Background
Damaged mitophagy and impaired angiogenesis involve in the pathogenic development of ischemic stroke. Active fraction of Polyrhachis vicina (Roger) (AFPR) showed great potential on neurological disease with it's remarkable anti-inflammatory and anti-oxidative effects.
Conclusion
AFPR promoted angiogenesis through activating mitophagy after cerebral ischemia reperfusion, which might partially involved in the amelioration of SIRT3-mediated regulation on Pink1/Parkin axis. Our study will shed new light on the role of SIRT3 in ischemic brain, especially in regulating mitophagy and angiogenesis after stroke.
Methods
CIR rats were orally treated with AFPR (8 and 4 g raw drug /kg) and Nimodipine (10.8 mg/kg) for 12 days to mimic the recovery phase post-stroke. The neurological function assessment, TTC staining, HE staining, TUNEL staining and Nissl staining were performed to assess neuroprotective effects of AFPR against CIR. Then CD31-labeled microvessel density in brain was visualized and quantified by immunofluorescence staining. Mitochondrial ultrastructure was assessed by transmission electron microscope scanning. Expressions of relative proteins,e.g. SIRT3, Pink1, Parkin, LC3-II, p62, VEGFA, involving in mitophagy and angiogenesis, were detected by Western blotting analysis. In vitro, bEnd.3 cells were cultured with AFPR or in combination of autophagy inhibitor 3-MA during the reoxygenation. Then cell viability, and LDH releasing were measured. Angiogenic indicators,such as migration and tube formation activity, VEGFA level were determined. To assess effects of AFPR on mitophagy, mitophagy-related proteins were detected, as well as the autophagosome engulfment and lysosome degradation of mitochondria. To address the role of SIRT3, deacetylation activity of SIRT3 was validated by detecting acetylated FOXO3A level with co-immunoprecipitation (Co-IP) assay. Pre-treatment of siRNA or combination use of 3-MA were used to verify the detailed mechanism.
Purpose
This study designed to clarify the correlation between Pink1/Parkin-mediated mitophagy and angiogenesis after stroke, and to elucidate the role of SIRT3 in regulating mitophagy and angiogenesis, and to address the mechanism of AFPR on promoting mitophagy and angiogenesis in microvessels endothelium of ischemic brain. Study design: A cerebral ischemia/reperfusion (CIR) rat model was developed by middle cerebral artery occlusion procedure. bEnd.3 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic CIR process. Neurological function, mitophagy and angiogenesis related indicators were measured. SIRT3 siRNA and 3-MA were used to verify the interaction between SIRT3-mediated mitophagy and angiogenesis.
Results
AFPR remarkably reduced neurological scores and infarct size, alleviated neuron apoptosis in cortex, and increased Nissl density in hippocampus of CIR rats. In addition, AFPR significantly promoted angiogenesis by increasing microvessels density and VEGFA expressions, increased SIRT3 expression, and activated Pink1/Parkin mediated mitophagy. In bEnd.3 cells, the combination use of 3-MA and AFPR further demonstrated that AFPR might promote angiogenesis after OGD/R injury through activating Pink1/Parkin mediated mitophagy. Co-IP assay suggested AFPR reduced acetylated FOXO3A level. This might be correlated with an elevation of SIRT3 expression and it's deacetylation activity. SIRT3 siRNA pretreatment significantly abolished the activation of mitophagy through Pink1/Parkin axis, eventually inhibited angiogenesis.