Hypoxia-inducible Factor Signaling Research Articles

FG-4592 protected haematopoietic system from ionising radiation in mice.

Ionising radiation exposure can lead to acute haematopoietic radiation syndrome. Despite significant advancements in the field of radioprotection, no drugs with high efficacy and low toxicity have yet been approved by the Food and Drug Administration. FG-4592, as a proline hydroxylase inhibitor, may play an important role in radioprotection of the haematopoietic system. Mice were peritoneal injected with FG-4592 or normal saline. After irradiation, the survival time, body weight, peripheral blood cell and bone marrow cell (BMC) count, cell apoptosis, pathology were analysed and RNA-sequence technique (RNA-Seq) was conducted to explore the mechanism of FG-4592 in the haematopoietic system. Our results indicated that FG-4592 improved the survival rate and weight of irradiated mice and protected the spleen, thymus and bone marrow from IR-induced injury. The number of BMCs was increased and protected against IR-induced apoptosis. FG-4592 also promoted the recovery of the blood system and erythroid differentiation. The results of RNA-Seq and Western blot showed that the NF-κB signalling pathway and hypoxia-inducible factor-1 (HIF-1) signalling pathway were upregulated by FG-4592. Meanwhile, RT-PCR results showed that FG-4592 could promote inflammatory response significantly. FG-4592 exhibited radioprotective effects in the haematopoietic system by promoting inflammatory response and targeting the NF-κB, HIF signalling pathway.

Analysis of mechanism of core points in acupuncture and moxibustion treatment for epilepsy based on data mining.

To explore the mechanism of core points in acupuncture and moxibustion treatment for epilepsy by using data mining technique, so as to provide a reference for clinical practice and experimental research. The data comes from relevant documents collected from CNKI, Wanfang, SinoMed, VIP, PubMed, Embase, Cochrane Library, EBSCO, Web of Science databases. The selected acupoints were analyzed in descriptive statistics, high-frequency acupoints group and core acupoint prescription. Further, potential target mining, "core acupoint prescription-target-epilepsy" network construction, protein-protein interactions (PPI) network establishment and core target extraction, gene ontology (GO) and KEGG gene enrichment analysis of the core acupoint prescription were carried out to predict its anti-epileptic potential mechanism. A total of 122 acupoint prescriptions were included. The core acupoint prescriptions were Baihui (GV20), Hegu (LI4), Neiguan (PC6), Shuigou (GV26) and Taichong (LR3). 277 potential targets were identified, among which 134 were shared with epilepsy. The core targets were extracted by PPI network topology analysis, including signal transducer and activator of transcription 3, tumor necrosis factor (TNF), interleukin (IL)-6, protein kinase B1, c-Jun N-terminal kinase, brain-derived neurotrophic factor, tumor protein 53, vascular endothelial growth factor A, Caspase-3, epidermal growth factor receptor, etc. The main anti-epileptic pathways of the core acupoints were predicted by KEGG enrichment, including lipid and atherosclerosis, neurodegeneration, phosphatidylinositol-3-kinase/protein B kinase signaling pathway, mitogen-activated protein kinase signaling pathway, cyclic adenosine monophosphate signaling pathway, TNF signaling pathway, IL-17 signaling pathway, hypoxia-inducible factor-1 signaling pathway, apoptosis, etc., involving neuronal death, synaptic plasticity, oxidative stress, inflammation and other related biological process. The core acupoint prescription of acupuncture and moxibustion intervention for epilepsy can act on multiple targets and multiple pathways to exert anti-epileptic effects, which can provide a theoretical basis for further clinical application and mechanism research.

Belzutifan: a novel therapeutic for the management of von Hippel-Lindau disease and beyond.

The identification of the VHL gene and its role in regulating the hypoxia-inducible factor signaling pathway has helped to revolutionize the treatment of renal cell carcinoma (RCC). Belzutifan is a novel small-molecule inhibitor of hypoxia-inducible factor 2α which has demonstrated efficacy in treating von Hippel-Lindau (VHL) disease, earning regulatory approvals for this indication. There is also early evidence for efficacy in sporadic RCC. Belzutifan has a favorable safety profile. Several clinical trials are currently ongoing, which should help in identifying this promising drug's role in RCC and beyond. This review summarizes the history, pharmacology and clinical evidence for belzutifan use to date, and also explores unanswered questions as they relate to this novel therapeutic agent.

