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Background: Paeoniae Radix Rubra (PRR) has been used widely to promote blood circulation and eliminate blood stasis in China clinical practice owing to its extensive pharmacological effects. However, the "quality markers" (Q-markers) of the antioxidant effects remains unknown. Object: To explore the Q-markers of antioxidant activity based on multiple strategies, which would provide reference for the quality evaluation of PRR based on specific pharmacodynamic-oriented. Methods: Firstly, the "fingerprint" profiles of 15 batches of PRR were acquired and identified by ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF MS/MS) and the common peaks extracted. Meanwhile, the MTT assay was used to evaluate the effect of 15 batches of PRR on H2O2-induced oxidative stress in HT-22 cells. The antioxidant activity of PRR was investigated simultaneously by superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) commercial kits. The relationship between common peaks and antioxidant indexes were constructed by grey relational analysis (GRA) and partial least squares-discriminant analysis (PLS-DA) for the identification of preselected Q-markers. Secondly, experimental verification was conducted to investigate the protective effect of the preliminary components on HT-22 cells undergoing oxidative stress. Finally, for the further validation of effectiveness of antioxidant Q-markers, network pharmacology was applied to explore potential targets, and the molecular docking technology was used to value the binding ability of the potential active components of PRR to the antioxidant targets. Results: Thirty-seven common peaks from 15 batches of PRR were identified qualitatively by UHPLC-Q-TOF MS/MS. The MTT assay showed that PRR could reduce the oxidative damage induced by H2O2 upon HT-22 cells according to the index of MDA, SOD and GSH. Eight potential antioxidant components were screened by spectrum-effect correlation analysis: paeoniflorin, galloylpaeoniflorin, albiflorin, 1,2,3,4,6-o-pentagalloylglucose, benzoylpaeoniflorin, pinocembrin, oleanic acid, and isorhamnetin-3-o-nehesperidine. Each of these preliminary components showed significant protections on cellular oxidative stress (P < 0.05). Interleukin-6 (IL-6), protein kinase B (AKT1), and tumor necrosis factor (TNF) were predicted to be the major potential targets of PRR, and the good binding ability were presented between the potential active components of PRR and each target as a whole. Conclusion: Eight components were identified as the antioxidant Q-markers of PRR based on an integrated multimodal strategy.

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AIMS: Ischemic stroke is a severe cerebrovascular disease in which neuronal death continually occurs through multiple forms, including apoptosis, autophagy, pyroptosis and ferroptosis. Quercetin (QRC), as a natural flavonoid compound, has been reported to have pharmacological effects on ischemic injury accompanied by unclear anti-ferroptotic mechanisms. This study is designed to investigate the therapeutic effects of QRC against ferroptosis in ischemic stroke. MATERIALS AND METHODS: In vivo, the model of MCAO rats were used to assess the protective effect of QRC on cerebral ischemic. Additionally, we constructed oxidative stressed and ferroptotic cell models to explore the effects and mechanisms of QRC on ferroptosis. The related proteins were analysed by western blotting, immunohistochemical and immunofluorescence techniques. RESULTS: The experiments demonstrated that QRC improves neurological deficits, infarct volume, and pathological features in MCAO rats, also increased the viability of HT-22 cells exposed to H2O2 and erastin. These results, including MDA, SOD, GSH, ROS levels and iron accumulation, indicated that QRC suppresses the generation of lipid peroxides and may involve in the regulatory of ferroptosis. Both in vitro and in vivo, QRC was found to inhibit ferroptosis by up-regulating GPX4 and FTH1, as well as down-regulating ACSL4. Furthermore, we observed that QRC enhances the nuclear translocation of Nrf2 and activates the downstream antioxidative proteins. Importantly, the effect of QRC on ferroptosis can be reversed by the Nrf2 inhibitor ML385. CONCLUSIONS: This study provides evidence that QRC has a neuroprotective effect by inhibiting ferroptosis, demonstrating the therapeutic potential for cerebral ischemic stroke.

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ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, but its effect on cerebral ischemia is still rarely reported. AIM OF THE STUDY: The present study aimed to assess the potential therapeutic possibilities of the extract of PRR (PRRE) on cerebral ischemia, further exploring the underlying mechanism, and preliminary screening of the corresponding active components. MATERIALS AND METHODS: The neuroprotective effects of PRRE in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO) injury and mouse hippocampal neuronal cells (HT22 cell line) following oxidative stress were confirmed. The mechanism was investigated using immunohistochemical staining, western blotting, transmission electron microscopy (TEM), and immunofluorescence. The active components of PRRE were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking. RESULTS: The in vivo study showed that PRRE reduced infarct volume and improved neurological deficits in rats, and the expression of GPX4, FTH1, Beclin1, LC3 II, and p-Akt was upregulated in the rat hippocampi. In addition, the vitro research indicated that PRRE can also alleviate H2O2-induced HT22 cell damage by regulating cytokines such as malondialdehyde (MDA), reduced glutathione (GSH) and reactive oxygen species (ROS), and the expressions of GPX4 and Beclin1 were observed to be elevated. The PI3K/Akt signalling pathway was inhibited by LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K). Furthermore, the effective components of PRRE in regulating ferroptosis and autophagy are mainly defined as albiflorin, paeoniflorin, benzoyl paeoniflorin, oleanolic acid, and hederagenin. CONCLUSION: PRRE exerts neuroprotective effects against cerebral ischaemic injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway. This study provides an experimental basis for the potential application of PRRE as a novel therapeutic drug, and PI3K/Akt-associated ferroptosis and autophagy as therapeutic targets for cerebral ischemia.

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Heat shock protein (HSP) 90, an important component of the molecular chaperone network, is closely concerned with cellular signaling pathways and stress response by participating in the process of maturation and activation of client proteins, playing a crucial role both in the normal and abnormal operation of the organism. In functionally defective tissues, programmed cell death (PCD) is one of the regulable fundamental mechanisms mediated by HSP90, including apoptosis, autophagy, necroptosis, ferroptosis, and others. Here, we show the complex relationship between HSP90 and different types of PCD in various diseases, and discuss the possibility of HSP90 as the common regulatory nodal in multiple PCD, which would provide a new perspective for the therapeutic approaches in disease.

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Traditional Chinese medicine (TCM) has attracted a great deal of attention in the treatment of cerebral ischemia is credited with the remarkable neuroprotective effects. However, the imperfect functional mechanism of TCM is a major obstacle to their application. Many studies have been conducted to illustrate the pathophysiology of post-ischemic cerebral ischemia by elucidating the neuronal cell death pathway. Meanwhile, a new type of cell death, ferroptosis, is gradually being recognized in various diseases and is becoming a new pathway of therapeutic intervention strategy to solve many health problems. Especially since ferroptosis has been found to be closely involved into the pathogenesis of cerebral ischemia, it has been considered as a key target in the treatment of cerebral ischemia. Therefore, this paper reviews the latest research findings about the treatment of cerebral ischemia with TCM focused on ferroptosis as a target. Also, in order to explores the possibility of a new approach to treat cerebral ischemia with TCM, we discusses the correlation between ferroptosis and other cell death pathways such as apoptosis and autophagy, which would provide references for the following researches.