Sirt1 Knockout by Crispr/Cas9 technique.

(A) Schematic illustration of the Sirt1 locus. Exon 1 of the Sirt1 gene was targeted. The CRISPR target site and PAM motif are indicated. (B) Sequence confirmation of four-nucleotide insertion mutations. (C) Predicted truncation of the SIRT1 protein. (D) Expression of the Sirt1 mutant was verified by western blot using an antibody of Sirt1; β-actin was used as a loading control.

Short term phenotype of Sirt1 ^-/- mutants.

Four-days post fertilization embryos were used to obtain representative images of short term phenotypes of wildtype and Sirt1^-/- mutants. (A) Phenotypes of wildtype and Sirt1^-/- zebrafish embryos. Note there are no differences in appearance between wildtype and Sirt1^-/- mutant embryos. (B) The embryo survival curves of wildtype and Sirt1^-/- mutants after treatment with 0.5 or 1 mM tBOOH. Note that wildtype embryos died earlier than Sirt1^-/- mutants. (C) ROS levels in 0.5 mM tBOOH-treated wildtype and Sirt1^-/- embryos were examined by H₂DCFDA fluorescence. (D) Detection of apoptosis in 0.5 mM tBOOH-treated wildtype and Sirt1^-/- mutant embryos by TdT-mediated dUTP nick end labeling (TUNEL). Note that Sirt1^-/- mutants showed higher levels of ROS and TUNEL than wildtype embryos. The effect of NAC treatment on ROS content and apoptosis incidence was studied by H₂DCFDA fluorescence (E) and the TUNEL assay (F). Embryos were exposed to treatment with NAC treatment for 24 h. Results showed that TUNEL-positive cells increased in both, tBOOH-treated wildtype and Sirt1^-/-groups; furthermore, NAC treatment was effective for both groups.  

Quantitative RT-PCR and western blot analysis.

Samples were prepared from (A, C) 4-dpf whole embryos, and (B, D) internal organs from 3-month-old zebrafish. Data are means, and bars indicate standard error. (A, B) Quantitative RT-PCR showed higher upregulation of inflammation-related genes in Sirt1^-/- than in controls. (C, D) Western blot hybridization showed upregulation of IL-1b, active-caspase 3 and TGF-β. Control, wildtype; IL-1b, 18kDa; caspase 3a, 29 and 31 kDa; TGF-β, 45 kDa; β–actin, 43 kDa. *P < 0.05 versus control.

Microscopic observations of wildtype and Sirt1^-/- adult zebrafish.

H&E images of wildtype and Sirt1^-/- zebrafish aged 6, 12, and 18 months. Sirt1-deficiency caused intestinal atrophy and inflammatory cell infiltration

Expression of genes involved in inflammation and apoptosis induced by Sirt1-knockout induced inflammation.

All images were obtained from 12-month-old zebrafish. (A) Immunohistochemical staining for myeloperoxidase (MPO). (B) ISH for NF-kB and (C) TNF-α expression. (D) TUNEL assay with 18-month old zebrafish organ sections. Wildtype organ section with few apoptotic cells and Sirt1^-/- organ section with numerous apoptotic cells, as per the TUNEL assay. (E) Survival curves of wildtype and Sirt1^-/- mutant zebrafish. Kaplan–Meier analysis showed poor survival of Sirt1^-/- compared to the wildtype. (n = 100 zebrafish in each group). Survival curves lasted for 540 days (observation started when zebrafish were 3 months old.) and were statistically compared using the Log-Rank Test. (F) Schematic diagram of the major findings of this study. Sirt1^-/—induced chronic inflammation led to the activation of oxidative stress and apoptosis. The continuous repetition of these events reduced the lifespan of zebrafish.