy has no evident effect on thephotochemically induced cochlear microcirculation disturbance4.RRDO therapy can significantly improve the expression of VEGF and bFGFfor guinea pig with microcirculation disturbance of the cochlea, and the effectis better than those of activating blood to resolve stasis and promoting qi.5. RRDO therapy may promote angiogenesis and improve microcirculationthrough promoting the expression of VEGF and bFGF.6. Both RRDO therapy and activating blood to resolve stasis therapy caninhibit the expression of iNOS to protect cochlear tissue ischemia, and theyare better than qi-promoting therapy.7. therapies of RRDO, activating blood to resolve stasis, and promoting qican all inhibit the expression of MMP-2.8. Bcl-2and Bax genes and proteins are all involved in cochlearmicrocirculation damage. And the decreased Bcl-2/Bax ratio is an importantfactor in promoting apoptosis. 9. RRDO therapy and activating blood to resolve stasis therapy cansignificantly inhibit apoptosis through increasing the Bcl-2/Bax ratio.10. In inhibiting apoptosis, RRDO therapy is better than activating bloodto resolve stasis therapy.11. The expression of iNOS is closely related to Bc1-2, and iNOS expressionwas negatively correlated with the ratio of Bcl-2/Bax.