| Objective:We took advantage of C57 mice intraperitoneally injected with cisplatin alone to make drug-induced hearing loss model,combined with cisplatin and allicin to investigate whether allicin plays a protective role in vivo against cisplatin-induced hearing loss.Methods:Experiments were performed on age-and sex-matched 7-to 8-week-old mice weighing 17-23 g.The mice were divided into three groups(n = 30 each;group 1,0.9%physiological saline-injected controls;group 2,cisplatin-injected;group 3,cisplatin+allicin-injected).Mice in groups 1,2 and 3 received intraperitoneal(i.p.)injections of 0.9%physiological saline(0.6 ml/100 g),cisplatin(3 mg/kg)or allicin(18.2 mg/kg),respectively.Group 1 mice were administered with 0.9%physiological saline(0.6 ml/100 g i.p.)for seven consecutive days.Group 2 mice were administered with cisplatin(3 mg/kg i.p.)for seven consecutive days.To evaluate the effects of allicin on cisplatin-induced ototoxicity,groups 3 mice were given 18.2mg/kg allicin i.p.one day ahead and at 2 hours before the daily injection of cisplatin.The auditory brain stem response(ABR)of mice in three groups were measured before and after seven day drug injection.TUNEL apoptotic kits was used to check apoptosis of outer hair cells(OHCs),supporting cells(SCs)and spiral ganglion neurons(SGNs)in three groups.Counting the survival outer hair cells and spiral ganglion neurons in three groups.To observe the mitochondria of outer hair cells and spiral ganglion neurons in three groups by scanning transmission electron microscope.To test the expression of Bax,Bcl-2,cleaved-caspase-9,cleaved-caspase-3 of cochlear tissues in three groups by Western blot.To test the expression of P53 in outer hair cells and supporting cells in three groups by immunofluorescence.To test the expression of cytochrome-C,cleaved-caspase-9 and cleaved-caspase-3 in spiral ganglion neurons in three groups by immunofluorescence.Detection the level of malondialdehyde(MDA)and superoxide dismutase(SOD)of the cochlea in three groups.Results:The result of ABR showed that the ABR threshold shifts in cisplatin group were increased compared to the control group.However,in pre-treated with allicin group,the ABR threshold shifts were obviously decreased in most frequencies except 32kHz.The survival number of outer hair cells and the mean density of SGNs in middle and basal turns were decrease in cisplatin group and allicin could protect the damage of outer hair cells and spiral neuron cells caused by cisplatin.TUNEL detection results showed that positive stainings of TUNEL could be found in OHCs,SCs,SGNs in middle and basal turns in cisplatin group,allicin could significantly reduce the apoptosis induced by cisplatin.Transmission scanning electron microscopy results showed that allicin significantly protected the damage of mitochondria in spiral neuron cells induced by cisplatin.The results of the Western blot showed that cisplatin increased the expression of Bax,cleaved-caspase-9,cleaved-caspase-3 and reduced the expression of Bcl-2.However,allicin could significantly reduce the expression of Bax,cleaved-caspase-9,cleaved-caspase-3 and increase the expression of Bcl-2.Immunofluorescence results showed that cisplatin induced the release of cytochrome-C,the expression of cleaved-caspase-9 and cleaved-caspase-3 in the spiral neurons cells,induced the expression of P53 in outer hair cells and supporting cells.Allicin could significantly reduce the release of cytochrome-C,the expression of cleaved-caspase-9 and cleaved-caspase-3 in the spiral neurons cells.Allicin also could reduce the expression of P53 in nucleus of OHCs in basal and middle turns,reduce the expression of P53 in nucleus of SCs in middle turns.In addition,allicin reduced the level of malondialdehyde(MDA)in the cochlear tissue,but increased the level of the superoxide dismutase(SOD).Conclusions:Allicin is effective in alleviating hearing loss casued by cisplatin,even though its protective effect is incomplete.Allicin protects SGNs from apoptosis induced by cisplatin through mitochondrial pathway while protects OHCs and SCs from apoptosis through p53 pathway.In the meantime,allicin protects the cochlear injury induced by cisplatin through antioxidant effect.Part nAllicin protects against cisplatin-induced vestibular dysfunction by inhibiting the apoptotic pathwayObjective:The purpose of this study was to investigate the effects of allicin against cisplatin-induced vestibular dysfunction in mice and to make clear the mechanism underlying the protective effects of allicin on oto-vestibulotoxicity.Methods:Experiments were performed on age-and sex-matched 7-to 8-week-old mice weighing 17-23 g.The mice were divided into three groups(n = 30 each;group 1,0.9%physiological saline-injected controls;group 2,cisplatin-injected;group 3,cisplatin+allicin-injected.Mice in groups 1,2 and 3 received intraperitoneal(i.p.)injections of 0.9%physiological saline(0.6 ml/100 g),cisplatin(3 mg/kg)or allicin(18.2 mg/kg),respectively.Group I mice were administered with 0.9%physiological saline(0.6 ml/100 g i.p.)for seven consecutive days.Group 2 mice were administered