NR5A2 gene affects the overall survival of LUAD patients by regulating the activity of CSCs through SNP pathway by OCLR algorithm and immune score.

Objective: Differentially expressed genes (DEGs) in lung adenocarcinoma (LUAD) tumor stem cells were screened, and the biological characteristics of NR5A2 gene were investigated. Methods: The expression and prognosis of NR5A2 in human LUAD were predicted and analyzed through bioinformatics analysis from a human cancer database. Gene expression and clinical data of LUAD tumor and normal lung tissues were obtained from The Cancer Genome Atlas (TCGA) database, and DEGs associated with lung cancer tumor stem cells (CSCs) were screened. Univariate and multivariate Cox regression models were used to screen and establish prognostic risk prediction models. The immune function of the patients was scored according to the model, and the relative immune functions of the high- and low-risk groups were compared to determine the difference in survival prognosis between the two groups. In addition, we calculated the index of stemness based on the transcriptome of the samples using one-class linear regression (OCLR). Results: Bioinformatics analysis of a clinical cancer database showed that NR5A2 was significantly decreased in human LUAD tissues than in normal lung tissues, and the decrease in NR5A2 gene expression shortened the overall survival and progression-free survival of patients with LUAD. Conclusion: The NR5A2 gene may regulate LUAD tumor stem cells through selective splicing mutations, thereby affecting the survival and prognosis of patients with lung cancer, and the NR5A2 gene may regulate CSCs through single nucleotide polymorphism.

The Role of Ferroptosis and Cuproptosis in Curcumin against Hepatocellular Carcinoma.

BACKGROUND: Among cancer-related deaths, hepatocellular carcinoma (HCC) ranks fourth, and traditional Chinese medicine (TCM) treatment is an important complementary alternative therapy for HCC. Curcumin is a natural ingredient extracted from Curcuma longa with anti-HCC activity, while the therapeutic mechanisms of curcumin remain unclear, especially on ferroptosis and cuproptosis. METHODS: Differentially expressed genes (DEGs) of curcumin treatment in PLC, KMCH, and Huh7 cells were identified, respectively. The common genes among them were then obtained to perform functional enrichment analysis and prognostic analysis. Moreover, weighted gene co-expression network analysis (WGCNA) was carried out for the construction of the co-expression network. The ferroptosis potential index (FPI) and the cuproptosis potential index (CPI) were subsequently used to quantitatively analyze the levels of ferroptosis and cuproptosis. Finally, single-cell transcriptome analysis of liver cancer was conducted. RESULTS: We first identified 702, 515, and 721 DEGs from curcumin-treated PLC, KMCH, and Huh7 cells, respectively. Among them, HMOX1, CYP1A1, HMGCS2, LCN2, and MTTP may play an essential role in metal ion homeostasis. By WGCNA, grey60 co-expression module was associated with curcumin treatment and involved in the regulation of ion homeostasis. Furthermore, FPI and CPI assessment showed that curcumin had cell-specific effects on ferroptosis and cuproptosis in different HCC cells. In addition, there are also significant differences in ferroptosis and cuproptosis levels among 16 HCC cell subtypes according to single-cell transcriptome data analysis. CONCLUSIONS: We developed CPI and combined it with FPI to quantitatively analyze curcumin-treated HCC cells. It was found that ferroptosis and cuproptosis, two known metal ion-mediated forms of programmed cell death, may have a vital effect in treating HCC with curcumin, and there are significant differences in various liver cancer cell types and curcumin treatment which should be considered in the clinical application of curcumin.

