Tongqiao Huoxue Decoction inhibits ferroptosis by facilitating ACSL4 ubiquitination degradation for neuroprotection against cerebral ischemia-reperfusion injury.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear. PURPOSE: This study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms. METHODS: The active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe2+, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: TQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment. CONCLUSION: This work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.

A polypeptide derived from pilose antler ameliorates CUMS-induced depression-like behavior by SENP2-PLCß4 signaling axis.

Neurogenesis is known to be closely associated with depression. We aimed to investigate whether a polypeptide monomer derived from pilose antler (polypeptide sequence LSALEGVFYP, PAP) exerts an antidepressant effect by influencing neurogenesis, and to elucidate the mechanism of its antidepressant action. Behavioral tests were performed to observe the antidepressant effect of PAP. Neurogenesis in the dentate gyrus (DG) region of hippocampus was observed by immunofluorescence. The expression of key proteins of Sentrin/SUMO-specific proteases 2 (SENP2)- Phosphoinositide-specific phospholipase C beta 4 (PLCß4) pathway was accessed by co-immunoprecipitation (Co-IP), and the calcium homeostasis associated proteins were observed via Western blot (WB). Subsequently, temozolomide (TMZ) pharmacologically blocked neurogenesis to verify the antidepressant effect of PAP on neurogenesis. The mechanism of PAP antidepressant effect was verified by constructing a sh-SENP2 virus vector to silence SENP2 protein. Finally, corticosterone (CORT)-induced PC12 cell model was used to verify whether PAP was involved in the process of deconjugated PLCß4 SUMOylated. The results showed that PAP improved depression-like behavior and neurogenesis induced by chronic unpredictable mild stimulation (CUMS). In addition, PAP acted on SENP2-PLCß4 pathway to deconjugate the SUMOylation of PLCß4 and affect calcium homeostasis. Pharmacological blockade of neurogenesis by TMZ treatment impaired the antidepressant efficacy of PAP. Knockout of SENP2 in the CUMS model attenuated the antidepressant response of PAP, and the impaired neurogenesis was not ameliorated by PAP treatment. In summary, PAP acted on the SENP2-PLCß4 signaling pathway to inhibit the SUMOylation of PLCß4 and maintain calcium homeostasis, thereby protecting neurogenesis and playing an antidepressant role.