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During their lifespan, dendritic cells (DCs) are exposed to different pO 2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO 2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.

Objectives Evidences demonstrate that sorafenib alleviates liver fibrosis via inhibiting HSC activation and ECM accumulation. The underlying mechanism remains unclear. Ferroptosis, a novel programmed cell death, regulates diverse physiological/pathological processes. In this study, we aim to investigate the functional role of HSC ferroptosis in the anti‐fibrotic effect of sorafenib. Materials and Methods The effects of sorafenib on HSC ferroptosis and ECM expression were assessed in mouse model of liver fibrosis induced by CCl4. In vitro, Fer‐1 and DFO were used to block ferroptosis and then explored the anti‐fibrotic effect of sorafenib by detecting α‐SMA, COL1α1 and fibronectin proteins. Finally, HIF‐1α siRNA, plasmid and stabilizers were applied to assess related signalling pathway. Results Sorafenib attenuated liver injury and ECM accumulation in CCl4‐induced fibrotic livers, accompanied by reduction of SLC7A11 and GPX4 proteins. In sorafenib‐treated HSC‐T6 cells, ferroptotic events (depletion of SLC7A11, GPX4 and GSH; accumulation iron, ROS and MDA) were discovered. Intriguingly, these ferroptotic events were not appeared in hepatocytes or macrophages. Sorafenib‐elicited HSC ferroptosis and ECM reduction were abrogated by Fer‐1 and DFO. Additionally, both HIF‐1α and SLC7A11 proteins were reduced in sorafenib‐treated HSC‐T6 cells. SLC7A11 was positively regulated by HIF‐1α, inactivation of HIF‐1α/SLC7A11 pathway was required for sorafenib‐induced HSC ferroptosis, and elevation of HIF‐1α could inhibit ferroptosis, ultimately limited the anti‐fibrotic effect. Conclusions Sorafenib triggers HSC ferroptosis via HIF‐1α/SLC7A11 signalling, which in turn attenuates liver injury and fibrosis. Sorafenib triggers hepatic stellate cell ferroptosis by inhibiting the HIF‐1α/SLC7A11 pathway to attenuate liver fibrosis. Treatment with sorafenib induces a decrease of HIF‐1α, which in turn reduces SLC7A11 expression in HSCs. Then leads to GPX4, GSH depletion and ROS excess, and ultimately induces HSC ferroptosis and ECM reduction.

Ferroptosis is an iron‐dependent cell death that has been found to aggravate the progression of osteoarthritis (OA) and gut microbiota‐ OA axis refers to the bidirectional information network between the gut microbiota and OA, which may provide a new way to protect the OA. However, the role of gut microbiota‐derived metabolites in ferroptosis‐relative osteoarthritis remains unclear. The objective of this study was to analyze the protective effect of gut microbiota and its metabolite capsiate (CAT) on ferroptosis‐relative osteoarthritis in vivo and in vitro experiments. From June 2021 to February 2022, 78 patients were evaluated retrospectively and divided into two groups: The health group (n = 39) and the OA group (n = 40). Iron and oxidative stress indicators were determined in peripheral blood samples. And then in vivo and in vitro experiments, a surgically destabilized medial meniscus (DMM) mice model was established and treated with CAT or Ferric Inhibitor‐1 (Fer‐1). Solute Carrier Family 2 Member 1 (SLC2A1) short hairpin RNA (shRNA) was utilized to inhibit SLC2A1 expression. Serum iron was increased significantly but total iron binding capacity was decreased significantly in OA patients than healthy people (p < 0.0001). The least absolute shrinkage and selection operator clinical prediction model suggested that serum iron, total iron binding capacity, transferrin, and superoxide dismutase were all independent predictors of OA (p < 0.001). Bioinformatics results suggested that SLC2A1, Metastasis‐Associated Lung Adenocarcinoma Transcript 1 (MALAT1), and HIF‐1α (Hypoxia Inducible Factor 1 Alpha)‐related oxidative stress signaling pathways play an important role in iron homeostasis and OA. In addition, gut microbiota 16s RNA sequencing and untargeted metabolomics were used to find that gut microbiota metabolites CAT in mice with osteoarthritis were negatively correlated with Osteoarthritis Research Society International (OARSI) scores for chondrogenic degeneration (p = 0.0017). Moreover, CAT reduced ferroptosis‐dependent osteoarthritis in vivo and in vitro. However, the protective effect of CAT against ferroptosis‐dependent osteoarthritis could be eliminated by silencing SLC2A1. SLC2A1 was upregulated but reduced the SLC2A1 and HIF‐1α levels in the DMM group. HIF‐1α, MALAT1, and apoptosis levels were increased after SLC2A1 knockout in chondrocyte cells (p = 0.0017). Finally, downregulation of SLC2A1 expression by Adeno‐associated Virus (AAV) ‐SLC2A1 shRNA improves osteoarthritis in vivo. Our findings indicated that CAT inhibited HIF‐1a expression and reduced ferroptosis‐relative osteoarthritis progression by activating SLC2A1. Our findings indicated that CAT inhibited HIF‐1a expression and reduced ferroptosis‐dependent osteoarthritis progression by activating SLC2A1.

