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. 2022 Aug 2;27(11):4575–4589. doi: 10.1038/s41380-022-01711-7

Fig. 6. Silencing of circIgfbp2 regulates circIgfbp2/miR-370-3p/BACH1 axis to alleviate mitochondrial dysfunction and oxidative stress-induced synapse dysfunction.

Fig. 6

A Western blot analysis of BACH1, HO-1, PSD95 and Syn in HT22 cells transduced with oe-circIgfbp2 lentivirus for 7 days, and then transduced with miR-NC or miR-370-3p mimics for 48 h, finally H2O2 (600 µmol/L) treated HT22 cells for 6 h. The results showed that transfection with oe-circIgfbp2 alone aggravated H2O2-induced mitochondrial oxidative stress-mediated synapse dysfunction, the expression of BACH1was upregulated, and HO-1, PSD95 and Syn were downregulated. The co-transfection with miR-370-3p mimics reversed these effects. n = 3 replications. BACH1:H2O2 + oe-circIgfbp2+miR-NC vs. H2O2 + miR-NC, ****p < 0.0001; H2O2 + oe-circIgfbp2+miR-370-3p mimics vs. H2O2 + oe-circIgfbp2+miR-NC, **p < 0.01; HO-1:H2O2 + oe-circIgfbp2+miR-NC vs. H2O2 + miR-NC, ****p < 0.0001;H2O2 + oe-circIgfbp2+miR-370-3p mimics vs. H2O2 + oe-circIgfbp2+miR-NC, ****p < 0.0001; PSD95: H2O2 + oe-circIgfbp2+miR-NC vs. H2O2 + miR-NC, ***p < 0.001, H2O2 + oe-circIgfbp2+miR-370-3p mimics vs. H2O2 + oe-circIgfbp2 + miR-NC, ***p < 0.001; Syn: H2O2 + oe-circIgfbp2+miR-NC vs. H2O2 + miR-NC, ***p < 0.001, H2O2 + oe-circIgfbp2+miR-370-3p mimics vs. H2O2 + oe-circIgfbp2+miR-NC, ***p < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test. B Western blot analysis of BACH1, HO-1, PSD95, Syn in HT22 cells transduced with circIgfbp2 shRNA lentivirus cells for 7 days, and then transduced with anti-miR-NC or anti-miR-370-3p for 48 h, finally treated with H2O2 (600 µmol/L) for 6 h. The transfection with sh-circIgfbp2 alone attenuated H2O2-induced mitochondrial oxidative stress-mediated synapse dysfunction, the expression of BACH1was downregulated; HO-1, PSD95 and Syn were upregulated, while co-transfection with anti-miR-370-3p reversed these effects, n = 3 replications. BACH1: H2O2 + sh-circIgfbp2+anti-miR-NC vs. H2O2 + anti-miR-NC, ****p  <  0.0001, H2O2 + sh-circIgfbp2+anti-miR-370-3p vs. H2O2 + sh-circIgfbp2 + anti-miR-NC, ***p < 0.001; HO-1: H2O2 + sh-circIgfbp2+anti-miR-NC vs. H2O2 + anti-miR-NC, ****p < 0.0001, H2O2 + sh-circIgfbp2+anti-miR-370-3p vs. H2O2 + sh-circIgfbp2+anti-miR-NC, ***p < 0.001; PSD95: H2O2 + sh-circIgfbp2+anti-miR-NC vs. H2O2 + anti-miR-NC, **p < 0.01, H2O2 + sh-circIgfbp2+anti-miR-370-3p vs. H2O2 + sh-circIgfbp2+anti-miR-NC, *p < 0.05; Syn: H2O2 + sh-circIgfbp2+anti-miR-NC vs. H2O2 + anti-miR-NC, ****p < 0.0001, H2O2 + sh-circIgfbp2+anti-miR-370-3p vs. H2O2 + sh-circIgfbp2+anti-miR-NC,****p < 0.0001 one-way ANOVA followed by Tukey’s multiple comparisons test. C Transduction of HT22 cells with the miR-370-3p mimics significantly reversed the oe-circIgfbp2 -induced decrease the content of ATP, n = 3 replications. H2O2 + oe-circIgfbp2+miR-NC vs. H2O2 + miR-NC, **p < 0.01, H2O2 + oe-circIgfbp2+miR-370-3p mimics vs. H2O2 + oe-circIgfbp2+miR-NC, *p < 0.05; one-way ANOVA followed by Tukey’s multiple comparisons test. D Transduction of HT22 cells with the anti-miR-370-3p mimics significantly attenuated the sh-circIgfbp2 -induced increase the content of ATP. n = 3 replications. H2O2 + sh-circIgfbp2+anti-miR-NC vs. H2O2 + anti-miR-NC, ***p < 0.001, H2O2 + sh-circIgfbp2+anti-miR-370-3p vs. H2O2 + sh-circIgfbp2+anti-miR-NC, *p < 0.05.One-way ANOVA followed by Tukey’s multiple comparisons test. E Transduction of HT22 cells with the miR-370-3p mimics significantly reversed the oe-circIgfbp2-induced increase the content of mitochondrial ROS; transduction of HT22 cells with the anti-miR-370-3p significantly attenuated the sh-circIgfbp2-induced the decrease of mitochondrial ROS, n = 3 replications. H2O2 + oe-circIgfbp2+miR-370-3p mimics vs. H2O2 + oe-circIgfbp2+miR-NC, ****p < 0.0001, H2O2 + sh-circIgfbp2+anti-miR-370-3p vs. H2O2 + sh-circIgfbp2+anti-miR-NC, ****p < 0.0001;one-way ANOVA followed by Tukey’s multiple comparisons test. ns: no significance. All data were represented as mean ± SEM.