The human testis-enriched HSPA2 interacts with HIF-1α in epidermal keratinocytes, yet HIF-1α stability and HIF-1-dependent gene expression rely on the HSPA (HSP70) activity

The Hypoxia-Inducible Factor 1 (HIF-1) is essential for cellular adaptation to reduced oxygen levels. It also facilitates the maintenance and re-establishment of skin homeostasis. Among others, it is involved in regulating keratinocyte differentiation. The stability of the oxygen-liable HIF-1α subunit is regulated by various non-canonical oxygen-independent mechanisms, which among others involve Heat Shock Proteins of the A family (HSPA/HSP70). This group of highly homologous chaperones and proteostasis-controlling factors includes HSPA2, a unique member crucial for spermatogenesis and implicated in the regulation of keratinocyte differentiation. HIF-1 can control the HSPA2 gene expression.In this study, we revealed that HIF-1α is the first confirmed client of HSPA2 in human somatic cells. It colocalises and interacts directly with HSPA2 in the epidermis in situ and immortalised keratinocytes in vitro. Using an in vitro model based on HSPA2-overexpressing and HSPA2-deficient variants of immortalised keratinocytes we showed that changes in HSPA2 levels do not affect the levels and intracellular localisation of HIF-1α or influence the ability of HIF-1 to modulate target gene expression. However, HIF-1α stability in keratinocytes appears critically reliant on HSPAs as a group of functionally overlapping chaperones. In addition to HSPA2, HIF-1α colocalises and forms complexes with HSPA8 and HSPA1, representing housekeeping and stress-inducible HSPA family paralogs, respectively. Chemical inhibition of HSPA activity, but not paralog-specific knockdown of HSPA8 or HSPA1 expression reduced HIF-1α levels and HIF-1-dependent gene expression. These observations suggest that pharmacological targeting of HSPAs could prevent excessive HIF-1 signalling in pathological skin conditions.

EPAS1 expression contributes to maintenance of the primordial follicle pool in the mouse ovary

Oxygen availability can have profound effects on cell fate decisions and survival, in part by regulating expression of hypoxia-inducible factors (HIFs). In the ovary, HIF expression has been characterised in granulosa cells, however, any requirement in oocytes remains relatively undefined. Here we developed a Hif2a/Epas1 germline-specific knockout mouse line in which females were fertile, however produced 40% fewer pups than controls. No defects in follicle development were detected, and quality of MII oocytes was normal, as per assessments of viability, intracellular reactive oxygen species, and spindle parameters. However, a significant diminishment of the primordial follicle pool was evident in cKO females that was attributed to accelerated follicle loss from postnatal day 6 onwards, potentially via disruption of the autophagy pathway. These data demonstrate the importance of HIF signalling in oocytes, particularly at the primordial follicle stage, and lend to the importance of controlling oxygen tension in the development of in vitro growth and maturation approaches for assisted reproduction.

Uncovering Chemical Interactions between Danshen and Danggui Using Liquid Chromatography–Mass Spectrometry and Network Pharmacology-Based Research on Stroke

Abstract Objective: The objective of this study was to decipher chemical interactions between Danshen and Danggui using liquid chromatography–mass spectrometry (LC-MS) and explore the mechanisms of Danshen–Danggui against stroke using network pharmacology and molecular docking. Materials and Methods: First, the chemical compounds of Danshen–Danggui were profiled using ultra-high-performance liquid chromatography (HPLC)-quadrupole time-of-flight MS. Accurately characterized compounds in various proportions of Danshen–Danggui were quantified using HPLC combined with triple quadrupole electrospray tandem MS. Network pharmacology was used to uncover the essential mechanisms of action of Danshen–Danggui against stroke. Discovery Studio Software was used for the molecular docking verification of key active chemicals and stroke-related targets. Results: A total of 53 compounds were characterized, and 22 accurately identified constituents (10 phenolic acids, 8 phthalides, and 4 tanshinones) were quantified in 15 proportions of Danshen–Danggui. The quantification results showed that Danggui significantly increased the dissolution of most phenolic acids (compounds from Danshen), whereas Danshen promoted the dissolution of most phthalides (compounds from Danggui). Overall, the combination of Danshen and Danggui at a 1:1 ratio resulted in the maximum total dissolution rate. Further network pharmacology and molecular docking results indicated that Danshen–Danggui exerted anti-stroke effects mainly by regulating inflammation-related (tumor necrosis factor, hypoxia-inducible factor, and toll-like receptor) signaling pathways, which ranked among the top three pathways based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Conclusion: The chemical compounds in Danshen–Danggui could interact with each other to increase the dissolution of the most active compounds, which could provide a solid basis for uncovering the compatibility mechanisms of Danshen–Danggui and Danshen–Danggui-based formulae.

