Mitochondria-derived H2O2 triggers liver regeneration via FoxO3a signaling pathway after partial hepatectomy in mice

Abstract

Reactive oxygen species (ROS) can induce oxidative injury and are generally regarded as toxic byproducts, although they are increasingly recognized for their signaling functions. Increased ROS often accompanies liver regeneration (LR) after liver injuries, however, their role in LR and the underlying mechanism remains unclear. Here, by employing a mouse LR model of partial hepatectomy (PHx), we found that PHx induced rapid increases of mitochondrial hydrogen peroxide (H₂O₂) and intracellular H₂O₂ at an early stage, using a mitochondria-specific probe. Scavenging mitochondrial H₂O₂ in mice with liver-specific overexpression of mitochondria-targeted catalase (mCAT) decreased intracellular H₂O₂ and compromised LR, while NADPH oxidases (NOXs) inhibition did not affect intracellular H₂O₂ or LR, indicating that mitochondria-derived H₂O₂ played an essential role in LR after PHx. Furthermore, pharmacological activation of FoxO3a impaired the H₂O₂-triggered LR, while liver-specific knockdown of FoxO3a by CRISPR-Cas9 technology almost abolished the inhibition of LR by overexpression of mCAT, demonstrating that FoxO3a signaling pathway mediated mitochondria-derived H₂O₂ triggered LR after PHx. Our findings uncover the beneficial roles of mitochondrial H₂O₂ and the redox-regulated underlying mechanisms during LR, which shed light on potential therapeutic interventions for LR-related liver injury. Importantly, these findings also indicate that improper antioxidative intervention might impair LR and delay the recovery of LR-related diseases in clinics.

Fig. 1. Time course of liver regeneration (LR) and reactive oxygen species (ROS) production after 70% partial hepatectomy (PHx) in mice.

Liver samples were taken, and LR and ROS production was determined at different time points (6 h, 1 d, 2 d, 3 d, 5 d, and 7 d) after the C57 mice were subjected to PHx. A Schematic representation of the experimental procedure and representative macroscopic images of remnant liver. B LR rate, n = 6. C Immunohistochemistry staining and quantification of Ki67, bar = 50 µm, n = 6. D Western blot analysis and quantification of PCNA and Cyclin D1 (three independent experiments). E Flow cytometry assay and quantification of cellular ROS (probed with DCFH-DA) and mitochondrial ROS (probed with MitoSOX) (three independent experiments). Data are shown as means ± SEM. *P < 0.05 vs Sham group.

Fig. 2. The mitochondrial-targeted antioxidant MitoQ inhibited LR after PHx in mice.

The C57 mice were intraperitoneally injected with MitoQ (2 mg/kg BW) immediately after PHx and followed once daily. The ROS production was detected at 6 hours, and LR was determined on the 2th day after PHx. A Schematic representation of the experimental procedure and flow cytometry assay and quantification of cellular ROS (probed with DCFH-DA) and mitochondrial ROS (probed with MitoSOX) (three independent experiments). B Western blot analysis and quantification of PCNA and Cyclin D1 (three independent experiments). C Immunohistochemistry staining and quantification of Ki67, bar = 50 µm, n = 6. D LR rate, n = 7. Data are shown as means ± SEM. *P < 0.05 vs Sham group and ^#P < 0.05 vs PHx group.

Fig. 3. Mitochondria-derived H₂O₂ promoted LR after PHx in mice.

