Exploring the effect of LncRNA DANCR to regulate the Keap1-Nrf2/ARE pathway on oxidative stress in rheumatoid arthritis.

Aberrant expression of long noncoding RNAs (LncRNAs) can regulate oxidative stress in rheumatoid arthritis (RA). This study focused on investigating the effects of LncRNA differentiation antagonizing nonprotein coding RNA (DANCR) regulation of Keap1-Nrf2/ARE pathway on inflammation and oxidative stress in RA. The levels of LncRNA DANCR/miR-486-3p/Keap1 in peripheral blood of 30 RA groups and 30 normal subjects were examined, and the association of LncRNA DANCR with inflammatory indicators of RA was investigated. We stimulated fibroblast-like synoviocytes (FLS) from RA patients with tumor necrosis factor α and subsequently performed in vitro cellular assays to construct overexpression plasmids and small interfering RNAs of LncRNA DANCR to investigate the relationship between LncRNA DANCR and FLSs viability and migration in RA, as well as the effects on cellular oxidative stress factors and Keap1-Nrf2/ARE pathway; molecular biology analysis was used to predict microRNAs that can bind LncRNA DANCR, and luciferase verified the binding sites of LncRNA DANCR with Keap1 and miR-486-3p; to further refine the gene and protein expression results, we used reverse transcription-quantitative polymerase chain reaction and immunoblotting assays. In both groups of peripheral blood mononuclear cells, the expression levels of LncRNA DANCR and Keap1 messenger RNA were higher in the RA group than in the normal control group, and the opposite was true for miR-486-3p; LncRNA DANCR was positively correlated with total antioxidant capacity (TAOC), IL6, IL17, malondialdehyde (MDA), but not with IL11, rheumatoid factor, cyclic citrullinated peptide, superoxide dismutase (SOD), with 28-joint disease activity score, reactive oxygen species, C-reactive protein, and erythrocyte sedimentation rate were negatively correlated; overexpression of LncRNA DANCR stimulated the Keap1-Nrf2/ARE pathway, decreased the expression of IL10, SOD, TAOC, and increased the expression levels of MDA, IL11, IL-17, PD-L1, and silencing of LncRNA DANCR was the opposite, but knockdown of miR-486-3p or overexpression of keap1 reversed the expression of the above-mentioned inflammatory and oxidative factors. In addition, pcDNA-DANCR clearly showed stronger cell invasion and migration ability and exacerbated its inflammatory response, which may be related to the regulatory role of miR-486-3p and Keap1-Nrf2/ARE signaling pathway, and we verified their targeting relationship using dual luciferase, showing that DANCR could regulate Keap1-Nrf2/ARE through miR-486-3p modulates the Keap1-Nrf2/ARE pathway and affects inflammatory and oxidative responses in RA patients. The low-expressed LncRNA DANCR may regulate the Keap1-Nrf2/ARE pathway and suppress the inflammatory and oxidative responses in RA patients.

Cai S ，Sun Y ，Wang Y ，Lin Z ... -  《Immunity Inflammation and Disease》

CircSpna2 attenuates cuproptosis by mediating ubiquitin ligase Keap1 to regulate the Nrf2-Atp7b signalling axis in depression after traumatic brain injury in a mouse model.

Depression is a common but often overlooked consequence in individuals with post-traumatic brain injury (TBI). Circular RNAs (circRNAs) play essential roles in the nervous system, yet their involvement in the cell death mechanism known as cuproptosis and in TBI-related depression remains unclear. This study aimed to investigate the role of circRNA, specifically circSpna2, in the regulation of cuproptosis and its association with depression in TBI patients. RNA sequencing (RNA-Seq) was used to assess the differential expression of circRNAs. Depression was evaluated using subjective and objective rating scales, and circSpna2 expression levels in plasma were measured. Further functional experiments were conducted in TBI mouse models, including knockdown and overexpression of circSpna2, to explore its impact on the Keap1-Nrf2-Atp7b pathway and cuproptosis. TBI patients exhibited decreased levels of circSpna2, which correlated with depression (p < 0.0001). Knocking down circSpna2 in TBI mice aggravated depression-like symptoms (p < 0.0001). Mechanistically, circSpna2 was found to bind ubiquitin ligase Keap1, modulating the Nrf2-Atp7b signaling pathway and influencing cuproptosis (docking score: -331.88). Overexpression of circSpna2 alleviated cuproptosis after TBI through the Keap1/Nrf2/Atp7b axis. CircSpna2 plays a regulatory role in cuproptosis and may serve as a novel biomarker and therapeutic target for depression following TBI. Enhancing circSpna2 expression could mitigate depression after TBI by modulating the Keap1/Nrf2/Atp7b pathway. This study explores the role of circSpna2 in depression following traumatic brain injury (TBI). It was found that circSpna2 is significantly downregulated in TBI patients, and its expression levels correlate with depressive symptoms. In TBI mouse models, overexpression of circSpna2 alleviated depression-like behaviours, while its knockdown exacerbated these symptoms, suggesting its potential as both a biomarker and a therapeutic target for post-TBI depression. Mechanistically, circSpna2 regulates the Nrf2-Atp7b signalling pathway by binding to the DGR domain of Keap1, which prevents Nrf2 ubiquitination and enhances Nrf2 activity. This in turn promotes the transcription of Atp7b, a copper transport protein, helping to maintain copper homeostasis and mitigate copper-induced oxidative stress, a key driver of cell death (cuproptosis). The overexpression of circSpna2 also improved mitochondrial function and synaptic integrity, which are typically impaired by copper dysregulation. These findings highlight the therapeutic potential of circSpna2 in managing TBI-related depression through the regulation of oxidative stress and copper homeostasis.