From Vessels to Neurons-The Role of Hypoxia Pathway Proteins in Embryonic Neurogenesis.

Embryonic neurogenesis can be defined as a period of prenatal development during which divisions of neural stem and progenitor cells give rise to neurons. In the central nervous system of most mammals, including humans, the majority of neocortical neurogenesis occurs before birth. It is a highly spatiotemporally organized process whose perturbations lead to cortical malformations and dysfunctions underlying neurological and psychiatric pathologies, and in which oxygen availability plays a critical role. In case of deprived oxygen conditions, known as hypoxia, the hypoxia-inducible factor (HIF) signaling pathway is activated, resulting in the selective expression of a group of genes that regulate homeostatic adaptations, including cell differentiation and survival, metabolism and angiogenesis. While a physiological degree of hypoxia is essential for proper brain development, imbalanced oxygen levels can adversely affect this process, as observed in common obstetrical pathologies such as prematurity. This review comprehensively explores and discusses the current body of knowledge regarding the role of hypoxia and the HIF pathway in embryonic neurogenesis of the mammalian cortex. Additionally, it highlights existing gaps in our understanding, presents unanswered questions, and provides avenues for future research.

428 Promoting Infant Gut Barrier Development Through Culturally Relevant Adoption of Fruit and Vegetable Intake.

OBJECTIVES/GOALS: To determine in vitro mechanisms by which fruits and vegetables (FV) contribute to colon barrier development in Latin American infants. We hypothesize that simulated colonic fermentation of FVs will stimulatein vitro cell barrier function by activating the hypoxia-inducible factor (HIF) pathway in colonocytes. METHODS/STUDY POPULATION: FVs consumed by US-based Latin American infants 6-12 months old (identified from NHANES-What We Eat in America Surveys) will be combined with human breast-milk samples from women self-identified as Hispanic or non-Hispanic, and then subjected to in vitro digestion and anaerobic colonic fermentation using human feces. FV fermenta will be incubated with Caco2 monolayers to measure in vitro cell permeability and protein levels of cellular tight junction, metabolic, and HIF signaling enzymes. To examine their effects in vivo, FVs identified to modulate in vitro barrier function, will be fed (5% freeze dried powder) to wild-type mice and the above parameters will be examined. If in vivo effects are found, intestinal specific HIF knockout mice will be used to examine the role of HIF signaling in mediating these effects. RESULTS/ANTICIPATED RESULTS: We expect that fermenta derived from human milk and FVs will reduce in vitro gut permeability in Caco2 monolayers by increasing gene and protein expression of the HIF signaling complex relative to fermenta of human milk alone. This will be reflected with higher cellular trans-epithelial resistance and greater expression levels of tight junction proteins. We expect FV powder consumption will similarly increase in vivo gut permeability and expression of related genes in mice as compared to mice fed diets without FVs. As we expect an increase in HIF signaling in the colon, we expect that FV powder consumption will not enhance in vivo gut permeability in mice colons with an intestinal specific knockout of HIF. DISCUSSION/SIGNIFICANCE: Data from this study will provide mechanistic evidence to help clinicians promote relevant FVs recommendations for Latin American infants and families. Due to the link between gut permeability and obesity, our next step will be to conduct a dietary intervention in this population.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor β1/Smad (TGF-β1/Smad) signaling pathway. Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway. A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-β signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

The Role of Hypoxia-inducible Factor in Mycobacterium tuberculosis-infected Macrophages

