The underlying pathophysiological mechanisms of cerebral ischemia reperfusion injury (CIRI) is intricate, and current studies suggest that neuron, astrocyte, microglia, endothelial cell, and pericyte ...all have different phenotypic changes of specific cell types after ischemic stroke. And microglia account for the largest proportion after CIRI. Previous transcriptomic studies of ischemic stroke have typically focused on the 24 hours after CIRI, obscuring the dynamics of cellular subclusters throughout the disease process. Therefore, traditional methods for identifying cell types and their subclusters may not be sufficient to fully unveil the complexity of single-cell transcriptional profile dynamics caused by an ischemic stroke.
In this study, to explore the dynamic transcriptional profile of single cells after CIRI, we used single-cell State Transition Across-samples of RNA-seq data (scSTAR), a new bioinformatics method, to analyze the single-cell transcriptional profile of day 1, 3, and 7 of transient middle cerebral artery occlusion (tMCAO) mice. Combining our bulk RNA sequences and proteomics data, we found the importance of the integrin beta 2 (Itgb2) gene in post-modeling. And microglia of Itgb2+ and Itgb2- were clustered by the scSTAR method. Finally, the functions of the subpopulations were defined by Matescape, and three different time points after tMCAO were found to exhibit specific functions.
Our analysis revealed a dynamic transcriptional profile of single cells in microglia after tMCAO and explored the important role of Itgb2 contributed to microglia by combined transcriptomics and proteomics analysis after modeling. Our further analysis revealed that the Itgb2+ microglia subcluster was mainly involved in energy metabolism, cell cycle, angiogenesis, neuronal myelin formation, and repair at 1, 3, and 7 days after tMCAO, respectively.
Our results suggested that Itgb2+ microglia act as a time-specific multifunctional immunomodulatory subcluster during CIRI, and the underlying mechanisms remain to be further investigated.
Celastrol plays a significant role in cerebral ischemia-reperfusion injury. Although previous studies have confirmed that celastrol post-treatment has a protective effect on ischemic stroke, the ...therapeutic effect of celastrol on ischemic stroke and the underlying molecular mechanism remain unclear. In the present study, focal transient cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in mice and celastrol was administered immediately after reperfusion. We performed lncRNA and mRNA analysis in the ischemic hemisphere of adult mice with celastrol post-treatment through RNA-Sequencing (RNA-Seq). A total of 50 differentially expressed lncRNAs (DE lncRNAs) and 696 differentially expressed mRNAs (DE mRNAs) were identified between the sham and tMCAO group, and a total of 544 DE lncRNAs and 324 DE mRNAs were identified between the tMCAO and tMCAO + celastrol group. Bioinformatic analysis was done on the identified deregulated genes through gene ontology (GO) analysis, KEGG pathway analysis and network analysis. Pathway analysis indicated that inflammation-related signaling pathways played vital roles in the treatment of ischemic stroke by celastrol. Four DE lncRNAs and 5 DE mRNAs were selected for further validation by qRT-PCR in brain tissue, primary neurons, primary astrocytes, and BV2 cells. The results of qRT-PCR suggested that most of selected differentially expressed genes showed the same fold change patterns as those in RNA-Seq results. Our study suggests celastrol treatment can effectively reduce cerebral ischemia-reperfusion injury. The bioinformatics analysis of lnRNAs and mRNAs profiles in the ischemic hemisphere of adult mice provides a new perspective in the neuroprotective effects of celastrol, particularly with regards to ischemic stroke.
Stroke is the second leading cause of death worldwide, and oxidative stress plays a crucial role. Celastrol exhibits strong antioxidant properties in several diseases; however, whether it can affect ...oxidation in cerebral ischemic-reperfusion injury (CIRI) remains unclear. This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms. Here, we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2 (Nrf2). Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry, we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4 (Nedd4) and then released Nrf2 from Nedd4 in astrocytes. Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI, which was significantly blocked by celastrol. Furthermore, by inhibiting oxidative stress and astrocyte activation, celastrol effectively rescued neurons from axon damage and apoptosis. Our study uncovered Nedd4 as a direct target of celastrol, and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.
