Study On The Mechanism Of NLRX1 Gene In Regulating Cisplatin-induced Ototoxity

PART 1:Study on the relatiohship of NLRX1 and ROS/JNK Signaling in Regulating Cisplatin-induced OtotoxityObjective:Cis-diamminedichloroplatinum(cisplatin)is an effective chemotherapeutic drug for cancer therapy,but,is limited to use due to its severe ototoxicity.Reactive oxygen species(ROS),apoptosis and inflammatory damage have been verified to contribute to the pathogenesis of cisplatin ototoxicity.However,the mechanism has not been fully elucidated yet.Nucleotide-binding domain and leucine-rich-repeat-containing family member X1(NLRX1),a recently discovered protein,is a cytoplasmic pattern recognition receptor.NLRX1 is predominantly located in mitochondria,which plays roles in regulating major cellular pathways that is tightly related to mitochondrial function,reactive oxygen species(ROS)production,inflammation and apoptosis.The role of NLRX1 in regulating hearing impairment has not been understood.This study is designed to explore whether NLRX1 expresses in HEI-OC1 cells and,if so,to investigate the possible correlations between NLRX1 and cisplatin-induced ototoxity in vitro.Methods:(1)The location and expression of NLRX1 were investigated by immunofluorescence,qRT-PCR and Western blot in HEI-OC1 cells.(2)MTT assay combined with TUNEL and flow cytometry assay were performed to detect cell viability and apoptosis in HEI-OC1 cells treated with cisplatin.(3)qRT-PCR and Western-blot were used to detect the expression of NLRX1 in HEI-OC1 cells induced by cisplatin.The level of ROS production was measured by immunofluorescence.(4)The NLRXI overexpression and NLRX1 silencing cells were constructed and the inner ear injury model was established by cisplatin treatment for 24 hours in primary culture of cochlear explants.(5)Flow cytometry was used to detect the apoptosis induced by cisplatin in both NLRX1 silencing and overexpression cells.Western-blot was performed to detect the expression levels of cleaved caspase3,bax and bcl-2 in both NLRX1 silencing and overexpression cells followed by cisplatin treatment.(6)The ROS induced by cisplatin in both NLRX1 silencing and overexpression cells were detected by flow cytometry.The effect of NLRX1 on activation of JNK signaling induced by cisplatin was detected by Western-blot.(7)The changes of NLRX1 expression,ROS/JNK activation and apoptosis in mice cochlea explants treated with cisplatin were detected by immunofluorescence and Western-blot.Results:(1)NLRX1 was expressed in mitochondria of HEI-OC1 cells.NLRX1 was spotted in the cytoplasm of HEI-OC1 cells and co-stained with mitochondrial marker Mitotraker.(2)Cisplatin reduced cell viability via inducing apoptosis in HEI-OC1 cells.Cisplatin-treatment decreased cell viability of HEI-OC1 cells in a concentration-and time-dependent manner.The number of TUNEL-positive HEI-OC1 cells was visibly increased in cisplatin-treated group than that in control group.Similar changes were obtained by flow cytometry of double Annexin V/PI staining cells,in which the proportion of apoptotic cells was significantly higher in cisplatin-treated cells than that in control group.(3)Results of real-time PCR and western-blot assay showed that,NLRX1 mRNA and protein expression in cisplatin-treated group was obviously enhanced at 18 h,24 h,48 h time points and peaked at 24 h.ROS generation was enhanced in a time-dependent manner after cisplatin treatment and peaked at 24 h,which coincided with the peak time of NLRX1 expression.(4)NLRX1-silenced(nlrx1-siRNA)and NLRX1-knock-in(nlrx1-KI)HEI-OC1 cells were successfully generated.NLRX1 deficiency decreased the apoptotic proportion in cisplatin-treated cells(stress condition)and overexpression presented an opposite role.(5)Mechanism study revealed that Bax and cleaved caspase-3(an activated form of caspases-3)expressions were significantly decreased in nlrxl-siRNA cells following cisplaitn treatment with an obvious increase of Bcl-2.Instead,NLRX1 overexpression resulted in opposite changes in these three proteins in nlrx1-Kl cells.(6)NLRX1 silencing attenuated cisplatin-induced ROS production and activation of JNK signaling,and overexpression of NLRX1 accelerated cisplatin-induced ROS production and activation of JNK signaling.(7)NLRX1 promoted cisplatin-induced apoptosis and ROS/JNK activation.Inhibition of ROS over-production or JNK signaling significantly attenuated NLRX1?induced apoptosis.