Tuberculosis is caused by Mycobacterium tuberculosis infection. During M. tuberculosis infection, there is a decrease in the partial pressure of oxygen in the granuloma microenvironment, which causes the hypoxia-inducible factor (HIF) to become stable. HIF functions as a transcription factor that regulates the expression of genes crucial for metabolic adaptation in hypoxic conditions. Recent research suggests that HIF plays a vital role in infectious and inflammatory conditions. Several studies have demonstrated that HIF signaling can enhance macrophages antimicrobial activity and bactericidal effect against M. tuberculosis, such as increasing macrophage autophagy, enhancing the effects of rifampicin, inhibiting p38 MAPK signaling, enhancing the regulation of effector antimicrobial pathways mediated by human β defensin 2 (hBD2) and vitamin D receptor (VDR), redirecting energy metabolism to glycolysis, and producing various cytokines. All these responses ultimately result in the inhibition of intracellular M. tuberculosis growth. HIF has therapeutic implications, potentially being a new candidate for host-directed therapy as a complement to existing antituberculosis drugs. Understanding the role of HIF in macrophages during M. tuberculosis infection and comprehending the host-pathogen relationship with M. tuberculosis is advantageous for developing future therapies.Keywords: Mycobacterium tuberculosis, macrophages, hypoxia-inducible factor

Abstract 4131: Combination therapeutic strategy to fight against triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBCs) lack estrogen-and progesteron receptors and human epidermal growth factor receptor-2 (HER2) which limits the therapeutic approaches. The high metastasizing capability of TNBCs is in correlation with the poor oxygenation of the cancerous breast tissue. This leads to the activation of the hypoxia-inducible-factor 1 (HIF-1), which induces multiple target genes that are involved in tumor progression and metastasis formation. This makes HIF-1 an attractive drug target, however, HIF-1 inhibitors failed if applied in monotherapy. Therefore, our aim is to investigate the combined therapeutic effect of HIF-1 inhibitor acriflavine and paclitaxel, a commonly used taxane in TNBC treatment. The synergy assessment of the two drugs was carried out by utilizing Sulforhodamine B assay on TNBC cell lines. The in vivo toxicity and efficacy of the drug combination was tested on NOD SCID mice with orthotopically inoculated MDA-MB-231 cell line. Quantitative and qualitative analysis of multiple genes involved in tumor progression was performed by mRNA and protein expression level assessment with qPCR, western blot and immunohistochemistry methods. HIF-1 stimulation was achieved by either incubating the cells at 1% oxygen level or through CoCl2 treatment. The in vitro mRNA expression studies demonstrated that acriflavin possesses a hypoxic-effect-mitigating property on the expression of metastasis-involved- and HIF-1 target genes. Acriflavine alleviated the epithelial-mesenchymal transition of TNBC cells by increasing the expression of the epithelial marker, E-cadherin. At protein level, we detected decrease in HIF-1alpha and Phospho-Protein kinase B (Akt) and increase in p53 tumor suppressor gene expression in response to acriflavine treatment. We further established MDA-MB-231 xenograft model in mice for investigating in vivo toxicity and efficacy. The drug combination efficiently reduced the tumor size and the macrometastasis formation compared to the control groups. The protein analysis of frozen tumor tissue samples indicated that the combination therapy decreased the HIF-1alpha expression and the amount of Phospho-Akt and Phospho-Mitogen-activated protein kinases (pMAPK/pERK) in CoCl2treated mice. Additionally, expression of epithelial markers increased and that of mesenchymal markers decreased. The combination therapy led to a decrease in the platelet-derived growth factor receptor beta (PDGFRB) as well. To conclude, we identified a novel drug combination which can efficiently combat TNBC by inhibiting HIF-1 signaling, mitigating cell migration and metastasis formation and suppressing the activation of the Phosphoinositide 3-kinase (PI3K) and MAPK pathways. This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Citation Format: Laura Svajda, Ivan Ranđelović, Sára Eszter Surguta, Marcell Baranyi, István Kenessey, Mihály Cserepes, József Tóvári. Combination therapeutic strategy to fight against triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4131.

Hypoxic Functional Regulation Pathways in the GI Tract: Focus on the HIF-1α and Microbiota's Crosstalk.