with cisplatin(3 mg/kg i.p.)for seven consecutive days.To evaluate the effects of allicin on cisplatin-induced ototoxicity,groups 3 mice were given 18.2mg/kg allicin i.p.one day ahead and at 2 hours before the daily injection of cisplatin.Swimming tests were conducted to observe the mice’s swimming strokes before and after seven day drug injection.The swimming device for test was a rectangular water tank with 70 cm long and 40 cm wide,with room temperature water to the depth of 10 cm,the mice were placed in the center of the water tank and the body was parallel to the long axis of water tank,let go,to observe the free swimming posture.The swimmer who headed up and out the water with stable posture was recorded 3 scores.The swimmer who headed up and out the water,the posture is not(?)not expose to the surface of the water was recorded 0 score,the mouse must be immediately rescued from the water to avoid drowning.5 mice were randomly selected in each group for swimming test,each mouse was tested 3 times,1 min each time,each time interval of 10 min.Test alternately,preventing excessive fatigue which might effect the results of test.Counted the average and performed statistic analysis.Tests were conducted at room temperature.The appearance and density of the hair bundle of hair cells in utricule,saccule and ampulla were observed in three groups by immunofluorescence method.TUNEL apoptotic kits was used to check apoptosis of hair cells in the utricule,saccule and ampulla in three groups.The expression and fluorescence density of cleaved-caspase-3 in hair cells layers and vascular layer cells in utricule,saccule and ampulla were checked by immunofluorescence method.The expression of AIF(Apoptosis-inducing factor)and AIF nuclear transfer rate in hair cells layers and vascular layer cells in utricule,saccule and ampulla were checked by immunofluorescence method.Results:In swimming test,vestibular function of C57 mice was damaged in the cisplatin group.Allicin could significantly improve the vestibular function of C57 mice.After 7 days of cisplatin treatment,the utricule,saccule and ampulla in the cisplatin group showed an obviously reduced number of hair bundles of hair cells with an uneven distribution and abnormal appearance.However,hair bundles of hair cells in the cisplatin+allicin group were well-preserved with normal appearance and a high density.There was no staining of TUNEL staining in vestibular cells in control group.Following 7 days of cisplatin treatment,positive TUNEL staining were found in neurons of vestibular ganglion,mesenchymal cells,hair cells and supporting cells in utricule,saccule and ampulla in cisplatin group.However,no TUNEL staining could be observed in vestibular ganglion,hair cells and supporting cells in cisplatin+allicin treatment group.Only some mesenchymal cells were marked by TUNEL staining.Immunofluorescence results showed that there was no staining of cleaved-caspase-3 in control group.However,in cisplatin treatment group,positive staining of cleaved-caspase-3 could be found in the cytoplasm of hair cells and vascular layer cells in utricule,saccule and ampulla.In cisplatin+allicin group,the expression of cleaved-caspase-3 was significantly decreased both in the cytoplasm of hair cells and vascular layer cells.The fluorescence density of cleaved-caspase-3 in hair cells layers and vascular layer cells in cisplatin group were significantly higher than that in control group,while allicin significantly reduced the fluorescence density of cleaved-caspase-3 in hair cells layers and vascular layer cells in cisplatin+allicin group.Immunofluorescence results of AIF showed that AIF was distributed surrounding the nucleus in the cytoplasm and no distribution within the nucleus in control group.After 7 days of cisplatin treatment,a strong fluorescence of AIF was detected within the nuclear region both in hair cells layers and vascular layer cells.The nuclear translocation ratio of AIF in hair cells layers and in vascular cells layers in cisplatin group were significantly higher than that in control group,while allicin significantly reduced the AIF nuclear translocation ratio in hair cells layers and allicin didn’t significantly reduced the AIF nuclear translocation ratio in vascular cells layers in cisplatin+allicin group.Conclusions:Mice intraperitoneally injected with cisplatin exhibited vetstibular dysfunction in swimming test,which agreed with impairment in vestibule.However,these impairments were significantly prevented by pre-treatment with allicin.Allicin markedly reduced cisplatin-activated expression of cleaved-caspase-3 in hair cells layers and vascular layer cells of utricule,saccule and ampulla,but also decreased AIF nuclear translocation of hair cells layers in utricule,saccule and ampulla.These results showed that allicin played an effective role in protecting vestibular dysfunction induced by cisplatin via inhibiting caspase-dependent and caspase-independent apoptotic pathways.Therefore,allicin may be useful in preventing oto-vestibulotoxicity mediated by cisplatin. |
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