Hydroxysafflor yellow a confers neuroprotection against acute traumatic brain injury by modulating neuronal autophagy to inhibit NLRP3 inflammasomes.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydroxysafflor yellow A (HSYA) is the principal bioactive compound isolated from the plant Carthamus tinctorius L. and has been reported to exert neuroprotective effects against various neurological diseases, including traumatic brain injury (TBI). However, the specific molecular and cellular mechanisms underlying HSYA-mediated neuroprotection against TBI are unclear. AIM OF THE STUDY: This study explored the effects of HSYA on autophagy and the NLRP3 inflammasome in mice with TBI and the related mechanisms. MATERIALS AND METHODS: Mice were subjected to TBI and treated with or without HSYA. Neurological severity scoring, LDH assays and apoptosis detection were first performed to assess the effects of HSYA in mice with TBI. RNA-seq was then conducted to explore the mechanisms that contributed to HSYA-mediated neuroprotection. ELISA, western blotting, and immunofluorescence were performed to further investigate the mechanisms of neuroinflammation and autophagy. Moreover, 3-methyladenine (3-MA), an autophagy inhibitor, was applied to determine the connection between autophagy and the NLRP3 inflammasome. RESULTS: HSYA significantly decreased the neurological severity score, serum LDH levels and apoptosis in mice with TBI. A total of 921 differentially expressed genes were identified in the cortices of HSYA-treated mice with TBI and were significantly enriched in the inflammatory response and autophagy. Furthermore, HSYA treatment markedly reduced inflammatory cytokine levels and astrocyte activation. Importantly, HSYA suppressed neuronal NLRP3 inflammasome activation, as indicated by decreased levels of NLRP3, ASC and cleaved caspase-1 and a reduced NLRP3+ neuron number. It increased autophagy and ameliorated autophagic flux dysfunction, as evidenced by increased LC3 II/LC3 I levels and decreased P62 levels. The effects of HSYA on the NLRP3 inflammasome were abolished by 3-MA. Mechanistically, HSYA may enhance autophagy through AMPK/mTOR signalling. CONCLUSION: HSYA enhanced neuronal autophagy by triggering the AMPK/mTOR signalling pathway, leading to inhibition of the NLRP3 inflammasome to improve neurological recovery after TBI.

Mesenchymal stem cells protect against TBI-induced pyroptosis in vivo and in vitro through TSG-6.

BACKGROUND: Pyroptosis, especially microglial pyroptosis, may play an important role in central nervous system pathologies, including traumatic brain injury (TBI). Transplantation of mesenchymal stem cells (MSCs), such as human umbilical cord MSCs (hUMSCs), has been a focus of brain injury treatment. Recently, MSCs have been found to play a role in many diseases by regulating the pyroptosis pathway. However, the effect of MSC transplantation on pyroptosis following TBI remains unknown. Tumor necrosis factor α stimulated gene 6/protein (TSG-6), a potent anti-inflammatory factor expressed in many cell types including MSCs, plays an anti-inflammatory role in many diseases; however, the effect of TSG-6 secreted by MSCs on pyroptosis remains unclear. METHODS: Mice were subjected to controlled cortical impact injury in vivo. To assess the time course of pyroptosis after TBI, brains of TBI mice were collected at different time points. To study the effect of TSG-6 secreted by hUMSCs in regulating pyroptosis, normal hUMSCs, sh-TSG-6 hUMSCs, or different concentrations of rmTSG-6 were injected intracerebroventricularly into mice 4 h after TBI. Neurological deficits, double immunofluorescence staining, presence of inflammatory factors, cell apoptosis, and pyroptosis were assessed. In vitro, we investigated the anti-pyroptosis effects of hUMSCs and TSG-6 in a lipopolysaccharide/ATP-induced BV2 microglial pyroptosis model. RESULTS: In TBI mice, the co-localization of Iba-1 (marking microglia/macrophages) with NLRP3/Caspase-1 p20/GSDMD was distinctly observed at 48 h. In vivo, hUMSC transplantation or treatment with rmTSG-6 in TBI mice significantly improved neurological deficits, reduced inflammatory cytokine expression, and inhibited both NLRP3/Caspase-1 p20/GSDMD expression and microglial pyroptosis in the cerebral cortices of TBI mice. However, the therapeutic effect of hUMSCs on TBI mice was reduced by the inhibition of TSG-6 expression in hUMSCs. In vitro, lipopolysaccharide/ATP-induced BV2 microglial pyroptosis was inhibited by co-culture with hUMSCs or with rmTSG-6. However, the inhibitory effect of hUMSCs on BV2 microglial pyroptosis was significantly reduced by TSG-6-shRNA transfection. CONCLUSION: In TBI mice, microglial pyroptosis was observed. Both in vivo and in vitro, hUMSCs inhibited pyroptosis, particularly microglial pyroptosis, by regulating the NLRP3/Caspase-1/GSDMD signaling pathway via TSG-6. Video Abstract.