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•Celastrol attenuates oxidative stress after CIRI by upregulating Nrf2 in astrocytes.•Celastrol suppresses ROS generation and reduces astrocytes activation and neuron damage.•Celastrol directly binds to Nedd4 and thus inhibits Nrf2 degradation in astrocytes.•Nedd4 is a novel E3 ligase of Nrf2 and mediated K48-linked ubiquitination of Nrf2.
Epigenetic changes in the spinal cord play a key role in the initiation and maintenance of nerve injury-induced neuropathic pain. N6-methyladenosine (m6A) is one of the most abundant internal RNA ...modifications and plays an essential function in gene regulation in many diseases. However, the global m6A modification status of mRNA in the spinal cord at different stages after neuropathic pain is unknown. In this study, we established a neuropathic pain model in mice by preserving the complete sural nerve and only damaging the common peroneal nerve. High-throughput methylated RNA immunoprecipitation sequencing results showed that after spared nerve injury, there were 55 m6A methylated and differentially expressed genes in the spinal cord. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway results showed that m6A modification triggered inflammatory responses and apoptotic processes in the early stages after spared nerve injury. Over time, the differential gene function at postoperative day 7 was enriched in "positive regulation of neurogenesis" and "positive regulation of neural precursor cell proliferation." These functions suggested that altered synaptic morphological plasticity was a turning point in neuropathic pain formation and maintenance. Results at postoperative day 14 suggested that the persistence of neuropathic pain might be from lipid metabolic processes, such as "very-low-density lipoprotein particle clearance," "negative regulation of cholesterol transport" and "membrane lipid catabolic process." We detected the expression of m6A enzymes and found elevated mRNA expression of Ythdf2 and Ythdf3 after spared nerve injury modeling. We speculate that m6A reader enzymes also have an important role in neuropathic pain. These results provide a global landscape of mRNA m6A modifications in the spinal cord in the spared nerve injury model at different stages after injury.
Forkhead Box F1 (FOXF1) has been recently implicated in cancer progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms of FOXF1 in the ...regulation of the progression of colorectal cancer (CRC) are largely unknown. We showed that FOXF1 was up-regulated in 93 paraffin-embedded archived human CRC tissue, and both high expression and nuclear location of FOXF1 were significantly associated with the aggressive characteristics and poorer survival of CRC patients. The GSEA analysis showed that the higher level of FOXF1 was positively associated with an enrichment of EMT gene signatures, and exogenous overexpression of FOXF1 induced EMT by transcriptionally activating SNAI1. Exogenous overexpression FOXF1 functionally promoted invasion and metastasis features of CRC cells, and inhibition of SNAI1 attenuates the invasive phenotype and metastatic potential of FOXF1-overexpressing CRC cells. Furthermore, the results of the tissue chip showed that the expression of FOXF1 was positively correlated with SNAI1 in CRC tissues chip. These results suggested that FOXF1 plays a critical role in CRC metastasis by inducing EMT via transcriptional activation of SNAI1, highlighting a potential new therapeutic strategy for CRC.
To clarify the suppressive effects of astragalus injection (AI) on different stages of early hepatocarcinogenesis induced by weak promotion, SD rats initiated with a single intraperitoneal (i.p.) ...injection of N-diethylnitrosamine (DEN) at 200 mg/kg body weight and promoted with 0.5% piperonyl butoxide (PBO) in diet were repeatedly administered AI at 5 ml/kg body weight/day in the early postinitiation (EPI) or late postinitiation (LPI) period for 2 or 8 weeks, respectively. The number and area of glutathione S-transferase placental form (GST-P)-immunoreactive (+) foci tended to increase in the DEN+PBO group compared with the DEN-alone group. Among the PBO-promoted groups, number and area of GST-P+ foci did not visibly change in the DEN+PBO+AI-EPI group compared with the DEN+PBO group. In contrast, number and area of GST-P+ foci tended to decrease in the DEN+PBO+AI-LPI group compared with the DEN+PBO group. Number of Ki67+ cells was increased in the DEN+PBO group compared with the DEN-alone group and was decreased in both AI-administered groups compared with the DEN+PBO group. Gene expression analysis revealed that the DEN+PBO+AI-LPI group showed increased transcript levels of Ccne1, Cdkn1b, Rb1, Bax, Bcl2, Casp3, and Casp9 compared with the DEN+PBO group; however, the DEN+PBO+AI-EPI group did not show changes in the transcript levels of any genes examined compared with the DEN+PBO. These results suggest that AI administration during the LPI period caused weak suppression of hepatocarcinogenesis under weak promotion with a low PBO dose by the mechanism involving facilitation of cell cycle suppression causing G1/S arrest and apoptosis via the mitochondrial pathway. In addition, the results suggest that AI administration during the EPI period has no effect on weakly promoted hepatocarcinogenesis.
