Elemene mitigates oxidative stress and neuronal apoptosis induced by cerebral ischemia-reperfusion injury through the regulation of glutathione metabolism.

Chinese materia medica (CMM) has a long history and extensive experience in treating ischemic stroke. Wen Ezhu, the rhizome of Curcuma wenyujin Y.H. Chen et C. Ling, is renowned for promoting blood circulation, dispersing blood stasis, alleviating pain, and eliminating masses. Promoting blood circulation and removing blood stasis are essential principles in Traditional Chinese Medicine for treating stroke. Consequently, Wen Ezhu is frequently used in clinical practice as a key CMM for treating stroke. The Elemene active fraction (ELE), a sesquiterpene compound extracted from Wen Ezhu, primarily consists of β-Elemene. It also contains β-Caryophyllene, γ-Elemene, and δ-Elemene isomers. ELE has shown potential pharmacological effects in various diseases, including ischemic stroke. However, its precise mechanism of action in treating stroke remains to be confirmed. To explore the therapeutic potential of ELE in acute ischemic stroke and elucidate its underlying mechanisms. A rat model of middle cerebral artery occlusion reperfusion (MCAO/R) was used to evaluate ELE's effects. Therapeutic efficacy was assessed through mNSS scoring, magnetic resonance imaging (MRI), tetrazolium chloride (TTC) staining, Hematoxylin and eosin (H&E), and Nissl staining. Non-targeted metabolomics identified key pathways, confirmed using biochemical analysis, immunohistochemistry, and Western blotting. ROS levels and apoptosis-related proteins were also evaluated. Our findings show that ELE administration significantly reduced the cerebral infarct area and lowered modified neurological severity scores (mNSS) in animals, indicating a strong neuroprotective effect. Metabolomics results highlight the glutathione (GSH) metabolic pathway as a key mechanism through which ELE exerts its therapeutic effects. Specifically, ELE upregulates glutathione reductase (GR) protein expression and downregulates glutathione peroxidase (GPX) expression. The regulatory process of ELE decreases oxidized glutathione (GSSG) levels and increases GSH levels, effectively reducing oxidative stress damage (lower reactive oxygen species levels) during CI/RI. This results in the downregulation of the pro-apoptotic protein Bax and the upregulation of the pro-survival protein Bcl-2, thus reducing neuronal apoptosis. ELE protects neurons in MCAO/R rats through the GSH metabolism pathway, balancing GSH and GSSG levels to mitigate oxidative stress and enhance neuroprotection in cerebral ischemia/reperfusion injury.

Wu P ，Cheng LH ，Liu YL ，Zhang JL ，Dong XM ，Chen L ，Xu YX ，Ren YY ，Zhang HM ，Liu ZQ ，Zhou JL ，Xie T ... -  《-》

β-elemene, a sesquiterpene constituent from Curcuma phaeocaulis inhibits the development of endometriosis by inducing ferroptosis via the MAPK and STAT3 signaling pathways.

The rhizome of Curcuma phaeocaulis Valeton, Curcuma wenyujin Y.H. Chen & C. Ling, or Curcuma kwangsiensis S. G. Lee et C. F. Liang, commonly known as Wen-E-Zhu and E'zhu, has been utilized in traditional Chinese medicine for the treatment of cancer and gynecological diseases since antiquity. This traditional medicinal herb is highly esteemed for its efficacy in promoting blood circulation, dissolving blood stasis, reducing swelling, and alleviating pain. β-Elemene (β-ELE), a sesquiterpene compound derived from Curcuma phaeocaulis, has demonstrated potential in inhibiting tumor cell proliferation and inducing ferroptosis, which have been extensively studied in various malignant neoplasms. Previous studies have confirmed that Sparganium stoloniferum-Curcuma phaeocaulis containing β-ELE may possess anti-endometriotic properties. However, the exact mechanism underlying β-ELE's anti-endometriosis activity remains largely unknown and requires further research and investigation. To identify the anti-endometriosis target of β-ELE and elucidate the underlying molecular mechanism of β-ELE in endometriosis, focusing on inducing ferroptosis. The target pathway of β-ELE in endometriosis treatment was predicted through network pharmacology and bioinformatics analysis. Surface plasmon resonance-high performance liquid chromatography-protein mass spectrometry (SPR-HPLC-MS) and molecular docking were used to further identify the potential targets of β-ELE in endometriosis. The immortalized endometriosis epithelial cell line 12Z was used for in vitro study. The effect of β-ELE on cell proliferation and migration was detected by CCK-8, EdU and wound healing assay, and ultrastructural changes were examined via transmission electron microscopy. The effect of β-ELE-induced ferroptosis was determined by western blot, immunohistochemistry staining and flow cytometry. SPR affinity analysis was performed to specific the direct interaction between β-ELE and FTH1, FTL, GPX4, STAT3 and MAPK14. To establish a mouse model of endometriosis and to assess the inhibitory effects of β-ELE and ELE injection on endometriosis in vivo as well as safety profile of administration, and investigate the effects and underlying mechanisms of β-ELE and ELE injection on ferroptosis in ectopic lesions. SPR-HPLC-MS was employed to identify 76 potential targets of β-ELE for endometriosis treatment, closely linked to ferroptosis. Molecular docking revealed that glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), ferritin light chain (FTL), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase 14 (MAPK14) are key action targets of β-ELE in endometriosis. Further investigations revealed that β-ELE inhibited the proliferation and migration of endometriotic cells in vitro while inducing ferroptosis, as evidenced by increased levels of iron, reactive oxygen species (ROS), and lipid peroxidation. In a mouse model, β-ELE inhibited the growth of endometriotic lesions, induced ferroptosis, suppressed fibrosis, and exhibited anti-endometriotic effects. Mechanistically, β-ELE downregulates the expression levels of GPX4, FTH1, and FTL and inhibited the phosphorylation of STAT3 and MAPK14, which may elucidate its underlying molecular mechanisms. This study demonstrates that the inhibitory effect of β-ELE on endometriosis by inducing ferroptosis in vitro and in vivo. Our results revealed that β-ELE exerts anti-endometriosis effects by inducing ferroptosis via the MAPK and STAT3 signaling pathways.