Du M ，Fu J ，Zhang J ，Zhu Z ，Huang X ，Tan W ，Liu L ，Huang Z ，Liu X ，Tan Q ，Liao Z ，Cheng Y ... -  《Clinical and Translational Medicine》

SUMOylation Inhibitors Exert a Protective Effect on Oxidative Damage in Retinal Pigment Epithelial Cells Through the Keap1/Nrf2/ARE Signaling Pathway.

To investigate the effect of the SUMOylation inhibitor TAK981 on hydrogen peroxide (H2O2)-induced oxidative damage in human retinal pigment epithelial cells (ARPE-19) and its regulatory mechanism. An oxidative damage model of ARPE-19 cells induced by H2O2 was established, and 1, 2, and 5 µM TAK981 solutions were administered for intervention respectively. Normal cells were used as the control group. The viability of the cells in each group was detected by the methyl thiazolyl tetrazolium (MTT) method. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in each group of cells were detected by biochemical methods. The levels of IL-1β and TNF-α produced by each group of cells were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of Nrf2, HO-1, NQO-1, Keap1, and Sumo1 in each group of cells were detected by Western blotting. In addition, 2 µM TAK981 and 2 µM TAK981 combined with 10 µM ML385 (an Nrf2 inhibitor) were administered to H2O2-induced ARPE-19 cells, and the levels of SOD and MDA, IL-1β and TNF-αwere detected again. The viability of the ARPE-19 cells decreased with increasing H2O2 concentration (F=19.158, P<0.001). H2O2 treatment at 350 µM was the concentration at which the cells essentially reached half inhibition (IC50), and the cell oxidative damage model was successfully established. After intervention with TAK981, cell survival increased significantly (F=0.098, P<0.001). The differences between the 2 µM and 5 µM TAK981 groups and the model group were statistically significant (all P<0.01). Compared with those in the normal group, the MDA content in the model group increased, the SOD activity decreased, and the release levels of IL-1β and TNF-α increased (all P<0.01). Compared with those in the model group, the MDA content in the TAK981 group decreased, the SOD activity increased, and the release levels of IL-1β and TNF-α decreased. The differences between the 2 µM and 5 µM TAK981 groups were statistically significant (P<0.05). Compared with those in the normal group, the protein expression levels of Nrf2, HO-1 and NQO-1 in the model group were greater, whereas the protein expression levels of Keap1 and Sumo1 were lower (all P<0.05). Compared with those in the model group, the protein expression levels of Nrf2, HO-1 and NQO-1 in the TAK981-treated group continued to increase, whereas the protein expression levels of Keap1 and Sumo1 continued to decrease. The differences in the 5 µM TAK981 group were statistically significant (P<0.05). In addition, after the combined intervention of TAK981 and ML385 on H2O2-induced cells, compared with the TAK981-only intervention on H2O2-induced cells, the cell viability increased, the MDA content increased, the SOD activity decreased, and the IL-1β and TNF-α release levels increased. The differences were statistically significant (P<0.05). The SUMOylation inhibitor TAK981 activates the Keap1/Nrf2/ARE signaling pathway, enhances the activity of antioxidant enzymes, and reduces the production of oxidative stress products and inflammatory factors, thereby exerting a protective effect on H2O2-induced oxidative damage in ARPE-19 cells. Therefore, it is suggested that intervention in SUMO regulation can be used as a new therapeutic target in the AMD disease model, in order to delay the development of AMD by reducing the oxidative damage of RPE.

