# Thonningianin A derived from Penthorum chinense Pursh alleviates cerebral ischemia/reperfusion-mediated apoptosis and pyroptosis through the activation of PINK1/Parkin-dependent mitophagy

## TL;DR

Thonningianin A, a compound from a traditional herb, reduces brain damage after stroke by activating a cellular cleanup process called mitophagy.

## Contribution

This study is the first to show that Thonningianin A alleviates stroke injury via PINK1/Parkin-dependent mitophagy.

## Key findings

- TA improved neurological function and reduced brain infarct volume in a rat stroke model.
- TA suppressed mitochondrial stress, apoptosis, and pyroptosis in brain cells under oxygen-glucose deprivation.
- TA's protective effects were blocked when mitophagy was inhibited, confirming its mechanism.

## Abstract

Cerebral ischemia/reperfusion injury (CI/RI) remains a critical barrier to effective ischemic stroke (IS) treatment. While mitophagy activation has been shown to attenuate apoptosis and pyroptosis, thereby ameliorating CI/RI, the therapeutic potential of natural compounds targeting this pathway remains underexplored. Penthorum chinense Pursh (PCP), a traditional hepatoprotective herb, contains Thonningianin A (TA), a bioactive compound with reported autophagic properties. However, the role and mechanisms of TA in CI/RI mitigation remain unclear.

In vivo, a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established to evaluate TA’s neuroprotective effects via TTC staining, Longa neurological scoring, and immunofluorescence staining. In vitro, oxygen–glucose deprivation/reoxygenation (OGD/R)-treated HT22 and BV2 cells were used to assess TA’s impact on cell viability (MTT, Hoechst/PI staining), mitochondrial oxidative stress (DHE, TMRM, JC-1, Mito-Tracker staining and Western blot), apoptosis (flow cytometry, immunofluorescence staining, Hochest and PI staining and Western blot), and pyroptosis (EthD-2/YO-PRO-1 staining and Western blot). Autophagy and mitophagy modulation was investigated using rapamycin (Rap), 3-MA (autophagy inhibitor), CCCP (mitophagy inducer), and AC220 (mitophagy inhibitor) in EGFP-LC3-U87 and mCherry-GFP-FIS1-293T cells. Co-localization immunofluorescence and Western blotting were employed to validate PINK1/Parkin pathway involvement.

TA administration significantly improved neurological function, reduced cerebral infarct volume, and attenuated neuronal damage in MCAO/R rats. In vitro, TA suppressed OGD/R-induced mitochondrial oxidative stress and apoptosis in HT22 cells while mitigating pyroptosis in BV2 microglia. Mechanistically, TA activated PINK1/Parkin-dependent mitophagy, as evidenced by enhanced LC3-II/I ratio, and increased mitochondrial-autophagosome co-localization. Crucially, TA’s anti-apoptotic and anti-pyroptotic effects were abolished upon mitophagy inhibition. These findings were corroborated in the MCAO/R model, where TA upregulated PINK1/Parkin signaling and mitigated cell damage.

This study identifies TA as a novel natural agent alleviating CI/RI by activating PINK1/Parkin-mediated mitophagy, thereby concurrently suppressing apoptosis and pyroptosis. These findings provide the first elucidating the molecular mechanis underlying TA's potential as a therapeutic candidate for IS.

The online version contains supplementary material available at 10.1186/s13020-025-01247-2.