Study On The Mechanism Underlying The Action Of A Novel Ligustrazine Chalcone Derivative On Cochlear Hair Cells

Part ⅠThe mechanism underpinning the effect of a newly-synthesized chalcone derivative of Ligustrazine in HEI-OC1 cellsObjective:A newly-synthesized chalcone derivative of ligustrazine,named as Z11,has shown a variety of promising biological activities.However,no study has been reported about the effects of Z11 on HEI-OC1 cell line,the utility model relates to a mouse auditory cell line for evaluating the ototoxicity of drugs,which can also be used to evaluate the ear protective properties of drugs.Therefore,the present study aimed to investigate the pharmacological activities of Z11 on the HEI-OC1 cells in vitro as well as the possible mechanism.Methods:The cell viability of each group was detected by cell counting kit-8,and the appropriate concentration of Z11 was selected according to the results of cell viability experiments.Then HEI-OC1 cells in the experimental group were treated with 5μM Z11 for 6 hours,12 hours,24 hours and 48 hours,respectively,and cell counting kit-8 was used to detect the viability of HEI-OC1 cells at different time points.After the HEI-OC1 cells in the experimental group were treated with Z11,TUNEL and flow cytometry were used to detect the apoptosis of HEI-OC1 cells induced by Z11.After the HEI-OC1 cells of the experimental group were treated with Z11,the expression of cleaved-Caspas-3 protein in the cells of the experimental and group was determined by immunofluorescence method.Results:CCK-8 assay showed that Z11 damaged HEI-OC1 cells,and induced HEI-OC1 cytotoxicity in a concentration-dependent and time-dependent mannerin vitro.TUNEL assay and flow cytometry showed that Z11 induced apoptosis in HEIOC1 cells.Immunofluorescence showed that Z11 exerted its cytotoxicity through apoptosis in HEI-OC1 in vitro.Conclusion:These findings suggest that Z11 has ototoxicity via triggering apoptosis via Caspase-dependent pathway in HEI-OC1 cells.Part ⅡThe mechanism underlying the action of a newly-synthesized chalcone derivative of Ligustrazine in primary cultured cochlear hair cells of miceObjective:Ligustrazine,as one of the major active components derived from the traditional Chinese medicine Ligusticum chuanxiong,has strong antioxidant activity.Ligustrazine can ameliorate neuron and endothelial cell damages caused by cerebral ischemia,and inhibit platelet aggregation.Ligustrazine injection has been clinically used in the treatment of stroke.However,the short half-time and rapid metabolism of ligustrazine in the body have limited its application in clinical practice.Therefore,there is a strong need to design and synthesize novel derivates of ligustrazine.Currently,the studies have found that a newly-synthesized chalcone derivative of ligustrazine(named as Z11)has protective effect on brain injury induced by cerebral ischemia-reperfusion.However,what effect of Z11 exerts on cochlear hair cells(HCs)has not been reported to date.Herein,we aim to explore the effects as well as the possible mechanisms of Z11 on HCs.Methods:The cochlear basement membrane of C57BL/6 mice on the 3rd day after birth was dissected under microscope,then moved to sterile Hank’s Balanced Salt Solution(HBSS).The basilar membrane including Corti’s organ of the cochlea was placed onto a glass of coverslip and incubated for 24 hours,then,the cultured cochleae at indicated time points were selected into experimental and control groups in a random fashion.Transmission electron microscopy(TEM)and immunofluorescence assay were used to detect the morphological changes of the cochlear basilar membrane containing the organ of Corti,and flow cytometry method and TUNEL technique for detecting apoptosis of the cultured cochleae HCs.The cultured cochlear explants were applied immunostaining with primary antibodies:rabbit anti-cleaved-Caspase-3 antibody,mouse anti-AIF antibody to detect the expression of cleaved-Caspase-3 and AIF.The expressions of Caspase-9,Caspase-3,Apaf-1,Bax and Bcl-2 were assayed by qRT-PCR,and the expressions of cleaved-Caspase-9 and cleaved-Caspase-3 were examined by Western-blot analysis.Reactive oxygen species(ROS)produced in mitochondria were analyzed by immunofluorescence using MitoSOX TM red mitochondrial superoxide indicator.Results:Immunostaining showed that Z11 was ototoxic to mouse cochlear hair cells and significantly triggered cell death in a concentration-,time-and locationdependent manner.TEM images showed the damages of HCs in cochlear explants treated with Z11.Apoptotic cells were stained by TUNEL positively in cochlear explants in response to Z11.Apoptotic cells were stained by cleaved-Caspase-3 positively,and AIF was detected in the HCs treated with Z11.The protein expressions of cleaved-Caspase-3 and cleaved-Caspase-9 were increased significantly,and qRTPCR data demonstrated that the mRNA expression of Caspase-9,Caspase-3,Bax and Apaf-1 was elevated significantly in cochleae upon exposure to Z11.As shown in immunofluorescence using MitoSOXTM red mitochondrial superoxide indicator,treatment of Z11 significantly elevated the level of ROS in mitochondria,which would lead to a series of damages in mitochondria function and morphology.Conclusion:These findings suggest that Z11 exhibits its ototoxicity through inducing apoptosis of HCs via both Caspase-dependent and AIF translocation pathways in mouse cochlear cultures.