Hypoxia is an essential gastrointestinal (GI) tract phenomenon that influences both physiologic and pathologic states. Hypoxia-inducible factors (HIFs), the primary drivers of cell adaptation to low-oxygen environments, have been identified as critical regulators of gut homeostasis: directly, through the induction of different proteins linked to intestinal barrier stabilization (ie, adherent proteins, tight junctions, mucins, integrins, intestinal trefoil factor, and adenosine); and indirectly, through the regulation of several immune cell types and the modulation of autophagy and inflammatory processes. Furthermore, hypoxia and HIF-related sensing pathways influence the delicate relationship existing between bacteria and mammalian host cells. In turn, gut commensals establish and maintain the physiologic hypoxia of the GI tract and HIF-α expression. Based on this premise, the goals of this review are to (1) highlight hypoxic molecular pathways in the GI tract, both in physiologic and pathophysiologic settings, such as inflammatory bowel disease; and (2) discuss a potential strategy for ameliorating gut-related disorders, by targeting HIF signaling, which can alleviate inflammatory processes, restore autophagy correct mechanisms, and benefit the host-microbiota equilibrium.

Framework Nucleic Acids-Based VEGF Signaling Activating System for Angiogenesis: A Dual Stimulation Strategy.

Angiogenesis is crucial for tissue engineering, wound healing, and regenerative medicine. Nanomaterials constructed based on specific goals can be employed to activate endogenous growth factor-related signaling. In this study, based on the conventional single-stranded DNA self-assembly into tetrahedral framework nucleic acids (tFNAs), the Apt02 nucleic acid aptamer and dimethyloxallyl glycine (DMOG) small molecule are integrated into a complex via a template-based click chemistry reaction and toehold-mediated strand displacement reaction. Thus, being able to simulate the VEGF (vascular endothelial growth factor) function and stabilize HIF (hypoxia-inducible factor), a functional whole is constructed and applied to angiogenesis. Cellular studies demonstrate that the tFNAs-Apt02 complex (TAC) has a conspicuous affinity to human umbilical vein endothelial cells (HUVECs). Further incubation with DMOG yields the tFNAs-Apt02-DMOG complex (TACD), which promotes VEGF secretion, in vitro blood vessel formation, sprouting, and migration of HUVECs. Additionally, TACD enhances angiogenesis by upregulating the VEGF/VEGFR and HIF signaling pathways. Moreover, in a diabetic mouse skin defect repair process, TACD increases blood vessel formation and collagen deposition, therefore accelerating wound healing. The novel strategy simulating VEGF and stabilizing HIF promotes blood-vessel formation in vivo and in vitro and has the potential for broad applications in the vascularization field.

Role of Protein Ubiquitination and HIF Signaling in the Evolution of Hypoxic Breast Cancer.

Alternations in protein ubiquitination along with hypoxia-inducible factor (HIF) signaling contribute to tumorigenesis and breast tumor advancement. Ubiquitination is an impulsive process, which is coordinately governed by E3 ligases and deubiquitinases (DUBs), that have come out as charismatic therapeutic targets. HIF expression, as well as the transcriptional process in malignancies, are frequently elevated, resulting in pitiable clinical outcomes. According to increasing research, multiple E3 ligases, in addition to UBDs work together to modulate HIF expression and activity, permitting breast cancer cells to make out a hypoxic milieu. On the other hand, hypoxia and HIF signaling regulate numerous E3 ligases as well as DUBs. Interpreting involved networks connecting E3 ligase, DUBS, and HIF will reveal profound mechanisms of physiological response to hypoxia and aid in the discovery of new molecular references for cancer management. The present state of knowledge about the entire kinship among E3 ligase, DUBs, and HIF signaling is reviewed here, emphasizing using E3 ligase or DUB inhibitors in breast cancer.

Identification of key genes and signalling pathways in clear cell renal cell carcinoma: An integrated bioinformatics approach.

Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent types of kidney cancer. Unravelling the genes responsible for driving cellular changes and the transformation of cells in ccRCC pathogenesis is a complex process. In this study, twelve microarray ccRCC datasets were chosen from the gene expression omnibus (GEO) database and subjected to integrated analysis. Through GEO2R analysis, 179 common differentially expressed genes (DEGs) were identified among the datasets. The common DEGs were subjected to functional enrichment analysis using ToppFun followed by construction of protein-protein interaction network (PPIN) using Cytoscape. Clusters within the DEGs PPIN were identified using the Molecular Complex Detection (MCODE) Cytoscape plugin. To identify the hub genes, the centrality parameters degree, betweenness, and closeness scores were calculated for each DEGs in the PPIN. Additionally, Gene Expression Profiling Interactive Analysis (GEPIA) was utilized to validate the relative expression levels of hub genes in the normal and ccRCC tissues. The common DEGs were highly enriched in Hypoxia-inducible factor (HIF) signalling and metabolic reprogramming pathways. VEGFA, CAV1, LOX, CCND1, PLG, EGF, SLC2A1, and ENO2 were identified as hub genes. Among 8 hub genes, only the expression levels of VEGFA, LOX, CCND1, and EGF showed a unique expression pattern exclusively in ccRCC on compared to other type of cancers.

