Rhein attenuates cerebral ischemia-reperfusion injury via inhibition of ferroptosis through NRF2/SLC7A11/GPX4 pathway.

BACKGROUND: Ischemic stroke, a major cause of death and disability worldwide, results from reduced blood flow to the brain, leading to irreversible neuronal damage. Recent evidence suggests that ferroptosis, a form of regulated cell death, plays a critical role in the pathogenesis of ischemic stroke. Rhein, a natural anthraquinone compound, has demonstrated neuroprotective effects; However, its role in ferroptosis and the underlying mechanisms remain unclear. Here, we investigated the protective effects of Rhein against ischemia/reperfusion (I/R) injury in a rat model of middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT22 cells. Rhein treatment dose-dependently ameliorated neurological deficits, reduced infarct volume, and attenuated blood-brain barrier (BBB) disruption in the MCAO model. Furthermore, Rhein suppressed oxidative stress, intracellular ROS generation, and ferroptosis-related protein expression in both in vivo and in vitro models. Mechanistically, Rhein protected against OGD/R-induced HT22 cell injury by regulating the NRF2/SLC7A11/GPX4 signaling pathway. This effect was abolished upon NRF2 inhibition, suggesting that Rhein's neuroprotective action is NRF2-dependent. Molecular docking and microscale thermophoresis analyses further supported the direct interaction between Rhein and the ferroptosis-related protein NRF2. Collectively, our findings reveal that Rhein confers neuroprotection against cerebral I/R injury by inhibiting ferroptosis via the NRF2/SLC7A11/GPX4 axis, providing a potential therapeutic avenue for ischemic stroke. AIMS: To investigate the neuroprotective effects of Rhein, a natural anthraquinone compound, against ischemia/reperfusion (I/R) injury and elucidate the underlying mechanisms involving ferroptosis and the NRF2/SLC7A11/GPX4 pathway. METHODS: A rat model of middle cerebral artery occlusion (MCAO) was employed for in vivo assessments, while oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT22 cells were used as an in vitro model. Comprehensive analyses, including neurological score assessment, triphenyl tetrazolium chloride staining, Evans Blue leakage assay, intracellular ROS detection, MTT assay, dual-luciferase reporter assay, oxidative stress and Fe2+ content assessment, immunofluorescence, Western blot, flow cytometry, molecular docking, and microscale thermophoresis, were performed to evaluate the effects of Rhein on I/R injury and ferroptosis. RESULTS: Rhein conferred dose-dependent neuroprotection against cerebral I/R injury, reducing infarct volume and blood-brain barrier (BBB) disruption in the MCAO model. In both in vivo and in vitro models, Rhein suppressed oxidative stress, intracellular ROS generation, and ferroptosis-related protein expression. Furthermore, Rhein protected HT22 cells from OGD/R-induced injury by regulating the NRF2/SLC7A11/GPX4 signaling pathway, with NRF2 inhibition abolishing these therapeutic effects. Molecular docking and microscale thermophoresis analyses supported a direct interaction between Rhein and NRF2, a ferroptosis-related protein. CONCLUSION: Rhein attenuates cerebral I/R injury by inhibiting ferroptosis via the NRF2/SLC7A11/GPX4 axis, highlighting its potential as a therapeutic agent for ischemic stroke.

GSPT1 Functions as a Tumor Promoter in Human Liver Cancer.

OBJECTIVE: This study analyzed the role of G1 to S phase transition 1 protein (GSPT1) in promoting progression of liver cancer cells. METHODS: A bioinformatics database was used to analyze the expression levels of GSPT1 in liver cancer tissues and the prognosis of patients. Subsequently, Western blotting and quantitative PCR were used to verify the expression levels of GSPT1 between normal hepatocytes and hepatoma cells. We used a CRISPR/Cas9 system to construct knockouts of GSPT1 in HepG2 and HCCLM9 liver cancer cells. The effect of GSPT1 on liver cancer cell migration and invasion was analyzed using flow cytometry, migration, and tumor formation assays. RESULTS: The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset indicated that GSPT1 expression was upregulated in liver cancer cell lines, and patients with liver cancer had poor prognosis. Knockout of GSPT1 in cells significantly inhibited tumor proliferation, cell migration, and growth in vivo. CONCLUSION: In this study, we found that GSPT1 promotes the migration and invasion of liver cancer cells.

