Fig 1: KLF6 binds to SIRT5 promoter to inhibit its transcription. (A) The expression of SIRT5 in the ICH-related microarray GSE149317. The box diagram represents the expression of SIRT5 gene. (B) SIRT5 colocalization with hippocampal neurons in ICH rats detected by immunofluorescence staining. (C) The enrichment of KLF6 in the SIRT5 promoter region detected by CistromeDB database ChIP-Seq data, and the binding site of KLF6 and SIRT5 promoter predicted by JASPAR database. (D) The enrichment of KLF6 in SIRT5 promoter -20/-220 (Prime2) and -700/-900 (Prime1) regions determined by ChIP assay. (E) The effect of KLF6 overexpression on the luciferase activity of SIRT5 promoter determined by dual luciferase reporter gene assay. (F) The level of SIRT5 in hippocampal neurons infected with sh-KLF6 determined by RT-qPCR. (G) The level of SIRT5 in hippocampal neurons infected with sh-KLF6 determined by Western blot analysis. *p < 0.05. The measurement data were expressed as mean ± standard deviation. Comparisons between two groups were analyzed using unpaired t-test, and comparisons among multiple groups were tested by one-way ANOVA followed by Dunnett post-hoc test. The experiment was repeated three times.
Fig 2: Silencing KLF6 causes inhibition of mitochondrial ROS and mitochondrial damage of OxyHb-induced hippocampal neurons via the SIRT5/Nrf2/HO-1 axis. (A) The expression of Nrf2 and HO-1 in the hippocampus of ICH rats detected by IHC (n = 6). (B) The expression of SIRT5 in hippocampal neurons after the silencing and overexpression of SIRT5 determined by RT-qPCR. (C) The expression of SIRT5, Nrf2 (total, cytoplasm, and nuclear), and HO-1 in hippocampal neurons after the silencing and overexpression of KLF6 determined by Western blot analysis. (D) The expression of Nrf2 (total, cytoplasm, and nuclear) and HO-1 in hippocampal neurons exposed to 20 µM OxyHb after the silencing of KLF6 determined by Western blot analysis. (E) OxyHb-induced hippocampal neuronal apoptosis after different infection determined by flow cytometry. (F) The changes of mitochondrial active oxygen determined by MitoSOX fluorescent staining. (G) Changes in mitochondrial membrane potential determined by JC-1 staining. *p < 0.05. The measurement data were expressed as mean ± standard deviation. Comparisons between two groups were analyzed using unpaired t-test, and comparisons among multiple groups were tested by one-way ANOVA followed by Dunnett post-hoc test. The experiment was repeated three times.
Fig 3: KLF6 accelerates neurological dysfunction and hippocampal neuronal apoptosis and degeneration in ICH rats through SIRT5/Nrf2/HO-1 axis. (A) Different infection of ICH model rats. ICH rats were treated with oe-NC, oe-SIRT5, sh-NC, sh-SIRT5, and sh-SIRT5 + sh-KLF6. (B) The mNSS score of ICH rats. (C) Water content in brain tissues of ICH rats. (D) Evans blue penetration of brain tissues of ICH rats. (E) Microglia activation of ICH rats detected by immunofluorescence staining. (F) The activation of neutrophils of ICH rats detected by immunofluorescence staining. (G) The apoptosis of hippocampal neurons of ICH rats detected by TUNEL staining. (H) The degeneration of hippocampal neurons in the cerebral cortex of ICH rats detected by FJC staining. (I) The levels of KLF6, SIRT5, Nrf2 (total, cytoplasm, and nucleus), and HO-1 in the hippocampus of ICH rats detected by Western blot analysis. n = 6 rats/group. *p < 0.05. The measurement data were expressed as mean ± standard deviation. Comparisons between two groups were analyzed using unpaired t-test, and comparisons among multiple groups were tested by one-way ANOVA followed by Dunnett post-hoc test.
Fig 4: dVB reduced the PA-induced mitochondrial dysfunction. (A) Representative images by fluorescence microscopy (scale bars = 100 µm) and (B,C) FACS analysis of mitochondrial ROS detection (MFI). Levels of mRNA and immunoblotting analysis of (D,E) SIRT2, (F,G) SIRT3, (H,I) SIRT4, and (J,K) SIRT5 in EC exposed for 48 h to 0.5 mM dVB, 0.5 mM PA (PA), or pretreated for 16 h with dVB before 48 h PA treatment (dVB+PA). Control cells (Ctr) were treated with the corresponding highest volume of HBSS-10 mM Hepes. Western blotting results (n = 5) are expressed as arbitrary units (AU). mRNA levels are reported as floating bars with line representing the median ± SD (n = 3). * p < 0.05 vs. Ctr; † p < 0.01 vs. Ctr; ‡ p < 0.001 vs. Ctr; § p < 0.05 vs. PA; # p < 0.01 vs. PA.
Fig 5: KLF6 silencing depresses oxidative stress in ICH rats by inhibiting SIRT5-mediated Nrf2/HO-1 signaling pathway. ICH rats were infected lentiviruses carrying oe-NC, oe-SIRT5, sh-NC, sh-SIRT5, and sh-SIRT5 + sh-KLF6. (A) The active oxygen in the hippocampus of ICH rats determined by DHE staining. (B) SOD activity in hippocampal tissues of ICH rats. (C) GSH-Px activity in hippocampal tissues of ICH rats. (D) MDA level in hippocampal tissues of ICH rats. (E) 3-NT concentration in hippocampus of ICH rats determined by ELISA. (F) 8-OHdG concentration in hippocampus of ICH rats determined by ELISA. n = 6 rats/group, *p < 0.05. The measurement data were expressed as mean ± standard deviation. Comparisons between two groups were analyzed using unpaired t-test, and comparisons among multiple groups were tested by one-way ANOVA followed by Dunnett post-hoc test.
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