Role Of Mitochondrial Permeability Transition In Gentamicin-Induced MDCK Cells Apoptosis And Protect Effect Of Edaravone

Gentamicin is a widely used in clinical aminoglycoside antibiotics,had strong antibacterial activity and wide antibacterial spectrum,improper use can cause multiple organ damage.Gentamycin into the body would accumulate in the kidneys,causing kidney damage.In recent years,apoptosis had been found to play an important role in gentamicin-induced renal injury,but the mechanisms of apoptosis have not been elucidated.The opening of MPTP causes MPT to activate the mitochondrial apoptotic pathway,but it is unclear whether gentamicin can induce the opening of MPTP.In this study,MDCK cells were cultured in vitro as a model to explore the mechanism of MPTP in gentamicin-induced mitochondrial apoptosis pathway and the protective effect of edaravone in vitro.1.Effect of gentamicin's cytotoxicity in MDCK cellsTo investigate the effect of gentamicin's cytotoxicity of MDCK cells,MDCK cells were treated with different concentrations of gentamycin(0,2,4,8 mmol/L)for 24 h and 4 mmol/L gentamycin treated cells with different times(0,6,12,24 h).The cell viability were measured by CCK-8 assay,The changes of KIM-1 and NGAL wre detected by ELISA.The MPTP opening and apoptosis rate were detected by flow cytometry,the changes of nuclear morphology were observed by Hochest 33258 staining.The expression of Bax and Bcl-2 protein were detected by Western blotting.The results showed that compared with the control group,the viability of MDCK cells decreased with the increase of gentamicin's concentrations and times(P<0.05 or P<0.01).The KIM-1 and NGAL contents increased firstly and then decreased with the increase of gentamycin's concentrations,and reached the peak of secretion when gentamycin was 4 mmol/L(P<0.05 or P<0.01).With the prolongation of times,the content of KIM-1 increased first and then decreased,while the content of NGAL increased(P<0.05 or P<0.01).Compared with the control group,with increase of gentamicin's concentration and the prolongation of times,the the opening of MPTP and apoptosis rate increased significantly(P<0.05 or P<0.01),Nuclei appeared pyknosis,thick stain even chipping and the ratio of Bcl-2/Bax was decreased(P<0.05 or P<0.01).These findings suggested that gentamicin has a toxic effect on MDCK cells,there is a dose effect relationship and a time effect relationship,and it can induce the opening of MPTP and apoptosis,so it's toxic mechanism may be related to the opening of MPTP and apoptosis.2.MPTP in gentamicin-induced apoptosis in MDCK cellsIn order to investigate the role of MPTP in gentamicin-induced mitochondrial apoptosis in MDCK cells,MDCK cells were treated with 4 mmol/L gentamycin and 2 ?mol/L inhibitor CsA for 24 h respectively or unitedly.Flow cytometry was used to detect MPTP open and apoptosis rate.Fluorescence was used to detect mitochondrial changes and mitochondrial matrix swelling.The changes of mitochondrial membrane potential were detected by JC-1 probe.The expression of Bax and Bcl-2,the activation of caspase-9,caspase-3 and PARP protein,the release of Cyt C and AIF were detected by Western blotting.The results showed that inhibitor CsA could significantly or very significantly inhibit the opening of MPTP induced by gentamicin in MDCK cells;the increase of apoptosis rate;the swelling of mitochondria;the decrease of ATP content;the decrease of mitochondrial membrane potential;Bcl-2/Bax ratio decreased,the activation of caspase-9,caspase-3 and PARP protein,the release of Cyt C and AIF(P<0.05 or P<0.01).These date suggested that MPTP positive regulation of gentamicin-induced mitochondrial apoptosis in MDCK cells.3.The protection of EDa on apoptosis induced by gentamicin in MDCK cellsIn order to investigate the protection of EDa on apoptosis induced by gentamicin in MDCK cells,MDCK cells were treated with 4 mmol/L GM and 40 ?mol/L EDa for 24 h respectively or unitedly.The apoptotic rate was detected by Annexin V-FITC/PI double staining.The changes of nuclear morphology were observed by Hochest 33258 staining.T-SOD,GSH-Px,CAT activity and MDA content were detected by colorimetric assay.ROS content was detected by fluorescent staining.The expression of Bax and Bcl-2,the activation of caspase-9,caspase-3 and PARP protein,the release of Cyt C and AIF were detected by Western blotting.The results showed that edaravone significantly or very significantly inhibited gentamicin-induced apoptosis in MDCK cells;changes in nuclear morphology;ROS content;T-SOD,GSH-Px activity increased,CAT activity Decreased MDA content,decreased Bcl-2/Bax ratio,activation of caspase-9,caspase-3 and PARP protein,and release of Cyt C and AIF(P<0.05 or P<0.01).These findings suggested that edaravone can relieve oxidative damage to supperess the gentamicin-induced apoptosis through mitochondrial apoptotic pathway.