Revealing the potential mechanism of Astragalus membranaceus improving prognosis of hepatocellular carcinoma by combining transcriptomics and network pharmacology.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death. Traditional Chinese medicine (TCM) has special advantages in relieving HCC, while Astragalus membranaceus is commonly used in TCM treatment. However, its underlying mechanisms for treatment of HCC are unclear. METHODS: Differentially expressed genes (DEGs) of Astragalus membranaceus treatment in HepG2 cells were identified, and Astragalus membranaceus-gene network was constructed. The hub genes were then obtained via protein-protein interaction (PPI) analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA) were subsequently performed. Furthermore, prognosis genes related to HCC from The Cancer Genome Atlas Program (TCGA) was identified to explore the correlation between Astragalus membranaceus treatment and prognosis of HCC. Finally, Astragalus membranaceus-component-target network was established through SymMap. RESULTS: Twenty five DEGs (15 up-regulated and 10 down-regulated) of Astragalus membranaceus treatment in HepG2 cells were identified. Among the 25 genes, MT1F, MT1G, MT1X and HMOX1 may play essential roles. Astragalus membranaceus mainly affects the Mineral absorption pathway in HCC. A total of 256 genes (p < 0.01) related to prognosis of HCC were identified, and MT1G is a common gene between prognosis genes and DEGs. Furthermore, Astragalus membranaceus may directly down-regulate MT1G through daidzein to promote ferroptosis of HCC cells and improve prognosis for HCC. CONCLUSION: Our study provided new understandings of the pharmacological mechanisms by which Astragalus membranaceus improves the prognosis of HCC, and showed that the combination of transcriptomics and network pharmacology is helpful to explore mechanisms of TCM and traditional medicines from other nations.

Delta Opioid Receptor Activation with Delta Opioid Peptide [d-Ala2, d-Leu5] Enkephalin Contributes to Synaptic Improvement in Rat Hippocampus against Global Ischemia.

Global cerebral ischemia induced by cardiac arrest usually leads to poor neurological outcomes. Numerous studies have focused on ways to prevent ischemic damage in the brain, however clinical therapies are still limited. Our previous studies revealed that delta opioid receptor (DOR) activation with [d-Ala2, d-Leu5] enkephalin (DADLE), a DOR agonist, not only significantly promotes neuronal survival on day 3, but also improves spatial memory deficits on days 5-9 after ischemia. However, the neurological mechanism underlying DADLE-induced cognitive recovery remains unclear. This study first examined the changes in neuronal survival in the CA1 region at the advanced time point (day 7) after ischemia/reperfusion (I/R) injury and found a significant amelioration of damaged CA1 neurons in the rats treated with DADLE (2.5 nmol) when administered at the onset of reperfusion. The structure and function of CA1 neurons on days 3 and 7 post-ischemia showed significant improvements in both the density of the injured dendritic spines and the basic transmission of the impaired CA3-CA1 synapses following DADLE treatment. The molecular changes involved in DADLE-mediated synaptic modulation on days 3 and 7 post-ischemia implied the time-related differential regulation of PKCα-MARCKS on the dendritic spine structure and of BDNF- ERK1/2-synapsin I on synaptic function, in response to ischemic/reperfusion injury as well as to DADLE treatment. Importantly, all the beneficial effects of DADLE on ischemia-induced cellular, synaptic, and molecular deficits were eliminated by the DOR inhibitor naltrindole (2.5 nmol). Taken together, this study suggested that DOR activation-induced protective signaling pathways of PKCα-MARCKS involved in the synaptic morphology and BDNF-ERK-synapsin I in synaptic transmission may be engaged in the cognitive recovery in rats suffering from advanced cerebral ischemia.

Delta opioid peptide [d-Ala2, d-Leu5] enkephalin confers neuroprotection by activating delta opioid receptor-AMPK-autophagy axis against global ischemia.

BACKGROUND: Ischemic stroke poses a severe risk to human health worldwide, and currently, clinical therapies for the disease are limited. Delta opioid receptor (DOR)-mediated neuroprotective effects against ischemia have attracted increasing attention in recent years. Our previous studies revealed that DOR activation by [d-Ala2, d-Leu5] enkephalin (DADLE), a selective DOR agonist, can promote hippocampal neuronal survival on day 3 after ischemia. However, the specific molecular and cellular mechanisms underlying the DOR-induced improvements in ischemic neuronal survival remain unclear. RESULTS: We first detected the cytoprotective effects of DADLE in an oxygen-glucose deprivation/reperfusion (OGD/R) model and observed increased viability of OGD/R SH-SY5Y neuronal cells. We also evaluated changes in the DOR level following ischemia/reperfusion (I/R) injury and DADLE treatment and found that DADLE increased DOR levels after ischemia in vivo and vitro. The effects of DOR activation on postischemic autophagy were then investigated, and the results of the animal experiment showed that DOR activation by DADLE enhanced autophagy after ischemia, as indicated by elevated LC3 II/I levels and reduced P62 levels. Furthermore, the DOR-mediated protective effects on ischemic CA1 neurons were abolished by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, the results of the cell experiments revealed that DOR activation not only augmented autophagy after OGD/R injury but also alleviated autophagic flux dysfunction. The molecular pathway underlying DOR-mediated autophagy under ischemic conditions was subsequently studied, and the in vivo and vitro data showed that DOR activation elevated autophagy postischemia by triggering the AMPK/mTOR/ULK1 signaling pathway, while the addition of the AMPK inhibitor compound C eliminated the protective effects of DOR against I/R injury. CONCLUSION: DADLE-evoked DOR activation enhanced neuronal autophagy through activating the AMPK/mTOR/ULK1 signaling pathway to improve neuronal survival and exert neuroprotective effects against ischemia.