Forkhead box F1 (FOXF1) has been recently implicated in the progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms by which FOXF1 ...regulates the progression of colorectal cancer (CRC) are largely unknown. As shown in our previous study, FOXF1 is upregulated in 182 CRC tissues, and elevated FOXF1 expression is significantly associated with microvessel density and advanced TNM (T = primary tumour; N = regional lymph nodes; M = distant metastasis) stages. In this study, 43 CRC tissues collected from patients who underwent treatment with first-line standard chemotherapeutic regimens in combination with bevacizumab were used to explore the correlation between FOXF1 expression and resistance to bevacizumab. In addition, FOXF1 regulated angiogenesis by inducing the transcription of vascular endothelial growth factor A1 (VEGFA) in vitro and in vivo. Furthermore, upregulation of FOXF1 enhanced bevacizumab resistance in CRC, and inhibition of VEGFA attenuated angiogenesis and bevacizumab resistance in FOXF1-overexpressing CRC cells. These results suggest that FOXF1 plays critical roles in CRC angiogenesis and bevacizumab resistance by inducing VEGFA transcription and that FOXF1 represents a potentially new therapeutic strategy and biomarker for anti-angiogenic therapy against CRC.
•FOXF1 could promote angiogenesis and tumorigenesis in CRC.•FOXF1 transcriptional activates VEGFA by directly binding to its promoter and then induces angiogenesis in CRC.•FOXF1 could promote bevacizumab resistance by targeting VEGFA during angiogenesis in CRC.•FOXF1-VEGFA axis could be a potential therapeutic target in selecting bevacizumab treatment for \ \CRC anti-angiogenic therapy.
Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide ...distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.
Ischemic stroke is one of the leading causes of death and disability for adults, which lacks effective treatments. Dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) exerts beneficial ...effects on ischemic stroke by attenuating neuron death and inflammation induced by microglial activation. However, the impact and mechanism of n-3 PUFAs on astrocyte function during stroke have not yet been well investigated. Our current study found that dietary n-3 PUFAs decreased the infarction volume and improved the neurofunction in the mice model of transient middle cerebral artery occlusion (tMCAO). Notably, n-3 PUFAs reduced the stroke-induced A1 astrocyte polarization both in vivo and in vitro. We have demonstrated that exogenous n-3 PUFAs attenuated mitochondrial oxidative stress and increased the mitophagy of astrocytes in the condition of hypoxia. Furthermore, we provided evidence that treatment with the mitochondrial-derived antioxidant, mito-TEMPO, abrogated the n-3 PUFA-mediated regulation of A1 astrocyte polarization upon hypoxia treatment. Together, this study highlighted that n-3 PUFAs prevent mitochondrial dysfunction, thereby limiting A1-specific astrocyte polarization and subsequently improving the neurological outcomes of mice with ischemic stroke.
Forkhead Box F1 (FOXF1) has been recently implicated in cancer progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms of FOXF1 in the ...regulation of the progression of colorectal cancer (CRC) are largely unknown. We showed that FOXF1 was up-regulated in 93 paraffin-embedded archived human CRC tissue, and both high expression and nuclear location of FOXF1 were significantly associated with the aggressive characteristics and poorer survival of CRC patients. The GSEA analysis showed that the higher level of FOXF1 was positively associated with an enrichment of EMT gene signatures, and exogenous overexpression of FOXF1 induced EMT by transcriptionally activating SNAI1. Exogenous overexpression FOXF1 functionally promoted invasion and metastasis features of CRC cells, and inhibition of SNAI1 attenuates the invasive phenotype and metastatic potential of FOXF1-overexpressing CRC cells. Furthermore, the results of the tissue chip showed that the expression of FOXF1 was positively correlated with SNAI1 in CRC tissues chip. These results suggested that FOXF1 plays a critical role in CRC metastasis by inducing EMT via transcriptional activation of SNAI1, highlighting a potential new therapeutic strategy for CRC.
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