A–D The mitochondrial H₂O₂ and total H₂O₂ were determined at different time points (6 h, 1 d, 2 d, 3 d, 5 d, and 7 d) after the C57 mice were subjected to PHx. A Representative liver fluorescence images and quantified data of radiant efficiency of Mito-LX probe (three independent experiments). B Flow cytometry assay and quantification of mitochondrial H₂O₂ probed with Mito-LX (three independent experiments). C and D Mitochondrial H₂O₂ and total H₂O₂ in liver tissue assessed by Amplex red kit, n = 6. E–H The C57 mice were intraperitoneally injected with O₂^•− scavenger SOD mimic (5 mg/kg BW) or H₂O₂ scavenger CAT (10 mg/kg BW) immediately after PHx and followed by once every day. The liver H₂O₂ was detected at 6 hours and LR was determined on the 2nd day after PHx. E Schematic representation of the experimental procedure and total H₂O₂ in liver tissue detected by Amplex red kit, n = 6. F Western blot analysis and quantification of PCNA and Cyclin D1 (three independent experiments). G Immunohistochemistry staining and quantification of Ki67, bar = 50 µm, n = 6. (H) LR rate, n = 7. Data are shown as means ± SEM. *P < 0.05 vs Sham group and ^#P < 0.05 vs PHx group.

Fig. 4. The changes of FoxO3a signaling pathway at different time points after PHx in mice.

After the C57 mice were subjected to PHx, the FoxO3a signaling pathway in the liver was determined at different time points (6 h, 1 d, 2 d, 3 d, 5 d, and 7 d). A Western blot analysis and quantification of FoxO3a and its phosphorylation forms (Ser253 and Ser294). B Western blot analysis and quantification of Akt and Erk with their phosphorylation forms. C Western blot analysis and quantification of FoxO3a protein levels in nucleus and cytoplasm. D Q-PCR determination of p27 expression, n = 5. E Western blot analysis and quantification of p27 protein level. Data are shown as means ± SEM of three independent experiments. *P < 0.05 vs Sham group.

Fig. 5. FoxO3a pathway activation attenuated mitochondria-derived H₂O₂ triggered LR after PHx in mice.

The C57 mice were injected with a recombinant AAV8 carrying CAT gene with mitochondria-targeted sequence to overexpress mitochondria-targeted CAT (mCAT) specifically in the liver. The mice were intraperitoneally injected with Akt and Erk inhibitor Tic10 (25 mg/kg BW) immediately after PHx and followed once daily. The Akt/Erk/FoxO3a/p27 pathway and LR were determined on the 2nd day after the mice were subjected to PHx. A Schematic representation of the experimental procedure, western blot analysis, and quantification of Akt and Erk with their phosphorylation forms (three independent experiments). B Western blot analysis and quantification of FoxO3a and its phosphorylation forms (Ser253 and Ser294) (three independent experiments). C Western blot analysis and quantification of FoxO3a protein level in the nucleus and its target p27 protein level (three independent experiments). D Western blot analysis and quantification of PCNA and Cyclin D1 (three independent experiments). E Immunohistochemistry staining and quantification of Ki67, bar = 50 µm, n = 6. F LR rate, n = 8. Data are shown as means ± SEM. Significance was designated with *P < 0.05.

Fig. 6. FoxO3a knockdown ameliorated the LR inhibition by overexpression of mCAT after PHx in mice.

The C57 mice were injected with a recombinant AAV8 carrying mitochondria-targeted CAT (mCAT) gene and/or a recombinant AAV8 carrying the sgRNA sequence for FoxO3a to overexpress mCAT and/or knockdown FoxO3a specifically in the liver. The LR were determined on the 2nd day after the mice were subjected to PHx. A Schematic representation of the experimental procedure and western blot analysis and quantification of p27 protein level (three independent experiments). B Western blot analysis and quantification of PCNA and Cyclin D1 (three independent experiments). C Immunohistochemistry staining and quantification of Ki67, bar = 50 µm, n = 6. D LR rate, n = 8. Data are shown as means ± SEM. Significance was designated with *P < 0.05.

Fig. 7. Schematic representation of mitochondria-derived H₂O₂ triggering liver regeneration via FoxO3a signaling pathway after partial hepatectomy in mice.

After PHx, the mitochondria-derived H₂O₂ increased the phosphorylation of FoxO3a at the specific sites (Ser253 and Ser294) by activating both Akt and Erk, thus reducing transcription and expression of its target p27 in the downstream, a key cell cycle inhibitor, leading to the cell proliferation and LR. (This artwork was created at http://BioRender.com).