The Interventive Effects Of Histone Deacetylase Inhibitor On Cisplatin-induced Ototoxicity Via Regulating Expression Of Caspase12

Cisplatin was widely used for clinical anti-cancer therapy, however, thecisplatin-induced ototoxicity limited its long-term application. Finding out aneffective solution to inhibit its Side effect of ototoxicity becomes urgent priority. Histonedeacetylases inhibitors（HDACIs）is a kind of compound regulating the expression of gene intranscriptional level, and it has a certain effect of anti-cancer,also has a synergism withcisplatin in anti-cancer therapy. Moreover,it has a certain Protective effect to nervoussystem.As the similarity between cochlea sensitive cells and neurons,it is supposed thatHDACIs has a certain Protective effect to cochlea sensitive cells.Objective：To investigate the occurrence and mechanism of cisplatin ototoxicity andtheantagonism of HDACIs to cisplatin-induced ototoxicity from the perspectieof morphology, molecular biology, immunohistochemistry andchemistry using ratmodels in vivo and in vitro.Methods：In vivo: For a control study, the rats were divided into different groups as follows:control group （physiological saline）, three different doses of sodium butyrate groups(200、400、600mg·kg^-1), cisplatin group (2mg·kg^-1) and three different doses ofcisplatin combining with sodium butyrate groups.After7daysintraperitoneal injection of drugs, detect the changes of ABR values between injectionbefore and after, use HE and Hoechst staining to the rat cochlear slice, useimmunohistochemical staining to detect Caspase12.In vitro:Culture of the basilar membrane in vitro were divided into differentgroups as follows: control group, TSA200nM group, cisplatin150μM group andcisplatin combining with TSA group.Use Real-Time PCR, Western Blot,andimmunofluorescence technique to detect the changes of Caspase12in cochleatissues.Use Caspase activity kits to detect the activity of Caspase9and Caspse3.Results:ABR values shows: Compared with the control group, each of sodium butyrate groups (200、400、600mg·kg^-1) has no remarkable differences,but cisplatin group(2mg·kg^-1) has remarkable differences（P<0.05）; Compared with the cisplatin group(2mg·kg^-1), each of cisplatin combining with sodium butyrate groups(200mg·kg^-1、400mg·kg^-1、600mg·kg^-1) has remarkable differences（P<0.05）,and taking on adose-dependent manner. HE staining of the rat cochlear slice shows: cisplatin group(2mg·kg^-1):The cochlear spiral distorted;the inner and outer hair cells were scatteredaccompanying with loss;the spiral ganglion neuron（SGN）were scattered,and takingon the characteristics of apoptosis;the morphous of both the cochlear spiral and cellsof cisplatin combining with sodium butyrate group (600mg·kg^-1) get significantimproved. Hoechst staining shows: Compared with the control group,the hair cells ofcisplatin group (2mg·kg^-1) have a significant loss, especially in the bottom back ofcochlea（P＜0.05）; the survival rate of hair cells of cisplatin combining with sodiumbutyrate group (600mg·kg^-1) has been improved. Gene chips shows: Caspase12hasremarkable differences between each groups.The expression of Caspase12increased1.2times in cisplatin150μM group than that of control group,but the expression ofCaspase12decreased4.01times in cisplatin combining with TSA200nM group thanthat of cisplatin group. Real-Time PCR shows:the mRNA levels of Caspase12startedto increase after adding cisplatin150μM6hours（P＜0.05）, the mRNA levels ofCaspase12in cisplatin combining with TSA200nM group decreased significantlythan that of cisplatin150μM group （P＜0.05）. Western Blot shows:Compared with the control group, the protein levels of Caspase12after addingcisplatin150μM6hours has no remarkable differences, the protein levels ofCaspase12after adding cisplatin150μM12hours has significantly increased（P＜0.05）,and the protein levels of Caspase12recovered after adding cisplatin150μMnM group decreased significantly than that of cisplatin150μM group（P＜0.05）.Immunofluorescence shows: Caspase12expresses in nucleus and cytoplasm of haircells.The expression of Caspase12increased in cisplatin150μM group,and theexpression of Caspase12decreased in cisplatin combining with TSA200nM groupthan that of cisplatin150μM group. Immunohistochemisty of cochlea slices shows: the expression of Caspase12in cisplatin group (2mg·kg^-1) takes on positive（+++）, theexpression of Caspase12in cisplatin combining with sodium butyrategroup(600mg·kg^-1) takes on positive （+）.The activity of Caspase shows:compared with the control group,the activity of Caspase9in cisplatin150μM groupafter adding cisplatin6hours increased（P＜0.05）; the activity of Caspase9incisplatin150μM group after adding cisplatin12hours significantly increased（P＜0.05）; the activity of Caspase9in cisplatin150μM group after adding cisplatin24hours decreased a little,but remained above the normal levels （P＜0.05）.Compared with the control group,the activity of Caspase3in cisplatin150μM groupafter adding cisplatin6hours has no remarkable differences; the activity of Caspase3in cisplatin150μM group after adding cisplatin12hours significantly increased（P＜0.05）; the activity of Caspase3in cisplatin150μM group after adding cisplatin24hoursdecreased a little,but remained above the normal levels （P＜0.05）.Compared with the cisplatin150μM group,both the activity of Caspase9and Caspase3incisplatin combining with TSA200nM group significantly decreased（P＜0.05）.Conclusions:1.HDACIs does not have the injury effect to rats hearing;2.HDACIs can antagonize cisplatin induced hearing loss, the antagonisticeffect was dose-dependent;3.HDACIs play an antagonistic effect on cisplatin-induced ototoxicity viainhibiting Caspase12expression of both mRNA levels and protein levels;4. HDACIs play an antagonistic effect on cisplatin-induced ototoxicity viainhibiting Caspase12expression to depress the activity of Caspase9and Caspase3.