Abnormal metabolism of tumour cells is closely related to the occurrence and development of breast cancer, during which the expression of NF‐E2‐related factor 2 (Nrf2) is of great significance. Metastatic breast cancer is one of the most common causes of cancer death worldwide; however, the molecular mechanism underlying breast cancer metastasis remains unknown. In this study, we found that the overexpression of Nrf2 promoted proliferation and migration of breast cancers cells. Inhibition of Nrf2 and overexpression of Kelch‐like ECH‐associated protein 1 (Keap1) reduced the expression of glucose‐6‐phosphate dehydrogenase (G6PD) and transketolase of pentose phosphate pathway, and overexpression of Nrf2 and knockdown of Keap1 had opposite effects. Our results further showed that the overexpression of Nrf2 promoted the expression of G6PD and Hypoxia‐inducing factor 1α (HIF‐1α) in MCF‐7 and MDA‐MB‐231 cells. Overexpression of Nrf2 up‐regulated the expression of Notch1 via G6PD/HIF‐1α pathway. Notch signalling pathway affected the proliferation of breast cancer by affecting its downstream gene HES‐1, and regulated the migration of breast cancer cells by affecting the expression of EMT pathway. The results suggest that Nrf2 is a potential molecular target for the treatment of breast cancer and targeting Notch1 signalling pathway may provide a promising strategy for the treatment of Nrf2‐driven breast cancer metastasis.

After skin injury, wound repair involves a complex process in which angiogenesis plays a crucial role. Previous research has indicated that fucoidan may aid in wound healing; we therefore hypothesised that fucoidan may speed up the process by promoting angiogenesis. In this study, we investigated the potential molecular mechanism underlying fucoidan's ability to accelerate wound healing by promoting angiogenesis. Using a full‐cut wound model, we observed that fucoidan significantly intensified wound closure and promoted granulation formation and collagen deposition. Immunofluorescence staining revealed that fucoidan also promoted wound angiogenesis, specifically by accelerating the migration of new blood vessels to the middle area of the wound. Furthermore, fucoidan demonstrated the ability to enhance the proliferation of human umbilical vein endothelial cells (HUVECs) damaged by hydrogen peroxide (H2O2) and to improve the formation of endothelial tubes. Mechanistic studies revealed that fucoidan upregulated the protein levels of the AKT/Nrf2/HIF‐1α signalling pathway, which plays a crucial role in angiogenesis. This was further confirmed using the inhibitor LY294002, which reversed the promotion of endothelial tube formation by fucoidan. Overall, our findings suggest that fucoidan can promote angiogenesis via the AKT/Nrf2/HIF‐1α signalling pathway and accelerate wound healing.