Exploring the Molecular Mechanism by which Kaempferol Attenuates Sepsis-related Acute Respiratory Distress Syndrome Based on Network Pharmacology and Experimental Verification.

Sepsis-related acute respiratory distress syndrome (ARDS) is a fatal disease without effective therapy. Kaempferol is a flavonoid compound extracted from natural plant products; it exerts numerous pharmacological effects. Kaempferol attenuates sepsis-related ARDS; however, the underlying protective mechanism has not been elucidated completely. This study aimed to use network pharmacology and experimental verification to investigate the mechanisms by which kaempferol attenuates sepsis-related ARDS. We screened the targets of kaempferol by PharMapper, Swiss Target Prediction, and CTD database. We identified the targets of sepsis-related ARDS by GeneCards, DisGeNet, OMIM, and TTD. The Weishengxin platform was used to map the targets of both kaempferol and sepsis-related ARDS. We created a Venn diagram to identify the intersection targets. We constructed the "component-intersection targets-disease" network diagram using Cytoscape 3.9.1 software. The intersection targets were imported into the STRING database for developing the protein-protein interaction network. Metascape was used for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. We selected the leading 20 KEGG pathways to establish the KEGG relationship network. Finally, we performed experimental verification to confirm our prediction results. Through database screening, we obtained 502, 360, and 78 kaempferol targets, disease targets of sepsis-related ARDS, and intersection targets, respectively. The core targets consisted of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, albumin (ALB), IL-1β, and AKT serine/ threonine kinase (AKT)1. GO enrichment analysis identified 426 items, which were principally involved in response to lipopolysaccharide, regulation of inflammatory response, inflammatory response, positive regulation of cell migration, positive regulation of cell adhesion, positive regulation of protein phosphorylation, response to hormone, regulation of reactive oxygen species (ROS) metabolic process, negative regulation of apoptotic signaling pathway, and response to decreased oxygen levels. KEGG enrichment analysis identified 151 pathways. After eliminating the disease and generalized pathways, we obtained the hypoxia-inducible factor 1 (HIF-1), nuclear factor κB (NF-κB), and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Our experimental verification confirmed that kaempferol blocked the HIF-1, NF-κB, and PI3K-Akt signaling pathways, diminished TNF-α, IL-1β, and IL-6 expressions, suppressed ROS production, and inhibited apoptosis in lipopolysaccharide (LPS)-induced murine alveolar macrophage (MH-S) cells. Kaempferol can reduce inflammatory response, ROS production, and cell apoptosis by acting on the HIF-1, NF-κB, and PI3K-Akt signaling pathways, thereby alleviating sepsis- related ARDS.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- tofivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphataseand osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1αand increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2 ) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Loss of SLC30A10 manganese transporter alters expression of neurotransmission genes and activates hypoxia-inducible factor signaling in mice.

The essential metal manganese (Mn) induces neuromotor disease at elevated levels. The manganese efflux transporter SLC30A10 regulates brain Mn levels. Homozygous loss-of-function mutations in SLC30A10 induce hereditary Mn neurotoxicity in humans. Our prior characterization of Slc30a10 knockout mice recapitulated the high brain Mn levels and neuromotor deficits reported in humans. But, mechanisms of Mn-induced motor deficits due to SLC30A10 mutations or elevated Mn exposure are unclear. To gain insights into this issue, we characterized changes in gene expression in the basal ganglia, the main brain region targeted by Mn, of Slc30a10 knockout mice using unbiased transcriptomics. Compared with littermates, >1000 genes were upregulated or downregulated in the basal ganglia sub-regions (i.e. caudate putamen, globus pallidus, and substantia nigra) of the knockouts. Pathway analyses revealed notable changes in genes regulating synaptic transmission and neurotransmitter function in the knockouts that may contribute to the motor phenotype. Expression changes in the knockouts were essentially normalized by a reduced Mn chow, establishing that changes were Mn dependent. Upstream regulator analyses identified hypoxia-inducible factor (HIF) signaling, which we recently characterized to be a primary cellular response to elevated Mn, as a critical mediator of the transcriptomic changes in the basal ganglia of the knockout mice. HIF activation was also evident in the liver of the knockout mice. These results: (i) enhance understanding of the pathobiology of Mn-induced motor disease; (ii) identify specific target genes/pathways for future mechanistic analyses; and (iii) independently corroborate the importance of the HIF pathway in Mn homeostasis and toxicity.