GMFG Has Potential to Be a Novel Prognostic Marker and Related to Immune Infiltrates in Breast Cancer.

A growing amount of evidence has indicated immune genes perform a crucial position in the development and progression of breast cancer microenvironment. The purpose of our study was to identify immunogenic prognostic marker and explore potential regulatory mechanisms for breast cancer. We identified the genes related to ImmuneScore using ESTIMATE algorithm and WGCNA analysis, and we identified the differentially expressed gene (DEGs). Then, Glia maturation factor γ (GMFG) was determined as a predictive factor by intersecting immune-related genes with DEGs and survival analysis. We found the expression of GMFG was lower in breast cancer tissues compared with normal breast tissues, which was further verified by immunohistochemical (IHC). Moreover, the decreased expression of GMFG was significantly related to the poor prognosis. Besides, the expression of GMFG was related to the age, ER status, PR status, HER2 status and tumor size, which further suggested that the expression of GMFG was correlated with the subtype and the growth of tumor. The univariate and multivariate Cox regression analyses revealed that age, stage, the expression level of GMFG and radiotherapy were independent factors for predicting the prognosis of breast cancer patients. Subsequently, a prognostic model to predict the 3-year, 5-year and 10-year overall survival rate was developed based on the above four variables, and visualized as a nomogram. The values of area under the curve of the nomogram at 3-year, 5-year and 10-year were 0.897, 0.873 and 0.922, respectively, which was higher than stage in prognostic accuracy. In addition, we also found that GMFG expression level was correlated with sensitivity of some breast cancer chemotherapy drugs. Furthermore, the results of GSEA indicated immune-related pathways were mainly enriched in GMFG-high-expression group. CIBERSORT analysis for the proportion of tumor-infiltrating immune cells (TIICs) suggested that expression of GMFG was positively association with multiple kinds T-cell in BC. Among them, CD8+ T cells had the strongest correlation with GMFG expression, which revealed that GMFG might has an antitumor effect by increasing the infiltration of CD8+ T cells in breast cancer. Accordingly, GMFG has the potential to become a novel immune biomarker for the diagnosis and treatment of breast cancer.

Hypoxia-Associated Prognostic Markers and Competing Endogenous RNA Co-Expression Networks in Breast Cancer.

Objective: Many primary tumors have insufficient supply of molecular oxygen, called hypoxia. Hypoxia is one of the leading characteristics of solid tumors resulting in a higher risk of local failure and distant metastasis. It is quite necessary to investigate the hypoxia associated molecular hallmarks in breast cancer. Materials and Methods: According to the published studies, we selected 13 hypoxia related gene expression signature to define the hypoxia status of breast cancer using ConsensusClusterPlus package based on the data from The Cancer Genome Atlas (TCGA). Subsequently, we characterized the infiltration of 24 immune cell types under different hypoxic conditions. Furthermore, the differentially expressed hypoxia associated microRNAs, mRNAs and related signaling pathways were analyzed and depicted. On this basis, a series of prognostic markers related to hypoxia were identified and ceRNA co-expression networks were constructed. Results: Two subgroups (cluster1 and cluster2) were identified and the 13 hypoxia related gene signature were all up-regulated in cluster1. Thus, we defined the cluster1 as "hypoxic subgroup" compared with cluster2. The infiltration of CD8+ T cell and CD4+ T cell were lower in cluster1 while the nTreg cell and iTreg cell were higher, indicating that there was immunosuppressive status in cluster1. We observed widespread hypoxia-associated dysregulation of microRNAs and mRNAs. Next, a risk signature for predicting prognosis of breast cancer patients was established based on 12 dysregulated hypoxia associated prognostic genes. Two microRNAs, hsa-miR-210-3p and hsa-miR-190b, with the most significant absolute logFC value were related to unfavorable and better prognosis, respectively. Several long non-coding RNAs were predicted to be microRNA targets and positively correlated with two selected mRNAs, CPEB2 and BCL11A. Predictions based on the SNHG16-hsa-miR-210-3p-CPEB2 and LINC00899/PSMG3-AS1/PAXIP-AS1-hsa-miR-190b-BCL11A ceRNA regulation networks indicated that the two genes might act as tumor suppressor and oncogene, respectively. Conclusion: Hypoxia plays an important role in the initiation and progression of breast cancer. Our research provides potential mechanisms into molecular-level understanding of tumor hypoxia.

