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》

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 ... -  《-》

Icariin promoted ferroptosis by activating mitochondrial dysfunction to inhibit colorectal cancer and synergistically enhanced the efficacy of PD-1 inhibitors.

A controlled type of cell death called ferroptosis is linked to increased reactive oxygen species (ROS), lipid peroxidation, and iron buildup. Furthermore, evidence indicates that ferroptosis may act as an immunogenic form of cell death with potential physiological functions in tumors and immunosuppression. Inducing ferroptosis in tumor cells may have the potential to complement cancer immunotherapy strategies. The development of colorectal cancer (CRC) and the poor efficacy of immunotherapy are associated with the crosstalk of cellular ferroptosis. Currently, Icariin (ICA), the main bioactive component extracted from Epimedium, has been shown to inhibit a variety of cancers. However, the specific role and potential mechanism of ICA in regulating ferroptosis in CRC remains unclear. The aim of this investigation was to clarify the mechanism underlying the anti-CRC cancer properties of ICA and how it induces ferroptosis to enhance immunotherapy. To evaluate cell viability, the Cell Counting Kit-8 (CCK-8) test was utilized. The transwell test and the wound healing assay were used to assess cell migration. A subcutaneous graft tumor model was constructed with C57BL/6 mice using MC38 colorectal cancer cell lines. The inhibitory effect of ICA on CRC, ferroptosis level and immunomodulatory effects were detected by serum biochemical assay, cytokine assay, hematoxylin-eosin (H&E) staining, immunofluorescence staining, CyTOF mass spectrometry flow screening and Western blotting. Western blotting, proteomics, molecular docking and microscale thermophoresis (MST) were used to forecast and confirm ICA's binding and interaction with HMGA2, STAT3, and HIF-1α. Moreover, the levels of lipid peroxidation and ferroptosis were assessed through the use of the C11-BODIPY fluorescent probe, the FerroOrange fluorescent probe, the iron level, the malondialdehyde (MDA) and reduced glutathione (GSH) assay kit, and Western blotting analysis. To assess alterations in mitochondrial structure and membrane potential, transmission electron microscopy (TEM) and JC-1 immunofluorescence were employed. It was demonstrated in the current study that ICA treatment inhibits CRC and enhances anti-PD-1 therapy efficacy by inciting ferroptosis. As shown in vitro, ICA inhibits CRC cell proliferation, migration, and apoptosis. As demonstrated in vivo, ICA has a dose-dependent tumor suppressor effect when combined with anti-PD-1, it can significantly inhibit tumor growth, increase the expression of serum TNF-α, IFN-γ, and granzyme B, and promote CD69+CD8+ T, CD69+CD8+Tem, CD69+CD8+Teff, TCRβ+CD8+ T, TCRβ+CD8+ T, TCRβ+CD8+Tem, TCRβ+CD8+Teff. The inhibitory effect of ICA on CRC was associated with the binding of HMGA2, STAT3, and HIF-1α proteins, which inhibited CRC by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), promoting the accumulation of iron (Fe2+), depletion of reduced glutathione (GSH), inhibiting SLC7A11 and GPX4 expressions, thereby inducing ferroptosis in CRC. As a consequence of ICA-induced ferroptosis, mitochondria are dysfunctional, with increased ROS production, membrane potential depolarization (MMP), and ATP production reduced. This process can be efficiently reversed by the mitochondria-targeted antioxidant Mito-Q. It is noteworthy that the ferroptosis inhibitor liproxstatin-1 (lip-1), anti-CD8, and anti-IFN-γ exhibited a significant inhibitory effect on the level of ferroptosis and antitumor capacity of ICA combined with anti-PD-1. This finding suggests that the antitumor immunopotentiating effect of ICA on anti-PD-1 is dependent on the secretion of IFN-γ-induced ferroptosis of CRC cells by the CD8+ T cell. Our study represents the inaugural demonstration of the mechanism whereby ICA exerts anti-CRC effects and synergistically enhances the efficacy of anti-PD-1, inducing mitochondrial damage and leading to ferroptosis. ICA promotes ferroptosis of CRC cells by inducing mitochondrial dysfunction, and ICA combined with anti-PD-1 significantly promotes CD69, TCRβ signalling, activates effector CD8+ T cells to secrete IFN-γ, and achieves immunopotentiation by promoting ferroptosis of CRC cells, thus inhibiting CRC development. This study is built upon existing research into the pharmacodynamic mechanisms of ICA in the context of CRC, and offers a novel therapeutic approach in addressing the issue of CRC immunotherapy potentiation.

Haoyue W ，Kexiang S ，Shan TW ，Jiamin G ，Luyun Y ，Junkai W ，Wanli D ... -  《-》

Cryptotanshinone alleviates immunosuppression in endometriosis by targeting MDSCs through JAK2/STAT3 pathway.

Endometriosis (EMS), a well-recognized chronic inflammatory disorder, characterized by significant immune dysregulation, in which myeloid-derived suppressor cells (MDSCs) are essential for facilitating immunosuppression and driving to disease progression. Cryptotanshinone (CTS) is an active compound capable of modulating MDSC-mediated immunosuppression; however, its therapeutic effects and mechanisms in the treatment of EMS remain unclear. This study aims to investigate the therapeutic potential of CTS in modulating MDSCs through JAK2/STAT3 signaling pathway and to evaluate its effects on immune microenvironment and endometriotic lesion growth in EMS. Transcriptomic data (GSE141549) and single-cell RNA sequencing data (GSE213216) were analyzed to compare immune cell populations in control endometrium (CE), eutopic endometrium (EuE) and ectopic endometrium (EcE) of patients with EMS. Network pharmacology analysis, surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) were utilized to explore the molecular mechanism of CTS's effects on MDSCs. A C57BL/6J EMS mice model was established to evaluate CTS's influence on MDSC-mediated immune response in vivo. Flow cytometry and immunofluorescence were used to analyze the immune cell populations, particularly MDSCs and CD8+ T cells. Ex vivo bone marrow (BM)-derived MDSCs were prepared to investigate the modulatory activities of CTS on the frequency and function of MDSCs. The impacts of CTS on JAK2/STAT3 pathway were further examined by western blot. Bioinformatic analysis revealed that, among the three progression stages (CE, EuE, and EcE), the EcE stage exhibited a relatively elevated level of MDSCs and a reduced level of CD8+ T cells. Network pharmacological analysis, along with SPR and CETSA identified that CTS potentially modulates MDSCs in EMS by targeting the JAK2/STAT3 pathway. In vivo studies demonstrated that a relatively high dose of CTS treatment (60mg/kg) effectively inhibited lesion growth, reduced the population of MDSCs, and enhanced CD8+ T cell infiltration. Ex vivo experiments showed that CTS decreased the BM-derived MDSC frequency and rescued the suppressive ability of MDSC upon CD8+ T cells in a dose-dependent manner. Further mechanism analysis confirmed that CTS modulates the expression of immunosuppressive genes and proteins associated with MDSCs through JAK2/STAT3 pathway. This study is the first to demonstrate that CTS is a promising natural compound for EMS treatment by inhibiting MDSC accumulation and modulating MDSC-mediated immune responses. Its therapeutic efficacy is linked to the modulation of the JAK2/STAT3 signaling pathway.

Xie L ，Zhong Y ，Chen Y ，Wang Y ，Xian P ，Liu S ，Xin X ，Chen Y ，Guan Y ，Li K ... -  《-》