Fig 2: Ebselen and overexpression of PRDX1 protect against cisplatin-induced ROS accumulation and apoptosis and reduce SGN loss by activating autophagy in SGNs. The cultured cochleae from WT mice were treated with cisplatin (50 μM) alone, cotreated with ebselen (30 μM, pretreated for 1 h), cotreated with Anc80-Prdx1 (4 × 1010 GC/mL, preincubation for 24 h), or cotreated with 3-MA (5 mM, pretreated for 6 h) for 48 h. (A) RT-PCR results showed that the mRNA expression of genes associated with autophagic flux, including Atg5, Becn1, Lc3b, Ctsb, and Lamp1, was upregulated in SGNs induced by cisplatin, and the overexpression of PRDX1 by ebselen or Anc80-Prdx1 further enhanced the above gene transcriptions compared to the cisplatin-only group. (B) Ebselen and Anc80-Prdx1 both significantly increased the protein expression of LC3B-II compared to the cisplatin-only group, whereas the co-treatment with 3-MA effectively prevented the upregulation of LC3B-II expression induced by ebselen or Anc80-Prdx1. (C-G) The expressions of 4-HNE (C and D) and cleaved-CASP3 (F and G) were significantly decreased, and the number of surviving SGNs (E) was significantly increased in the Cis + ebselen group and in the Cis + Anc80- Prdx1 group compared to the cisplatin-only group, while co-treatment with 3-MA significantly reduced the effects of both ebselen and Anc80-Prdx1 on SGN ROS production, cell survival, and apoptosis. Scale bars: 12.5 μm in (C). Scale bars: 25 μm in (F). n = 6 for each subgroup. All data are presented as the mean ± SEM, * P < 0.05, ** P < 0.01

Fig 3: PRDX1 regulates the PTEN-AKT signaling pathway to activate autophagy in SGNs after cisplatin treatment. (A and B) The cultured cochleae and SGNs from WT mice or prdx1-/- mice were incubated with cisplatin (50 µM) for 48 h or cotreated with ebselen (30 µM, pretreated for 1 h) and cisplatin (50 µM) for 48 h. The protein expression of PTEN was significantly increased and the ratio of p-AKT:AKT was significantly decreased in cultured SGNs after cisplatin injury. Treatment with ebselen caused increased expression of PTEN and a lower ratio of p-AKT:AKT in SGNs compared to the cisplatin-only group, while the lack of PRDX1 in SGNs from prdx1-/- mice led to an opposite changing pattern. (C and D) The cultured cochleae and SGNs from WT mice were incubated with cisplatin (50 µM) alone, cotreated with ebselen (30 µM, pretreated for 1 h), or cotreated with SF1670 (15 µM) for 48 h. SF1670 effectively inhibited the expression of PTEN and enhanced the phosphorylation of AKT in SGNs after cisplatin treatment, and co-treatment with ebselen reversed the effect of SF1670 as the expression of PTEN was increased and the ratio of p-AKT:AKT was decreased in the Cis + ebselen + SF1670 group compared to the Cis + SF1670 group. In contrast, treatment with SF1670 did not change the expression of PRDX1 in SGNs after cisplatin incubation. (E-I) The inhibition of PTEN by SF1670 significantly decreased the expression of LC3B-II (E and F), increased cleaved-CASP3 expression (G and H), and reduced the number of surviving SGNs (I) compared to the cisplatin-only group, whereas co-treatment with ebselen rescued the above effect of SF1670 in SGNs. Scale bars: 25 µm. n = 6 for each group. All data are presented as the mean ± SEM, * P < 0.05, ** P < 0.01