Protective Effect Of Puerarin On Cisplatin-Induced Ototoxicity In Mice

ObjectiveTo investigate the protective effect of puerarin on cisplatin-induced ototoxicity in mice,and to provide a new way for clinical prevention and treatment of cisplatin-induced deafness.Methods1?In vivo study100 adult BALB/c mice were randomly divided into four groups:control group,cisplatin group,cisplatin plus puerarin group and puerarin group?n=25?.In cisplatin group,the mice were continuously given cisplatin 4.5 mg/kg/day by intraperitoneal injection for 5 days.The mice in Puerarin group were injected with puerarin 200 mg/kg/day for 14 days.In cisplatin plus puerarin group,the mice were injected puerarin 200mg/kg/day intraperitoneally for 9 days,and cisplatin 4.5mg/kg/d was injected intraperitoneally for 5 days at the same time on the 10th day.The mice in control group were injected with the same amount of saline.Auditory brainstem response test and hair cell count were used to observe the hearing changes and hair cell damage in mice before and after treatment.Expression of stromal interaction molecule 1?STIM1?,Orai1,transient receptor potential vanilloid receptor 1?TRPV1?and calpain 2 in mouse cochlea was detected by using fluorescence staining,Western blot and Real-time PCR technique.2?In vitro studyThe basal membrane of newborn 3-day-old BALB/c mice cochlea was cultured in serum-free fresh medium for 24h,then the original medium was discarded.The basal membrane was randomly divided into 4 groups.Control group was given fresh serum-free medium for 2 ml.Cisplatin group was treated with fresh serum-free medium containing 16?g/ml cisplatin for 2 ml;In cisplatin plus puerarin group,the serum-free fresh medium was given puerarin(concentration of 10^-6M)for 30min in advance,and then added cisplatin to 2ml;Puerarin group was treated with 2 ml serum-free fresh medium containing 10^-6M puerarin;All groups were cultured for 24 hours under the same condition.Morphological changes of cochlear hair cells were observed by fluorescence staining with FITC or TRITC and the number of hair cells in each group was counted.Expression of STIM1 and Orai1 in each group was detected by using Western blot technique,and the level of Ca²⁺was detected by the Fluo-4 AM Ca²⁺fluorescence probe.Results1.After continuous administration,the ABR threshold shift in cisplatin group was significantly higher than that in control group?P<0.01?,and the ABR threshold shift in cisplatin plus puerarin group was significantly lower than that in cisplatin group?P<0.01?.There was no significant difference in ABR threshold shift between puerarin group and control group?P>0.05?.2.Both in vivo and in vitro hair cell counts showed that the rate of hair cell deletion in cisplatin group was significantly higher than that in control group?P<0.01?.Compared with cisplatin group,the loss rate of outer hair cells in basal turn and middle turn of cochlear basal membrane was significantly lower than that in cisplatin plus puerarin group?P<0.01?.The deletions of outer hair cells and inner hair cells in cisplatin plus puerarin group were significantly lower than those in cisplatin group?P<0.01?.There was no significant difference between control group and puerarin group?P>0.05?.3.Fluo-4 AM Ca²⁺fluorescence probe detection showed that the level of Ca²⁺in the hair cells of cisplatin group was significantly higher than that of control group?P<0.01?,while the level of Ca²⁺in cisplatin plus puerarin group was significantly lower than that of cisplatin group?P<0.01?.There was no significant difference in fluorescence intensity between control group and puerarin group?P>0.05?.4.The results of Real-time PCR showed that the mRNA levels of STIM1and Orai1 in cisplatin group were significantly higher than those in control group?P<0.01?,while the mRNA in cisplatin plus puerarin group was significantly lower than that in cisplatin group?P<0.05,P<0.01?.There was no significant difference between control group and puerarin group?P>0.05?.5.The results of immunofluorescence test showed that the positive reaction of STIM1,Orai1 in cisplatin group was significantly higher than that in control group?P<0.01?,and the positive reaction of STIM1,Orai1 in cisplatin-puerarin group was significantly lower than that in cisplatin group?P<0.01?.There was no significant change in protein expression between puerarin group and control group?P>0.05?.6.The results of Western blot detection in vivo and in vitro experiment show that the expression of STIM 1 and Orai 1 protein in cisplatin group was significantly higher than that in control group?P<0.01?,while the expression of STIM 1 and Orai 1 protein in cisplatin plus puerarin group was significantly lower than that in cisplatin group?P<0.01?.There was no significant change in protein expression between puerarin group and control group?P>0.05?.The results of in vitro experiment were the same as those of in vivo experiment.7.The results of immunofluorescence showed that the positive expression of TRPV1 and calpain2 protein in cisplatin group was significantly higher than that in control group?P<0.01?,while that in cisplatin plus puerarin group was significantly lower than that in cisplatin group?P<0.01?.There was no significant change in fluorescence intensity between the control group and puerarin group?P>0.05?.ConclusionsPuerarin can inhibit the occurrence of calcium overload in cochlear cells by reducing the expression of STIM1,Orai1,TRPV1 and calpain2 proteins,thus effectively protecting mice from hearing impairment induced by cisplatin.