A Newly-Synthesized Chalcone Derivative of Ligustrazine Induces Caspase-Dependent and Apoptosis-Inducing Factor-Dependent Apoptosis in Cochlear Hair Cells

Abstract

Objective: A newly synthesized derivative of ligustrazine chalcone, named as Z11, has shown a variety of promising biological activities. Here we aim to explore the effects of Z11 on the cochlear hair cells (HCs). Methods: Immunostaining and transmission electron microscopy (TEM) were used to examine the survival of HCs and their morphological changes. Furthermore, apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and the mRNA expression of apoptosis related genes including Caspase-9, Caspase-3, Bcl-2, Bax and Apaf1 were measured by RT-PCR. In addition, the protein expression of cleaved-Caspas-3 and cleaved-Caspase-9 were analyzed by Western blot respectively, and the protein expressionof AIF and cleaved-Caspase-3 were assessed by immunofluorescence as well. Results: Immunostaining showed that Z11 was ototoxic to mouse cochlear hair cells and significantly triggered cell death in a concentration-, time- and location-dependent manner. TUNEL assays evidenced that Z11 exerts its cytotoxicity through induction of apoptosis of cochlear hair cells in vitro. Immunofluorescence and western blot assay showed that Z11 activated the translation of apoptosis-inducing factor (AIF) and Caspase-9/Caspase-3 dependent apoptotic pathway in cochlear hair cells (HCs). 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.