The nucleus pulposus is in a hypoxic environment in the human body, and when intervertebral disc degeneration (IVDD) occurs, the hypoxic environment is disrupted. Nucleus pulposus cell (NPC) ferroptosis is one of the causes of IVDD. N6‐methyladenosine (m6A) and its reader protein YTHDF1 regulate cellular activities by affecting RNA metabolism. However, the regulation of ferroptosis in NPCs by m6A‐modified RNAs under hypoxic conditions has not been as well studied. In this study, through in vitro and in vivo experiments, we explored the underlying mechanism of HIF‐1α and YTHDF1 in regulating ferroptosis in NPCs. The results indicated that the overexpression of HIF‐1α or YTHDF1 suppressed NPC ferroptosis; conversely, the knockdown of HIF‐1α or YTHDF1 increased ferroptosis levels in NPCs. Luciferase reporter assays and chromatin immunoprecipitation demonstrated that HIF‐1α regulated YTHDF1 transcription by directly binding to its promoter region. Polysome profiling results showed that YTHDF1 promoted the translation of SLC7A11 and consequently the expression of the anti‐ferroptosis protein GPX4 by binding to m6A‐modified SLC7A11 mRNA. In conclusion, HIF‐1α‐induced YTHDF1 expression reduces NPC ferroptosis and delays IVDD by promoting SLC7A11 translation in a m6A‐dependent manner. HIF‐1α regulated YTHDF1 transcription by directly binding to its promoter region. YTHDF1 promoted the translation of SLC7A11 and consequently the expression of the anti‐ferroptosis protein GPX4 by binding to m6A‐modified SLC7A11 mRNA. HIF‐1α‐induced YTHDF1 expression reduces NPC ferroptosis and delays IVDD by promoting SLC7A11 translation in a m6A‐dependent manner.

Background As a rate‐limiting enzyme of glycolysis, pyruvate kinase muscle isozyme M2 (PKM2) participates in tumor metabolism and growth. The regulatory network of PKM2 in cancer is complex and has not been fully studied in bladder cancer. The 5‐methylcytidine (m5C) modification in PKM2 mRNA might participate in the pathogenesis of bladder cancer and need to be further clarified. This study aimed to investigate the biological function and regulatory mechanism of PKM2 in bladder cancer. Methods The expression of PKM2 and Aly/REF export factor (ALYREF) was measured by Western blotting, qRT‐PCR, and immunohistochemistry. The bioprocesses of bladder cancer cells were demonstrated by a series of experiments in vitro and in vivo. RNA immunoprecipitation, RNA‐sequencing, and dual‐luciferase reporter assays were conducted to explore the potential regulatory mechanisms of PKM2 in bladder cancer. Results In bladder cancer, we first demonstrated that ALYREF stabilized PKM2 mRNA and bound to its m5C sites in 3′‐untranslated regions. Overexpression of ALYREF promoted bladder cancer cell proliferation by PKM2‐mediated glycolysis. Furthermore, high expression of PKM2 and ALYREF predicted poor survival in bladder cancer patients. Finally, we found that hypoxia‐inducible factor‐1alpha (HIF‐1α) indirectly up‐regulated the expression of PKM2 by activating ALYREF in addition to activating its transcription directly. Conclusions The m5C modification in PKM2 mRNA in the HIF‐1α/ALYREF/PKM2 axis may promote the glucose metabolism of bladder cancer, providing a new promising therapeutic target for bladder cancer. Our data suggested that m5C may play a critical role in hypoxia/glycolysis network and targeting HIF‐1α/ALYREF/PKM2 signaling may be a promising therapeutic strategy to treat bladder cancer.

