Quercetin attenuates cisplatin-induced mitochondrial apoptosis via PI3K/Akt mediated inhibition of oxidative stress in pericytes and improves the blood labyrinth barrier permeability.

Cisplatin (CDDP) is broadly employed to treat different cancers, whereas there are no drugs approved by the Food and Drug Administration (FDA) for preventing its side effects, including ototoxicity. Quercetin (QU) is a widely available natural flavonoid compound with anti-tumor and antioxidant properties. The research was designed to explore the protective effects of QU on CDDP-induced ototoxicity and its underlying mechanisms in male C57BL/6 J mice and primary cultured pericytes (PCs). Hearing changes, morphological changes of stria vascularis, blood labyrinth barrier (BLB) permeability and expression of apoptotic proteins were observed in vivo by using the auditory brainstem response (ABR) test, HE staining, Evans blue staining, immunohistochemistry, western blotting, etc. Oxidative stress levels, mitochondrial function and endothelial barrier changes were observed in vitro by using DCFH-DA probe detection, flow cytometry, JC-1 probe, immunofluorescence and the establishment in vitro BLB models, etc. QU pretreatment activates the PI3K/AKT signaling pathway, inhibits CDDP-induced oxidative stress, protects mitochondrial function, and reduces mitochondrial apoptosis in PCs. However, PI3K/AKT specific inhibitor (LY294002) partially reverses the protective effects of QU. In addition, in vitro BLB models were established by coculturing PCs and endothelial cells (ECs), which suggests that QU both reduces the CDDP-induced apoptosis in PCs and improves the endothelial barrier permeability. On the whole, the research findings suggest that QU can be used as a novel treatment to reduce CDDP-induced ototoxicity.

[Role of Cav1.2 in cisplatin induced apoptosis of cochlear spiral ganglion neurons in C57BL/6J mice].

Objective: To investigate the role of Cav1.2 and its possible mechanism in the apoptosis of cochlear spiral ganglion neurons(SGNs) induced by cisplatin (CDDP) in C57BL/6J mice. Methods: Animal experiment: 8-week-old male C57BL/6J mice were randomly divided into the following two groups (10 mice/group) : normal saline group (Control group) and Cisplatin group (Cisplatin group). The Control group received daily intraperitoneal injections of normal saline, Cisplatin group was injected with cisplatin intraperitoneally at a dose of 3 mg/kg at the first 4 days of each cycle, and normal saline was injected daily at the last 10 days,repeat for 3 cycles. After administration, auditory threshold was detected by auditory brainstem response (ABR). Blood samples were collected from inner canthus of mice, and cochlea was cut off from neck. SOD and MDA kits were used to detect SOD activity and MDA content in serum and cochlea tissues. The expressions of apoptosis proteins in cochlear tissues were detected by Western blot. Morphological changes of spiral ganglion in mouse cochlea were observed by hematoxylin-eosin (HE) staining. TUNEL staining was used to observe the apoptosis of SGNs in cochlea of mice. The distribution and expression of Cav1.2 in SGNs of cochlea were observed by immunofluorescence. Cell experiment: Primary cultured SGNs were randomly divided into: control group (Control), solvent group (DMSO), Cav1.2 blocker group (N), cisplatin group, cisplatin and Cav1.2 blocker co-incubation group (Cisplatin+N). 5 µmol/L cisplatin was selected to treat SGNs based on the results of CCK8. Western blot was used to detect the protein expressions of Cav1.2.and apoptotic proteins. Hoechst33342 staining was used to observe the apoptosis of each group. Flow cytometry was used to detect the apoptosis rate of each group. Mitochondrial superoxide indicator (MitoSOXTM-Red) was used to detect the ROS release of mitochondria. Results: Animal experiments: Compared to the Control group, the hearing threshold was increased in Cisplatin group (P<0.01), the content of MDA in serum and cochlea tissues, apoptosis protein Cleaved caspase-3, Bax protein level, TUNEL positive rate, Cav1.2 protein expression level were increased significantly (P<0.05, P<0.01); the activity of SOD in serum and cochlear tissue, anti-apoptotic protein bcl-2 protein level and SGCs density in cochlear tissue were decreased significantly (P<0.05, P<0.01). Cell tests: Compared with the Control group, the expression of Cav1.2, apoptosis rate, Cleaved caspase-3, Bax protein level, intracellular calcium ion concentration, and ROS release were increased significantly only in Cisplatin group (P<0.05, P<0.01). The levels of bcl-2 protein and mitochondrial membrane potential were decreased significantly (P<0.01). Cav1.2 blockers could partially reverse the above changes (P<0.05). Conclusion: Cisplatin may increase intracellular Ca2+ concentration through up-regulation of Cav1.2, and then damage mitochondria, causing oxidative stress injury of SGNs and inducing neuronal apoptosis.