Thonningianin A from Penthorum chinense Pursh alleviates apoptosis and pyroptosis in cerebral ischemia-reperfusion injury through the activation of mitophagy by the PINK1/Parkin signaling pathway

Background Cerebral ischemia-reperfusion injury (CI/RI) poses a significant challenge in the treatment of ischemic stroke (IS). Activation of mitophagy can effectively inhibit cell apoptosis and necroptosis, thereby improving CI/RI. Penthorum chinense Pursh (PCP) has been used for many years as a hepatoprotective agent, and Thonningianin A (TA), a key bioactive compound in PCP, has shown autophagic activity. However, studies on the effect and mechanism of TA on CI/RI treatment are currently lacking. Methods A rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used as a vivo model. The preliminary therapeutic effects of TA on CI/RI were assessed through TTC staining, Longa neurological scoring, and immunofluorescence staining. And an vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was employed to examine the impact of TA on cell viability using MTT and Hoechst/PI staining in HT22 and BV2 cells. Furtherly, the effects of TA on mitochondrial oxidative damage, apoptosis in HT22 and pyroptosis in BV2 were evaluated by using Dihydroethidium (DHE) staining, flow cytometry, JC-1 and Tetramethylrhodamine methyl ester perchlorate (TMRM) staining, Mito-Tracker staining, plasmid transfection, EthD-2/YO-PRO-1 staining and Western blot. The effects of TA on autophagy was assessed by using the positive control drug rapamycin (Rap) and the inhibitor 3-MA, and mitophagy by using the positive control drug Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the inhibitor AC220 in EGFP-LC3-U87 and mCherry-GFP-FIS1-293T cells, Western blot and immunofluorescence co-localization were applied to determine whether TA inhibits apoptosis and pyroptosis through mitophagy. Finally, the molecular mechanism of TA on CI/RI were validated in the MCAO/R model through immunofluorescence co- localization and Western blot analysis. Results TA significantly improved neurological function, reduced infarct volume, and mitigated neuronal damage in the MCAO/R model. TA decreased OGD/Rinduced mitochondrial oxidative damage and apoptosis in HT22 cells and improved pyroptosis in BV2 cells, these effects were mediated via the PINK1/Parkin mitophagy signaling pathway, which was corroborated in the MCAO/R model. Conclusion This study is the first to confirm that TA is a potential natural active component for the treatment of CI/RI. Its therapeutic effect and mechanism are associated with the inhibition of apoptosis and pyroptosis through the activation of the PINK1/Parkin mitophagy signaling pathway.