Liang Y ，Jia X ，Zheng F ，Wang Y ，Fan Y ，Zhang H ，Dang Z ，Wang L ... -  《-》

The Keap1-Nrf2/ARE signaling pathway regulates redox balance and apoptosis in the small yellow croaker (Larimichthys polyactis) under hypoxic stress.

Hypoxic stress can result in redox imbalance and apoptosis in teleostean fishes; however, the precise molecular mechanisms underlying this process, including its regulation by the key signaling pathway Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 related factor (Nrf2)/antioxidant response element (ARE), remain elusive. Therefore, in this study, we chose the Keap1-Nrf2/ARE signaling pathway as the entry point and a combination of in vivo (target organ liver) and in vitro (small yellow croaker fry [SYCF] cell line) experiments to investigate the molecular mechanism by which Larimichthys polyactis (L. polyactis) adapts to hypoxic stress by regulating redox balance and apoptosis. As our previous study found that hypoxic stress could lead to redox imbalance and apoptosis in L. polyactis. First, we observed significant alterations in the expression of key genes Lpkeap1, Lpnrf2, Lpho-1, and Lpnqo1 within the Keap1-Nrf2/ARE signaling pathway in both liver tissue and SYCF cells of L. polyactis under hypoxic stress, indicating activation of this pathway in response to hypoxia. Subsequently, we elucidated the mechanism by which hypoxia activates this pathway, that is, hypoxia weakened the interaction between LpNrf2 and LpKeap1, promoting the nuclear translocation of LpNrf2 and enhancing its binding activity to ARE, thereby activating the transcription of target genes. Furthermore, we found that significant changes occurred in the redox balance and apoptosis-related indicators after LpNrf2 knockdown and exposure to hypoxic stress for 24 h in SYCF cells, indicating that this pathway can regulate redox balance and apoptosis regulation under hypoxic stress in L. polyactis. Additionally, we used DNA affinity purification sequencing (DAP-seq) to identify the ARE sequence (ATGATTTAGC) that bound to LpNrf2 and its target genes. Finally, we conducted a combined analysis of DAP-seq and RNA-seq to identify six key target genes involved in the process: haeme oxygenase-1 (Ho-1), B-cell lymphoma-2 (Bcl2), pituitary homeobox 2 isoform X1 (Pitx2), aquaporin-4 isoform X1 (Aqp4), stress-induced phosphoprotein 1-like isoform X1 (Stip1), and guanine nucleotide-binding protein G (i) subunit alpha-2-like (Gnai2). In summary, hypoxic stress induced by weakening LpNrf2 and LpKeap1 interaction promoted LpNrf2 nuclear entry and enhanced its binding activity to ARE, thereby activating the transcription of multiple target genes to regulate redox balance and apoptosis. The research results not only help deepen our understanding of the adaptive mechanisms of L. polyactis and even marine fish to hypoxic stress and its survival strategies but also provide new ideas and potential targets for breeding new hypoxia-tolerant strains.

Wang J ，Wang L ，Liu Y ，Hou C ，Xie Q ，Tang D ，Liu F ，Lou B ，Zhu J ... -  《-》

Upregulated let-7 expression in the follicular fluid of patients with endometriomas leads to dysfunction of granulosa cells through targeting of IGF1R.