Hydroxysafflor Yellow A Confers Neuroprotection from Focal Cerebral Ischemia by Modulating the Crosstalk Between JAK2/STAT3 and SOCS3 Signaling Pathways.

Natural bioactive compounds have increasingly proved to be promising in evidence- or target-directed treatment or modification of a spectrum of diseases including cerebral ischemic stroke. Hydroxysafflor yellow A (HSYA), a major active component of the safflower plant, has drawn more interests in recent year for its multiple pharmacological actions in the treatment of cerebrovascular and cardiovascular diseases. Although the Janus kinase signaling, such as JAK2/STAT3 pathway, has been implicated in the modulation of the disease, the inhibition or activation of the pathway that contributed to the neuronal prevention from ischemic damages remains controversial. In this study, a series of experiments were performed to examine the dose- and therapeutic time window-related pharmacological efficacies of HSYA with emphasis on the HSYA-modulated interaction of JAK2/STAT3 and SOCS3 signaling in the MCAO rats. We found that HSYA treatment significantly rescued the neurological and functional deficits in a dose-dependent manner in the MCAO rats within 3 h after ischemia. HSYA treatment with a dosage of 8 mg/kg or higher markedly downregulated the expression of the JAK2-mediated signaling that was activated in response to ischemic insult, while it also promoted the expression of SOCS3 coordinately. In the subsequent experiments with the use of the JAK2 inhibitor WP1066, we found that the treatment of WP1066 alone or combination of WP1066/HSYA all exhibited inhibitory effects on JAK2-mediated signaling, while there was no influence on the SOCS3 activity of corresponding efficacious data in the MCAO rats, suggesting that excessive activation of JAK2/STAT3 might be necessary for HSYA to provoke SOCS3-negative feedback signaling. Taking together, our study demonstrates that HSYA might modulate the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways that eventually contributed to its therapeutic roles against cerebral ischemic stroke.

The coordinating role of the human norovirus minor capsid protein VP2 is essential to functional change and nuclear localization of the major capsid protein VP1.

Global outbreaks of norovirus (NOV) gastroenteritis are associated with the most prevalent genotype, GII.4. Mutations in the protruding domain 2 (P2 domain) of the norovirus major capsid protein (VP1) result in the emergence of various NOV variants, however, it is unclear whether the minor capsid protein (VP2) also affects the generation of VP1 variants. In this study, using a human 293T expression system, we investigated the interactions of VP1 and VP2 of three GII.4 strains, focusing on the changes in expression and cellular localization. We found that co-transfection with VP1 and VP2 leads to a significant increase in expression of both proteins compared to that in cells transfected with VP1 or VP2 alone. In contrast to VP1 expressed in the absence of VP2, which was dispersed throughout the cytosol, VP2 expressed in the absence of VP1 was found to be located in the nucleus. This could be attributed to a predicted specific nuclear localization signal found in this gene. When both proteins were expressed, VP1 was found together with VP2 in the nucleus. These results thus suggest that the VP2 of GII.4 NOVs affects the function and cellular location of VP1 and that, with the cooperation of VP2, VP1 could play a critical role in affecting cell functions by impairing the downstream transcriptional signaling and chromatin remodeling in the cell nuclei.

Curcumin synergizes with 5-fluorouracil by impairing AMPK/ULK1-dependent autophagy, AKT activity and enhancing apoptosis in colon cancer cells with tumor growth inhibition in xenograft mice.

BACKGROUND: Chemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells' fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu's cytotoxicity in vitro and in vivo. METHODS: Cell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice. RESULTS: Curcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells. CONCLUSION: Pre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.