Radix Sanguisorbae Improves Intestinal Barrier in Septic Rats via HIF-1 α/HO-1/Fe2+ Axis.

To investigate whether Radix Sanguisorbae (RS, Diyu) could restore intestinal barrier function following sepsis using a cecal ligation and puncture (CLP)-induced septic rat model and lipopolysaccharide (LPS)-challenged IEC-6 cell model, respectively. Totally 224 rats were divided into 4 groups including a control, sham, CLP and RS group according to a random number table. The rats in the control group were administrated with Ringer's lactate solution (30 mL/kg) with additional dopamine [10 µ g/(kg·min)] and given intramuscular injections of cefuroxime sodium (10 mg/kg) 12 h following CLP. The rats in the RS group were administrated with RS (10 mg/kg) through tail vein 1 h before CLP and treated with RS (10 mg/kg) 12 h following CLP. The rats in the sham group were only performed abdominal surgery without CLP. The rats in the CLP group were performed with CLP without any treatment. The other steps were same as control group. The effects of RS on intestinal barrier function, mesenteric microvessels barrier function, multi-organ function indicators, inflammatory response and 72 h survival window following sepsis were observed. In vitro, the effects of RS on LPS-challenged IEC-6 cell viability, the expressions of zona occludens-1 (ZO-1) and ferroptosis index were evaluated by cell counting kit-8, immunofluorescence and Western blot analysis. Bioinformatic tools were applied to investigate the pharmacological network of RS in sepsis to predict the active compounds and potential protein targets and pathways. The sepsis caused severe intestinal barrier dysfunction, multi-organ injury, lipid peroxidation accumulation, and ferroptosis in vivo. RS treatment significantly prolonged the survival time to 56 h and increased 72-h survival rate to 7/16 (43.75%). RS also improved intestinal barrier function and relieved intestinal inflammation. Moreover, RS significantly decreased lipid peroxidation and inhibited ferroptosis (P<0.05 or P<0.01). Administration of RS significantly worked better than Ringer's solution used alone. Using network pharmacology prediction, we found that ferroptosis and hypoxia inducible factor-1 (HIF-1 α) signaling pathways might be involved in RS effects on sepsis. Subsequent Western blot, ferrous iron measurements, and FerroOrange fluorescence of ferrous iron verified the network pharmacology predictions. RS improved the intestinal barrier function and alleviated intestinal injury by inhibiting ferroptosis, which was related in part to HIF-1 α/heme oxygenase-1/Fe2+ axis.

Bisphenol A exposure stimulates prostatic fibrosis via exosome-triggered epithelium changes

Fibrosis is the pathological basis for the clinical progression of benign prostatic hyperplasia (BPH). Prostatic fibrosis is an important risk factor in patients with BPH who experience lower urinary tract symptoms. Bisphenol A (BPA) is an environmental endocrine disruptor (EED) that causes prostate defects. The effects of BPA on the prostate were investigated in this study using mouse and human prostate cell models. BPA-induced mouse prostatic fibrosis is characterized by collagen deposition and an increase in hydroxyproline concentration. Furthermore, BPA-exposed prostatic stromal fibroblasts exosomes promote the epithelial-mesenchymal transition of epithelial cells. High-throughput RNA sequencing and functional enrichment analyses show that substantially altered mRNAs, lncRNAs and circRNAs play roles in cellular interactions and the hypoxia-inducible factor-1 signaling pathway. The results showed that exosomes participated in the pro-fibrogenic effects of BPA on the prostate by mediating communication between stromal and epithelial cells and triggering epithelial changes.