What molecular mechanisms underlie the decline in ovarian reserve as the number and quality of oocytes decrease in patients with ovarian endometriomas (OEM)? Elevated expression of the let-7 micro(mi)RNAs in the follicular microenvironment of OEM-affected ovaries targets the expression of type 1 insulin-like growth factor receptor (IGF1R) in granulosa cell (GC) and disrupts their proliferation, steroid hormone secretion levels, adenosine triphosphate (ATP) energy metabolism, and reactive oxygen species (ROS) oxidative stress levels. Patients with OEM exhibit diminished ovarian reserve, characterized by reduced oocyte quantity and quality. Fibrotic changes in the ovarian tissue surrounding the OEM create a disruptive microenvironment for follicular growth and development. This is a cross-sectional study aimed to elucidate the molecular mechanisms underlying the impact of OEM on follicular development. Initially, miRNA expression profiles in follicular fluid (FF) samples were sequenced from patients with infertility related to OEM (N = 3) and male factor (MF) infertility (N = 3), with the latter serving as the control group. Differentially expressed miRNAs were validated in additional samples from each group (N = 55 in OEM group and N = 45 in MF group) to confirm candidate miRNAs. The study also investigated indicators associated with GCs dysfunction in vitro on rat GCs. Subsequently, rat models of OEM were established through endometrial allogeneic transplantation, and fertility experiments were conducted to assess the let-7/IGF1R axis response to OEM in vivo. Patient samples were collected between May 2018 and April 2019, and the mechanistic study was conducted over the subsequent three years. FF and GC samples were obtained from infertile patients undergoing IVF treatment for OEM and MF related infertility. miRNA expression profiles in FF samples were analyzed using second-generation high-throughput sequencing technology, and candidate miRNAs were validated through quantitative PCR (qPCR). In the in vitro experiments conducted with rat GCs, cell proliferation was assessed using the CCK-8 assay, while steroid hormone concentrations were measured using chemiluminescence. ATP content was determined with an ATP assay kit, and levels of ROS were quantified using flow cytometry. A dual luciferase reporter gene assay was employed to identify the target gene of let-7 based on the construction of a IGF1R reporter gene plasmid using 293T cells. Western blotting was utilized to evaluate the expression of IGF1R in GCs, as well as its downstream proteins, and changes in signaling pathways following let-7 agomir/antagomir transfection and/or Igf1r silencing. In the in vivo OEM rat models, alterations in ovarian structure and cyst morphology were observed using hematoxylin and eosin staining. The expressions of let-7 and Igf1r in GCs were evaluated through qPCR, while variations in IGF1R expression were investigated with immunohistochemistry. The cohort of patients with ovarian OEM in this study exhibited significantly decreased antral follicle counts, oocyte retrieval numbers, and normal fertilization rates compared to the control group with MF. The expression of the let-7 miRNA family was markedly upregulated in the FF and GCs of OEM patients. Transfection of rat GCs with let-7 agonists diminished the functions of GCs, including disrupted cell proliferation, mitochondrial oxidative phosphorylation, and steroid hormone secretion, while transfection of rat GCs with let-7 antagonists caused the opposite effects. Luciferase reporter gene experiments confirmed that let-7 complementarily bound to the 3'-untranslated regions of IGF1R. Stimulation of let-7 expression in rat GCs led to a significant decrease in IGF1R expression, while inhibition of let-7 increased IGF1R expression. The expression of IGF1R in the GCs of OEM patients was also significantly reduced compared to MF patients. Silencing of Igf1r led to the dysfunction of GCs, similar to the effects of let-7 agonization, as demonstrated by the downregulation of key proteins involved in cell proliferation (CCND2 and CCND3) and oestradiol synthesis, as well as an increase in progesterone synthesis (StAR), while implicating the PI3K-Akt and MAPK signaling pathways. The antagonistic effect of let-7 on GCs was ineffective when Igf1r was silenced. Conversely, the agonistic effect of let-7 on GCs could be reversed by stimulation with the IGF1R ligand IGF-1. These findings suggested that let-7 regulated the proliferation, differentiation, and ATP synthesis of GCs through targeting IGF1R. The OEM rat model demonstrated alterations in ovarian morphology and structure, along with reduced fertility. Let-7 expression was significantly upregulated in GCs of OEM rats compared to normal rats, while Igf1r and IGF1R expression in pre-ovulatory follicular GCs were notably downregulated, supporting the notion that elevated let-7 expression in the follicular microenvironment of OEM inhibited IGF1R, leading to abnormal GC function and impacting fertility at the molecular level. N/A. The synthesis and secretion mechanisms of steroid hormones are intricate and complex. Some enzymes that regulate oestrogen synthesis also play a role in progesterone synthesis. Moreover, certain receptors can respond to multiple hormone signals. Therefore, in this study, the expression patterns of key enzymes such as CYP17A, CYP11A1, HSD3B2, StAR, and receptors including AR, LHCGR, FSHR, ESR2, might be influenced by various factors and might not demonstrate complete consistency. Future research will concentrate on investigating the potential impact of ovarian stromal cells on the external microenvironment of follicle growth. Additionally, screening for small molecule drugs that target let-7 and IGF1R actions can be conducted to intervene and modify the ovarian microenvironment, ultimately enhancing ovarian function. This study received funding from the National Natural Science Foundation of China (grant number 82301851 to L.B.S., grant numbers U23A20403 and U20A20349 to S.Y.Z., and grant number 82371637 to Y.D.D.) and the Natural Science Foundation of Zhejiang Province (grant LTGY23H040010 to F.Z.). The authors have no conflicts of interest to declare.

Shi L ，Ying H ，Dai Y ，Rong Y ，Chen J ，Zhou F ，Wang S ，Xu S ，Tong X ，Zhang S ... -  《-》