(8)Immunoflourescence staining showed that 30 ?M cisplatin treatments for 24 h led to conspicuous loss of hair cells in the middle turn of cochlea.Cisplatin significantly increased NLRX1 expression,activation of ROS/JNK and induction of apoptosis in cochlear explants.Conclusions:The findings from this work revealed that NLRX1 sensitized HEI-OC1 cells to cisplatin-induced apoptosis via activation of ROS/JNK signaling pathway,suggesting that NLRX1 acted as an important regulator of the cisplatin-elicited ototoxity.PART 2:Study on the mechanism underlying the action of NLRX1-mediated autophagy in cisplatin-induced OtotoxityObjective:NLRX1,a member of the NLRs family,located in mitochondria,is tightly related to mitochondrial function,reactive oxygen species production and autophagy.The present study was designed to explore the possible correlations between NLRX1-mediated autophagy and cisplatin-induced ototoxity in vitro.Methods:(1)Immunofluorescence and Western-blot were used to detect the expression of NLRXI and LC3-? in HEI-OC1 cells treated with different doses of cisplatin.(2)Cell viabilities of HEI-OC1 cells treated with different doses of cisplatin were detected by MTT assay.(3)Transmission electron microscopy was used to detect ultrastructural changes of HEI-OC1 cells and the formation of autophagosomes in HEI-OC1 cells of different treatment groups.(4)The NLRX1 overexpression HEI-OC1 cells were constructed by plasmid transfection.The expression of NLRX1 and LC3-? were detected by western-blot.The formation of autophagosomes in NLRX1 overexpression cells and conroll cells were observed by transmission electron microscope.(5)Immunofluorescence and western-blot were used to detect the expression of NLRX1 and LC3-? in cells of NLRX1 silencing group(siRNA-nlrxl)and control group(SC)treated with cisplatin.MTT assay was used to detect the cell viability in different treatment groups.(6)Immunofluorescence and Western-blot were used to detect the expression of NLRX1 and LC3-?in cells of NLRX1 overexpression group(nlrxl-KI)and vector-control group treated with cisplatin,and the cell viability was detected by MTT assay.(7)The morphology and number of mitochondria in cells of NLRX1 overexpression group(nlrxl-KI)and vector-control group were observed by transmission electron microscope.The levels of mitochondria1 ROS in different transfected groups were detected by immunofluorescence and flow cytometry.(8)Inhibition of ROS or autophagy,the autophagy-related protein LC3-? and apoptosis-related factor cleaved-caspase3 in cells treated with cisplatin were dectected by western-blot.(9)NLRX1 expression,ROS level,autophagy activity,and cleaved-caspase3 expression were detected by immunofluorescence and western-blot in mice cochlear explants with different treatments.Results:(1)Cisplatin promoted LC3-? and NLRX1 expression in HEI-OC1 cells:Cisplatin promoted the expression of LC3-? in a concentration-dependent manner and had a positive correlation with NLRX1 expression and a negative correlation with cell viability.(2)NLRX1 promoted autophagosome accumulation in HEI-OC1 cells:Immunofluorescence and transmission electron microscopy showed that NLRX1 overexpression promoted autophagosome accumulation in HEI-OC1 cells under resting conditions.(3)Effect of NLRX1 on cisplatin-induced autophagy and cytotoxicity in HEI-OC1 cells:NLRX1 silencing attenuated cisplatin-induced autophagy aggregation and cytotoxicity,and NLRX1 overexpression promoted cisplatin-induced autophagy aggregation and increased cisplatin-induced cytotoxicity.(4)Overexpression of NLRX 1 altered mitochondrial morphology and promoted cisplatin-induced intracellular ROS production:overexpression of NLRX1 resulted in swelling mitochondria,reduction and fragmentation of the mitochondrial crest,and increased production of cisplatin-induced ROS.(5)Inhibition of ROS significantly attenuated cisplatin-induced autophagy and apoptosis,and reduced cisplatin-induced cytotoxicity in HEI-OC1 cells.(6)Cisplatin promoted the up-regulation of NLRX1 in the mice cochlear explants in vitro and,inhibition of ROS attenuated cisplatin-induced cytotoxicity.(7)Inhibition of ROS attenuated autophagy and apoptosis in mice cochlear explants treated with cisplatin in vitro.Conclusions:The findings from this work revealed that NLRX1 sensitized auditory cells in vitro to cisplatin-induced ototoxity via autophagic cell death pathway,providing another strategy against cisplatin-induced ototoxicity.