HIF1A Research Articles

Investigating the Expression and Function of HIF-1α in Neocaridina davidi During Embryo Cleavage Stage

Neocaridina davidi, a member of Decapoda within Crustacea, stands out due to its petite stature, robust reproductive capabilities and short molting cycle. Consequently, it has emerged as a valuable experimental model for investigations spanning ecology, development, physiology, and toxicology. Despite the pivotal role of Hypoxia Inducible Factor-1α (HIF-1α) in diverse physiological processes like biological hypoxia stress, cellular growth, and stress resistance, its functionality in crustaceans remains underexplored. Moreover, the role and function of HIF-1α in crustaceans, especially in embryonic stages, have been insufficiently addressed. This study delves into the function of HIF-1α during embryonic development. Initially, the complete sequence of the HIF-1α gene was acquired. Notably, the expression of HIF-1α during the cleavage stage surpassed that of subsequent phases. Subsequently, dsRNAHIF-1α was introduced into sexually mature shrimp, revealing that the inhibitory impact of dsRNA on gene expression in the parental generation could be inherited by the offspring, exerting a specific gene silencing effect in the embryo. The consequences of silencing HIF-1α in embryos manifested as varying degrees of premature division termination, besides, the glycolysis in the embryos was also affected by HIF-1α suppression. These results provide data support for understanding the early embryonic development of crustaceans and HIF-1α functions within it.

Chitosan nanoparticles loaded with metformin and digoxin synergistically inhibit MCF-7 breast cancer cells through suppression of NOTCH-1 and HIF-1α gene expression

This study investigated the potential anticancer efficacy of co-treating the MCF-7 breast cancer cell line with chitosan nanoparticles (Cs NPs) loaded with metformin (Met) and digoxin (Dig). The Cs NPs had a size range of 90.6–148.7 nm and a zeta potential of +11.7 to +11.9 mV, indicating a positive surface charge. Notably, the Cs NPs demonstrated high encapsulation efficiencies, with values of 90.97 ± 5.14 % for Met and 92.12 ± 3.81 % for Dig, indicating effective loading of both drugs. The results revealed that the co-delivery of Met and Dig via Cs NPs significantly enhanced the anticancer efficacy, outperforming the treatment with individual free drugs or their combination, thereby demonstrating the potential benefits of nanoparticle-mediated co-administration. The drugs-loaded Cs NPs induced a marked increase in apoptosis in MCF-7 cells, with a cell death rate of 67.56 %, and significantly reduced mammosphere size by 48.08 %, thereby demonstrating a superior therapeutic efficacy compared to treatment with individual free drugs or their combination. Notably, the drug-loaded Cs NPs exhibited potent anti-migratory and anti-angiogenic effects, significantly inhibiting cell migration and new blood vessel formation, which may contribute to overcoming the inherent resistance of tumors to conventional therapies. Mechanistically, the co-treatment with drugs-loaded Cs NPs was found to downregulate the expression of NOTCH-1 and HIF-1α, two key transcription factors involved in tumor cell survival and adaptation, suggesting that their inhibition is a crucial component of the therapeutic efficacy of this treatment strategy. Collectively, the findings of this study suggest that the co-delivery of Met and Dig via chitosan Cs NPs represents a promising therapeutic strategy for breast cancer, as it effectively targets key pathways involved in tumor growth and progression, and underscores the potential of Cs NPs as a versatile platform for cancer therapy.

Modulating the tumor microenvironment in a mouse model of colon cancer using a combination of HIF-1α inhibitors and Toll-Like Receptor 7 agonists.

