CGR8 neural differention to NSC and the changes of tRF-Glu-CTC level under different H₂O₂ concentrations. A Differentiation scheme of CGR8 into NSCs. B Immunofluorescence staining of neuronal markers(ßIII-tubulin and MAP2) at 7th day, images were taken 20 × magnification, Olympus IX61 fluorescent microscope). C Immunofluorescence staining of NSCs for neuronal marker (MAP2) after H₂O₂ exposure (images were taken 40 × magnification, Olympus IX61 fluorescent microscope) and intensity analysis (data are presented as mean ± SD, n = 5, Shapiro–Wilk test, **p < 0.01). D Changes in the levels of tRF-Glu-CTC in NSCs at different H2O2 concentrations (data are presented as mean ± SD, n = 5, one-way ANOVA Dunnett test, *p < 0.05, **p < 0.01)

Effect of in vivo HyPer zebrafish H₂O₂ exposure on tRF-Glu-CTC levels. A H₂O₂ levels in 72 hpf HyPer zebrafish embryos for 24 h. HyPer imaging for different concentrations of H₂O₂. The H₂O₂ levels are inferred from the F₅₀₀/F₄₂₀ excitation ratio of HyPer. Respectively, control, 1 mM, 1.5 mM, and 2 mM H₂O₂. B Quantification of the H₂O₂ levels in the control and H₂O₂ concentrations in 72 hpf HyPer zebrafish embryos; images were taken 20 × magnification, Zeiss LSM 880 confocal microscopy (data are presented as boxplot, n = 5, ns, Kruskal–Wallis test, p = 0.168). C Changes in levels of tRF-Glu-CTC fragment in HyPer 72 hpf embryos after 24 h H₂O₂ exposure (data are presented as boxplot, n = 6, Kruskal–Wallis test, *p < 0.05, **p < 0.01)

Effect of in vivo AB wild-type zebrafish H₂O₂ exposure on tRF-Glu-CTC neurogenesis. Role of H₂O₂ on neurogenesis. A In situ hybridization (Calb2a) and immunofluorescence (HuC/D) images of in wild-type AB zebrafish 72 hpf embryos after 24 h H₂O₂ exposure. Calb2a expression decreases in various cranial structures, including the olfactory bulb (yellow arrows), retina (red arrows), cerebellum (green arrows), and hindbrain (blue arrows). Anti-HuC/D (red) staining of the heads of zebrafish larvae obtained from confocal microscopy. Yellow labels indicate telencephalon and habenula, respectively. B Intensity analysis of Calb2a and HuC/D (Data are presented as mean ± SD, n = 5, t-test, for Calb2a ***p < 0.001 and HuC/D p = 0.057). C, control; 2, 2 mM H₂O₂

Role of tRF-Glu-CTC inhibition on neurogenesis of NSCs. A Expression levels of neurogenesis markers (ßIII-tubulin, MAP2, and GFAP) after transfection of SCR and tRF-Glu-CTC antisense 2′-OMe-RNA (inhibitor) in 50 μM H₂O₂ for 6 h for neurogenesis markers (data are presented as mean ± SD, n = 6, t-test, BIIITUB t-test, C-SCR vs. H₂O₂-SCR p = 0.097 and C-INH vs. H₂O₂-INH p = 0.171, MAP2 t-test, C-SCR vs. H₂O₂-SCR p = 0.067 and C-INH vs. H₂O₂-INH p = 0.054, GFAP t-test, C-SCR vs. H₂O₂-SCR p = 0.090, C-INH vs. H₂O₂-INH, Mann–Whitney test p = 0.180). B Immunofluorescence images of neurogenesis markers (ßIII-tubulin, MAP2, and GFAP) after transfection (images were taken 20 × magnification, Olympus IX61 fluorescent microscope). INH, inhibitor; SCR, scramble

Interaction network and GO analysis of tRF-Glu-CTC target genes. A Network visualization of the top 30 predicted target genes of tRF-Glu-CTC (tRFdb-5022a), generated using the igraph and ggraph packages in R. tRF-Glu-CTC is shown at the center of the network, with its connections (edges) to target genes displayed as dotted lines. B GO enrichment analysis results for the predicted target genes of tRF-Glu-CTC under three classifications: biological process (BP), molecular function (MF), and cellular component (CC). Results indicate significant enrichment of terms associated with neuronal function and synaptic processes