Fu Z ，Liu H ，Kuang Y ，Yang J ，Luo M ，Cao L ，Zheng W ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

Piezo1 Modulates Neuronal Autophagy and Apoptosis in Cerebral Ischemia-Reperfusion Injury Through the AMPK-mTOR Signaling Pathway.

Cerebral ischemia-reperfusion (I/R) injury is a complex pathophysiological process involving multiple mechanisms, including apoptosis and autophagy, which can lead to significant neuronal damage. PIEZO1, a stretch-activated ion channel, has recently emerged as a potential regulator of cellular responses to ischemic conditions. However, its role in neuronal cell survival and death during ischemic events is not well elucidated. This study aimed to ascertain the regulatory function of PIEZO1 in neuronal cell apoptosis and autophagy in an in vitro model of hypoxia-reoxygenation and an in vivo model of brain I/R injury. HT22 hippocampal neuronal cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemic conditions, with subsequent reoxygenation. In vitro, PIEZO1 expression was silenced using small interfering RNA (si-RNA) transfection. The effects on cell viability, apoptosis, and autophagy were assessed using CCK-8 assays, PI-Annexin/V staining combined with flow cytometry, and Western blot analysis. Additionally, intracellular Ca2+ levels in HT22 cells were measured using a Ca2+ probe. The involvement of the AMPK-mTOR pathway was investigated using rapamycin. For in vivo validation, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was employed. To determine the neuroprotective role of PIEZO1 silencing, sh-PIEZO1 adeno-associated virus was stereotaxically injected into the cerebral ventricle, and neurological and histological outcomes were assessed using neurological scoring, TTC staining, H&E staining, Nissl staining, and immunofluorescence. In HT22 cells, OGD/R injury notably upregulated PIEZO1 expression and intracellular Ca2+ levels. Silencing PIEZO1 significantly diminished OGD/R-induced Ca2+ influx, apoptosis, and autophagy, as indicated by lower levels of pro-apoptotic and autophagy-related proteins and improved cell viability. Additionally, PIEZO1 modulated the AMPK-mTOR signaling pathway, an effect that was counteracted by rapamycin treatment, implying its regulatory role. In vivo, PIEZO1 silencing ameliorated brain I/R injury in MCAO/R rats, demonstrated by improved neurological function scores and reduced neuronal apoptosis and autophagy. However, these neuroprotective effects were reversed through rapamycin treatment. Our findings indicate that PIEZO1 is upregulated following ischemic injury and facilitates Ca2+ influx, apoptosis, and autophagy via the AMPK-mTOR pathway. Silencing PIEZO1 confers neuroprotection against I/R injury both in vitro and in vivo, highlighting its potential as a therapeutic target for stroke management.

Yue Y ，Chen P ，Ren C 《-》

Buyang huanwu decoction inhibits the activation of the RhoA/Rock2 signaling pathway through the phenylalanine metabolism pathway, thereby reducing neuronal apoptosis following cerebral ischemia-reperfusion injury.

Buyang Huanwu Decoction (BYHWD) exerts its anti-cerebral ischemia effects through multiple pathways and targets, although its specific mechanisms remain unclear. Ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) metabolomics and other methods were employed to investigate the role of BYHWD in inhibiting neuronal apoptosis following cerebral ischemia-reperfusion by modulating the RhoA/Rock2 pathway. A rat model of exhaustion swimming combined with middle cerebral artery occlusion (ES + I/R) was established to evaluate the intervention effects of Buyang Huanwu Decoction on cerebral ischemia-reperfusion. This was assessed using neurological function scores, Qi deficiency and blood stasis syndrome scores, HE staining, Nissl staining and TT staining. Differential metabolites and metabolic pathways associated with cerebral ischemia-reperfusion were identified using UPLC-QTOF-MS metabolomics, with key differential metabolites validated through ELISA. Molecular docking techniques were employed to predict interactions between the key differential metabolite, hippuric acid, and its primary downstream pathways. Finally, the levels of neurocellular apoptosis, as well as key molecules in the RhoA/Rock2 signaling pathway and the mitochondrial apoptosis pathway, were measured. The results indicated that the primary differential metabolites associated with BYHWD's protective effects against ischemia-reperfusion injury were hippuric acid, lysophosphatidic acid, and lysophosphatidylethanolamine, with the main metabolic pathway being phenylalanine metabolism. Molecular docking studies demonstrated that malonic acid exhibited a strong affinity for proteins related to the RhoA/Rock2 signaling pathway and the mitochondrial apoptosis pathway.Furthermore, we found that BYHWD treatment significantly decreased the apoptosis rate of cells following cerebral ischemia-reperfusion and inhibited the expression of key molecules in both the RhoA/Rock2 signaling pathway and the mitochondrial apoptosis pathway in brain tissue. BYHWD ameliorated brain tissue injury after cerebral ischemia/reperfusion in rats with qi deficiency and blood stasis. The underlying mechanism may involve BYHWD's inhibition of the RhoA/Rock2 signaling pathway activation through modulation of the phenylalanine metabolism pathway, thereby reducing neuronal apoptosis mediated by the mitochondrial apoptosis pathway.

Li Y ，Hu Z ，Xie L ，Xiong T ，Zhang Y ，Bai Y ，Ding H ，Huang X ，Liu X ，Deng C ... -  《-》