The immunosuppressive tumor microenvironment (TME) plays a pivotal role in the response to various anticancer therapies, such as immune and chemotherapeutic agents. In this study, the synergistic effects of gene-targeting HIF-1α siRNA combined with Toll-Like Receptor 7 agonist on TME remodeling were investigated in a mouse model of colorectal cancer (CRC). A HIF-1α-specific siRNA duplex was formulated based on the ionic gelation of tripolyphosphate (TPP) with cationic chitosan (CH) as a nanoplex and evaluated in terms of size, charge, polydispersity index and gel retardation assay. MTT assay was conducted to assess the cytotoxicity of the specific siRNA duplex against CT26 cells. Hypoxic condition was generated to evaluate the gene and protein expression levels of HIF-1α, respectively. CT26 mouse model was established to assess the synergistic effect of silencing HIF-1α combined with oxaliplatin (OXA) and imiquimod (IMQ) on tumor growth. The mean diameter of the CH/siRNA nanoparticles was 243 ± 6 nm, as confirmed with Micrograph scanning electron microscope. There were no significant differences observed between the CT26 cells treated with nanoparticles alone and the untreated cells, indicating that these nanoparticles are safe and physiologically biocompatible (p ≥ 0.05). Triple combination therapy involving HIF-1α siRNA, OXA, and IMQ significantly retarded tumor growth and led to elevated levels of cytokines linked to cellular immunity (INF-γ and IL-12) compared with those in the other groups (P < 0.05). The positive correlation coefficient (r = 0.68) between tumor size and HIF-1α expression levels was statistically significant (P = 0.003). Compared with those in the control group, the expression levels of the anti-inflammatory cytokines IL-10 and IL-4 significantly decreased (P < 0.05). In conclusion, our findings suggest that inhibiting HIF-1α could serve as a rational strategy to enhance the antitumor response in the TME.

Morphological and Molecular-Biological Features of Lewis Lung Carcinoma Progression in Mice with Different Resistance to Hypoxia.

In adult male mice with high (HR) and low (LR) resistance to hypoxia, on days 21 and 28 after subcutaneous injection of Lewis lung carcinoma cells, a morphological and morphometric study of the primary tumor nodes and metastases in the lungs was carried out. Peripheral blood parameters and subpopulation composition of blood cells, the expression of genes responsible for the development of inflammation (Nfkb, Il1b, Il6, Tnfa, Il10, and Tgfb) and the response to hypoxia (Hif1a) in the liver were also assessed. The tumors were detected in 84.6% HR and 91.7% LR mice. The mitotic index of tumor cells in the subcutaneous nodes of HR animals was statistically significantly higher. The metastases area on days 21 and 28 did not differ. In animals of both groups, an increase in the absolute number of leukocytes, monocytes, and granulocytes, a decrease in the hemoglobin content and the absolute number of erythrocytes in the peripheral blood were detected on day 28 of the experiment. Only in LR animals, an increase in the absolute number of CD11b+ monocytes was found on day 28 of the experiment in comparison with the control group. The expression of Hif1a, Nfkb, Tnfa, and Tgfb genes in the liver of LR animals was higher than in HR mice, which attested to more pronounced systemic inflammatory response. These data should be taken into account when developing new approaches for the treatment of neoplastic disorders.

Enhancing Fracture Healing with 3D Bioprinted Hif1a-Overexpressing BMSCs Hydrogel: A Novel Approach to Accelerated Bone Repair.

Addressing the urgent need for effective fracture treatments, this study investigates the efficacy of a 3D bioprinted biomimetic hydrogel, enriched with bone marrow mesenchymal stem cells (BMSCs) and targeted hypoxia-inducible factor 1 alpha (Hif1a) gene activation, in enhancing fracture healing. A photocross-linkable bioink, gelatin methacryloyl bone matrix anhydride (GBMA) is developed, and selected its 5% concentration for bioink formulation. Rat BMSCs are isolated and combined with GBMA to create the GBMA@BMSCs bioink. This bioink is then used in 3D bioprinting to fabricate a hydrogel for application in a rat femoral fracture model. Through transcriptome sequencing, WGCNA, and Venn analysis, the hypoxia-inducible factor Hif1a is identified as a critical gene in the fracture healing process. In vitro studies showed that Hif1a promoted BMSC proliferation, chondrogenic differentiation, and cartilage matrix stability. The in vivo application of the GBMA@BMSCs hydrogel with Hif1a overexpression significantly accelerated fracture healing, evidenced by early and enhanced cartilage callus formation. The study demonstrates that 3D bioprinting of GBMA@BMSCs hydrogel, particularly with Hif1a-enhanced BMSCs, offers a promising approach for rapid and effective fracture repair.