Analysis of N6-Methyladenosine Methyltransferase Reveals METTL14 and ZC3H13 as Tumor Suppressor Genes in Breast Cancer.

OBJECTIVES: Recently, an increasing number of studies have revealed that N6-methyladenosine (m6A) functions as a significant post-transcriptional modification which plays a critical role in the occurrence and progression of enriched tumors by regulating coding and non-coding RNA biogenesis. However, the biological function of m6A in breast cancer remains largely unclear. MATERIALS AND METHODS: In this study, we used a series of bioinformatic databases and tools to jointly analyze the expression of m6A methylation transferases (METTL3, METTL14, WTAP, RBM15, RBM15B and ZC3H13) and investigate the prognostic value of METTL14 and ZC3H13 in breast cancer. Besides, we analyzed the downstream carcinogenic molecular mechanisms related to METTL14 and ZC3H13 and their relationship with immune infiltration in breast tumor tissues. RESULTS: The results showed that METTL14 and ZC3H13 were the down-regulated m6A methylation transferases in breast cancer. Survival outcome analysis suggested that abnormally low expression of METTL14 and ZC3H13 could predict unfavorable prognosis in four breast cancer subtypes. Moreover, their down-regulation was associated with ER-, PR- and triple-negative breast cancer patients, as well as tumor progression (increased Scarff, Bloom and Richardson grade status and Nottingham Prognostic Index classification). Co-expression analysis revealed that METTL14 and ZC3H13 had a strong positive correlation with APC, an antagonist of the Wnt signaling pathway, indicating they might cooperate in regulating proliferation, invasion, and metastasis of tumor cells. METTL14, ZC3H13, and APC expression levels had significant positive correlation with infiltrating levels of CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells, and negative correlation with Treg cells in breast cancer. CONCLUSIONS: This study demonstrated that down-regulation of METTL14 and ZC3H13 which act as two tumor suppressor genes was found in breast cancer and predicted poor prognosis. Their abnormal expression promoted breast cancer invasion by affecting pathways related to tumor progression and mediating immunosuppression.

Corrigendum: Hypoxia-Associated Prognostic Markers and Competing Endogenous RNA Co-Expression Networks in Breast Cancer.

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[The effect of exogenous glucose, fructose and NO donor sodium nitroprusside (SNP) on rice seed germination under salt stress].

The effects and the relationship between soaking rice seed of exogenous glucose, fructose and nitric oxide (NO) donor sodium nitroprusside (SNP) on the seed germination under salt stress were investigated. The results showed that employment of exogenous fructose, glucose and NO donor SNP could significantly promote the germination index and the early germination rate of rice seed under salt stress. Also, SNP pretreatment enhanced endogenous glucose and fructose contents. Effects of exogenous glucose and fructose combined with different concentrations of SNP on the rice seed germination and seedling growth under salt stress were surveyed. The results also showed that there existing positive effect of application of glucose plus SNP on the rice seed germination in comparison with existing a few negative effects of fructose plus SNP pretreatment. Additionally, the alleviation of inhibition of early rice seedling growth under salt stress by SNP could be modulated by glucose and fructose pretreatment, also the effect of glucose was stronger.