Objectives Exosomes, as important players in intercellular communication due to their ability to transfer certain molecules to target cells, are believed to take similar effects in promoting bone regeneration with their derived stem cells. Studies have suggested that umbilical cord mesenchymal stem cells (uMSCs) could promote angiogenesis. This study investigated whether exosomes derived from uMSCs (uMSC‐Exos) could enhance fracture healing as primary factors by promoting angiogenesis. Materials and Methods uMSCs were obtained to isolate uMSC‐Exos by ultrafiltration, with exosomes from human embryonic kidney 293 cells (HEK293) and phosphate‐buffered saline (PBS) being used as control groups. NanoSight, laser light scattering spectrometer, transmission electron microscopy and Western blotting were used to identify exosomes. Next, uMSC‐Exos combined with hydrogel were transplanted into the fracture site in a rat model of femoral fracture. Bone healing processes were monitored and evaluated by radiographic methods on days 7, 14, 21 and 31 after surgery; angiogenesis of the fracture sites was assessed by radiographic and histological strategies on post‐operative day 14. In vitro, the expression levels of osteogenesis‐ or angiogenesis‐related genes after being cultured with uMSC‐Exos were identified by qRT‐PCR. The internalization ability of exosomes was determined using the PKH67 assay. Cell cycle analysis, EdU incorporation and immunofluorescence staining, scratch wound assay and tube formation analysis were also used to determine the altered abilities of human umbilical vein endothelial cells (HUVECs) administered with uMSC‐Exos in proliferation, migration and angiogenesis. Finally, to further explore the underlying molecular mechanisms, specific RNA inhibitors or siRNAs were used, and the subsequent effects were observed. Results uMSC‐Exos had a diameter of approximately 100 nm, were spherical, meanwhile expressing CD9, CD63 and CD81. Transplantation of uMSC‐Exos markedly enhanced angiogenesis and bone healing processes in a rat model of femoral fracture. In vitro, other than enhancing osteogenic differentiation, uMSC‐Exos increased the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor‐1α (HIF‐1α). uMSC‐Exos were taken up by HUVECs and enhanced their proliferation, migration and tube formation. Finally, by using specific RNA inhibitors or siRNAs, it has been confirmed that HIF‐1α played an important role in the uMSC‐Exos‐induced VEGF expression, pro‐angiogenesis and enhanced fracture repair, which may be one of the underlying mechanisms. Conclusions These results revealed a novel role of exosomes in uMSC‐mediated therapy and suggested that implanted uMSC‐Exos may represent a crucial clinical strategy to accelerate fracture healing via the promotion of angiogenesis. HIF‐1α played an important role in this process. Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF‐1α‐mediated promotion of angiogenesis in a rat model.

The peritoneum, especially the omentum, is a common site for gastric cancer (GC) metastasis. Our aim was to expound the role and mechanisms of Piezo1 on GC omentum metastasis. A series of functional assays were performed to examine cell proliferation, clone formation, apoptosis, Ca2+ influx, mitochondrial membrane potential (MMP) and migration after overexpression or knockdown of Piezo1. A GC peritoneal implantation and metastasis model was conducted. After infection by si‐Piezo1, the number and growth of tumours were observed in abdominal cavity. Fibre and angiogenesis were tested in metastatic tumour tissues. Piezo1 had higher expression in GC tissues with omentum metastasis and metastatic lymph node tissues than in GC tissues among 110 patients. High Piezo1 expression was associated with lymph metastasis, TNM and distant metastasis. Overexpressed Piezo1 facilitated cell proliferation and suppressed cell apoptosis in GC cells. Moreover, Ca2+ influx was elevated after up‐regulation of Piezo1. Piezo1 promoted cell migration and Calpain1/2 expression via up‐regulation of HIF‐1α in GC cells. In vivo, Piezo1 knockdown significantly inhibited peritoneal metastasis of GC cells and blocked EMT process and angiogenesis. Our findings suggested that Piezo1 is a key component during GC omentum metastasis, which could be related to up‐regulation of HIF‐1α.

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.