Investigating the effect of Quercetin in the presence of CoCl2 as an inducing hypoxia agent on the biological characteristics of human telomerase reverse transcription-immortalized adipose tissue-derived MSCs

Studying the effect of small chemical molecules on stem cell characteristics under normoxia and hypoxia conditions is crucial to discovering the best conditions for effective biomedical applications. This study aimed to investigate the effect of Quercetin (QC; a flavonoid) in the presence of CoCl2 as a mimicking hypoxia chemical on the biological features of human telomerase reverse transcription-immortalized mesenchymal stem cell (hTERT-MSC) lines. The effect of CoCl2, QC, and their combination on the viability, proliferation, and migration of hTERT-MSCs were evaluated by MTT, Trypan-blue staining and cell counting by hemocytometer, and in vitro wound healing assays, respectively. Moreover, the effect of treatments on the reactive oxygen species (ROS) production, cell cycle, and HIF1a, c-MET, H19, and CASP3 gene expression was assessed by NBT, PI-staining and flow-cytometry, and real-time PCR assays, respectively. We found that CoCl2 and QC have different effects on the viability, proliferation, and migration of hTERT-MSCs in a dose-dependent manner. In addition, CoCl2 and QC affect ROS levels in cells in a dose- and time-dependent manner. While CoCl2 up-regulated HIF1a, QC and CoCl2 down-regulated CASP3 and c-MET in hTERT-MSCs. Moreover, QC reduced HIF1a and lncRNA-H19 expression in cells. Furthermore, in the presence of CoCl2, QC at low concentrations reduced hTERT-MSC survival, proliferation, and migration at 48 h; however, at high concentrations, it induced cell survival and proliferation. The combination treatment also up-regulated ROS levels and down-regulated the investigated genes in cells. Altogether, we conclude that QC at high concentrations under CoCl2-mediated hypoxia and short exposure time induces hTERT-MSCs survival and proliferation.

Integrative pharmacological analysis of modified Zuojin formula: Inhibiting the HIF-1α-mediated glycolytic pathway in chronic atrophic gastritis

Ethnopharmacological relevanceZuojin formula (ZJF) is a well-known herbal medicine in Pharmacopoeia of China, which is widely used for gastritis. Modified Zuojin formula (MZJF) was adapted based on traditional Chinese medicine (TCM) theories concerning gastric atrophy and dysplasia, along with extensive clinical experience, has been clinically employed to treat chronic atrophic gastritis (CAG). However, the underlying mechanisms by which MZJF intervenes in CAG remain to be fully elucidated. Aim of the studyThe aim of this study was to evaluate the effects of MZJF intervention in CAG and explore its potential mechanisms. MethodsFour induction factors were used to establish a CAG rat model. HE and AB-PAS staining was utilized to assess the effects of MZJF in the intervention of CAG. The stomach weight index and gastric acid pH was used to assess the overall state of stomach. ELISA was used to assess the gastric mucosal inflammatory response. Using transmission electron microscopy to observe chief cells and parietal cells, we evaluated the improvement of ultrastructure by MZJF. Through network pharmacology analysis, the possible regulatory mechanism of MZJF in CAG was preliminarily explored. Binding interactions between MZJF components and predicted targets were explored using molecular docking. Subsequently, quantitative real-time PCR (qRT-PCR), Western blot, biochemical analysis and TUNEL staining were applied to validate the effect of MZJF on predicted pathway. ResultsMZJF treatment ameliorated gastric mucosal pathology, inflammation, cellular ultrastructural damage and PG levels, halted the exacerbation of CAG in rats, along with a reduction in stomach weight index and gastric acid pH. A total of 79 compounds in MZJF targeting 203 CAG-related molecules were identified through network pharmacology. Enrichment analysis of the core targets was focused on the hypoxia inducible factor-1α (HIF-1α) signaling pathway. Molecular docking results identified HIF-1α as stable binding targets for MZJF primary components. Subsequently, PCR, WB, and biochemical results showed that MZJF suppressed the gene and protein expression levels of HIF-1α and its downstream molecules including glycolytic enzymes and transporters, modulated glucose, pyruvic acid and lactate levels in gastric mucosal tissue. Moreover, MZJF induced apoptosis of gastric epithelial cells, as evidenced by the upregulation of cleaved caspase-3, Bax, Bax/Bcl-2 and TUNEL positive cells ratio. ConclusionsMZJF suppressed the HIF-1α-mediated glycolytic pathway, and promoted cell apoptosis, thereby halting the malignant transformation of CAG. The study provides a valuable reference point for applying TCM in preventing and treating CAG.

