Fig 2: GABA co-release from SNcDA axons requires Gat1-mediated GABA uptake(A) Confocal images of SNc in Gat1 cKODA mice expressing Cre-dependent HA-tagged Gat1 (left) and Gat1(R69K) (right) immunolabeled for TH (green), HA (red), and DAPI (blue). Inset: detailed view (scale, 50 μm).(B–F) oIPSCs recorded from SPNs (Vh = 0 mV) upon stimulation of SNcDA axons (blue bar) in Gat1 cKODA mice (B) exogenously expressing Gat1 (C), Gat1(R69K) (D), Gad67 (E), or Aldh1a1 (F).(G) Fluorescence in situ hybridization for Gad67 (red) and TH (green) in Gat1 cKODA mice with (right) or without (left) Gad67 in DA neurons. Inset: detail (scale, 50 μm).(H) oIPSC amplitude in SPNs of Gat1 cKODA mice virally expressing ChR2 in SNcDA neurons (n = 22, p = 1 × 10−15), or ChR2 + Gat1 (n = 14; p = 0.07), ChR2 + Gat1(R69K) (n = 18; p = 1 × 10−15), ChR2 + Gad67 (n = 13; p = 0.018), or ChR2 + Aldh1a1 (n = 17; p = 2 × 10−15) normalized to control. All p values versus control, Mann-Whitney. nd, not detected. Individual values shown along with population mean ± SEM.(I) Same as (H) for latency (Gat1DA: p = 0.32; Gad67DA: p = 0.0007 versus control, Mann-Whitney).(J) Same as (I) for rise time (Gat1DA: p = 0.18; Gad67DA: p = 0.39 versus control, Mann-Whitney).

Fig 3: Knockdown of Nrf2 reduces the protective effect of shikonin on SGNs differentiation. (A) The specificity and efficiency of siRNA against Nrf2 were tested by quantitative PCR. (B) The knockout efficiency of Nrf2 siRNA was detected at the protein level by Western blot. SGNs were transfected with Nrf2 siRNA for 24 h, pretreated with shikonin, and then treated with ouabain for another 22 h. To detect the expression changes of Nrf2/ARE signal pathway (Nrf2, HO-1, and NQO1) protein (C) and mRNA (E) in SGNs. As well as the protein (D) and mRNA (E) expression changes of glutamatergic neurons VGLUT1 and GABAergic neurons GAT1. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Shikonin can rescue the effect of ouabain on the differentiation of SGNs. After pretreatment of SGNs with different concentrations of shikonin (0.5, 1, 5, and 10 μM) to separate the cells for 2 h, 0.5 mM ouabain continued treatment for another 22 h. (A) The morphology of SGNs was observed by immunofluorescence. Red represents NeuN staining, green fluorescence represents Nestin staining, blue fluorescence represents nuclear (DAPI) staining, bar = 50 μm. (B) Statistics of the average length of SGNs from the cell body to the apex of the longer synapse in different treatment groups. After collecting cells in different treatment groups, RNA was extracted, and the relative mRNA expression levels of VGLUT1 (C) and GAT1 (D) were detected by qPCR, and β-antin was used as an internal reference gene. (E) The cells of different treatment groups were subjected to protein extraction to detect the expression of VGLUT1 and GAT1 proteins. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Shikonin has protective effects on ouabain-induced hearing damage in mice. (A) Experimental procedure of drug injection in mice. The arrow represents the drug injection point. The red bars represent the time period of continuous drug injection. The mice in the control group were injected with the same amount of normal saline or drug solvent at the same time point. (B) The statistical histogram of the hearing brainstem response of mice at 7, 14, and 30 days after drug injection (n = 3). After 14 days of drug treatment, detect the expression of Nrf2/ARE signal pathway (Nrf2, HO-1, and NQO1) protein (C) and mRNA (E) in mouse SGN tissue protein. And detect the protein (D) and mRNA (E) expressions of glutamatergic neurons VGLUT1 and GABAergic neurons GAT1. ***P < 0.001.