Phosphocreatine attenuates Gynura segetum-induced hepatocyte apoptosis via a SIRT3-SOD2-mitochondrial reactive oxygen species pathway

Abstract

Purpose:

To investigate the mitochondria-related mechanism of Gynura segetum (GS)-induced apoptosis and the protective effect of phosphocreatine (PCr), a mitochondrial respiration regulator.

Methods:

First, the mechanism was explored in human hepatocyte cell line. The mitochondrial oxidative stress was determined by fluorescence assay. The level of sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), SOD2, and apoptosis were detected by Western blotting. Mito-TEMPO and cell lines of viral vector-mediated overexpression of SIRT3 and SIRT3^H248Y were used to further verify the mechanism of GS-induced apoptosis. GS-induced liver injury mice models were built by GS through intragastric administration and interfered by PCr through intraperitoneal injection. A total of 30 C57BL/6J mice were assigned to 5 groups and treated with either saline, PCr (100 mg/kg), GS (30 g/kg), or PCr (50 or 100 mg/kg)+GS (30 g/kg). Liver hematoxylin and eosin (HE) staining, immunohistochemical analysis, and blood biochemical evaluation were performed.

Results:

GS induced hepatocyte apoptosis and elevated levels of mitochondrial ROS in L-02 cells. The expression of SIRT3 was decreased. Downregulation of SIRT3 was associated with increased levels of Ac-SOD2, which is the inactivated enzymatic form of SOD2. Conversely, when overexpressing SIRT3 in GS-treated cells, SOD2 activity was restored, and mitochondrial ROS levels and hepatocyte apoptosis declined. Upon administration of PCr to GS-treated cells, they exhibited a significant upregulation of SIRT3 and were protected against apoptosis. In animal experiments, serum ALT level and mitochondrial ROS of the mice treated with GS and 50 mg/kg PCr were significantly attenuated compared with only GS treated. The changes in SIRT3 expression were also consistent with the in vitro results. In addition, immunohistochemical analysis of the mouse liver showed that Ac-SOD2 was decreased in the PCr and GS co-treated group compared with GS treated group.

Conclusion:

GS caused liver injury by dysregulating mitochondrial ROS generation via a SIRT3-SOD2 pathway. PCr is a potential agent to treat GS-induced liver injury by mitochondrial protection.

Keywords:

• Gynura segetum
• apoptosis
• mitochondrial
• ROS
• SIRT3
• phosphocreatine

Acknowledgments

We thank Ronggui Hu (Professor at Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for providing research instruction and technical support. This study was funded by the Shanghai Committee of Science and Technology, People’s Republic of China (Grant 12401907400) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (KEF152006).

Abbreviation list

GS, Gynura segetum; PAs, pyrrolizidine alkaloids; ROS, reactive oxygen species; SIRT3, sirtuin 3; SOD2, superoxide dismutase 2; Ac-SOD2, acetyl-SOD2; PCr, phosphocreatine; ATP, adenosine triphosphate; ALT, alanine aminotransferase; AST, aspartate aminotransaminase.

Supplementary materials

Table S1 The quantification of Gynura segetum

Table S2 qPCR primers used in the study

Figure S1 Representative images of liver sections from patients for immunostaining analysis using an Ac-SOD2 antibody. Scale bar, 100 μm. **p<0.01.

Figure S2 Quantitative analysis of Western blot bands. (A) Quantitative analysis of Ac-SOD2 in cells overexpressing SIRT3. (B) Quantitative analysis of apoptosis-related protein in cells overexpressing SIRT3. (C) Quantitative analysis of apoptosis-related protein in cells overexpressing SIRT3^H248Y. Values are the mean±SEM. *p<0.05, **p<0.01, and ***p<0.001.

Disclosure

The authors report no conflicts of interest in this work.