Association between the variations in metabolic pathways and oral cancer risk: results from a Pakistani case-control study.

Oral cancer (OC) is a significant global health concern, with Pakistan ranking 5th worldwide in OC incidence. Given the poor prognosis, early detection of at-risk individuals is crucial. Genetic factors, particularly single nucleotide polymorphisms (SNPs) in metabolic genes, may influence OC susceptibility. This study investigated the association between SNPs in CYP1A1, COX2, SOD2, and HIF1a genes and OC risk in the Pakistani population. A prospective study was conducted from October 2019 to March 2022, enrolling 215 newly diagnosed OC patients and 410 controls. Genetic variations were analyzed using High-Resolution Melting (HRM)analysis and Sanger sequencing, with protein expression evaluated by ELISA. No significant associations were found between the studied SNPs and OC risk. However, a non-significant trend was observed for the SOD2 variant (rs4880), where the G allele was associated with a higher OC risk than the A allele (p = 0.20). Elevated COX2 and HIF1α levels (p-values of 0.014 and <0.001, respectively) and reduced SOD2 levels (p < 0.0001) were observed in OC patients, while CYP1A1 levels remained similar in both controls and cases. Although SNPs in CYP1A1, COX2, SOD2, and HIF1α were not significantly associated with OC risk in the Pakistani population, altered protein expression levels of COX2, HIF1α and SOD2 suggest additional regulatory mechanisms. Further investigation into post-transcriptional modifications and epigenetic factors could lead to novel biomarkers and therapeutic targets for OC in Pakistan.

Hypoxia and collagen deposition in the kidneys infected with Acanthamoeba sp.

Acanthamoeba spp. are facultative, opportunistic pathogens that are found in diverse environments. In the hosts, they lead to multi-organ disease. Recent studies reported that they may induce changes in the kidneys of hosts. The aim of the study was to determine the influence of Acanthamoeba sp. on hypoxia and collagen deposition in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba sp. The results strongly suggest that Acanthamoeba sp. induces hypoxia in mice with normal and reduced immune response by increasing gene and/or protein expression of HIF1α as well as HIF2α. Additionally, the activation of these factors is probably induced via NOX2/ROS. Hypoxia promotes vessel formation, and we found that angiogenesis occurs in the kidneys of mice infected with the parasite regardless of their immunological status. The proangiogenic factors released in hypoxic conditions cause modulation and inflammation in the kidney cells, which in turn leads to collagen deposition via TGF-β. This work reveals mechanisms occurring in the hosts infected with Acanthamoeba sp., highlights as well as supports the relevance of pathophysiology in the kidneys in hosts with systematic acanthamoebiasis.

TNFAIP2 promotes HIF1α transcription and breast cancer angiogenesis by activating the Rac1-ERK-AP1 signaling axis

Angiogenesis is well known to play a critical role in breast cancer. We previously reported that TNFAIP2 activates Rac1 to promote triple-negative breast cancer (TNBC) cell proliferation, migration, and chemoresistance. However, the potential contribution of TNFAIP2 to tumor angiogenesis remains unknown. In this study, we demonstrated that TNFAIP2 promotes TNBC angiogenesis by activating the Rac1-ERK-AP1-HIF1α signaling axis. Under hypoxia, TNFAIP2 activates Rac1 and ERK sequentially. Following that, ERK activates the AP-1 (c-Jun/Fra1) transcription factor. By employing chromatin immunoprecipitation and luciferase reporter assays, we showed that AP-1 directly interacts with the HIF1α gene promoter, thereby enhancing its transcription. The combined application of ERK inhibitors, U0126 or trametinib, with the VEGFR inhibitor Apatinib, additively suppresses angiogenesis and tumor growth of HCC1806 in nude mice. These findings provide new therapeutic strategies for TNBC.

TMIC-41. RBBP4/P300 COMPLEX CONTROLS GENES MODULATING ANGIOGENESIS AND IMMUNOSUPPRESSION IN GLIOBLASTOMA MICROENVIRONMENT

Abstract The RBBP4/p300 complex was recently shown to control genes critical for DNA damage repair and may play a role in glioblastoma (GBM) therapy responsiveness, especially to temozolomide (TMZ). However, the role of this complex in the modulation of the GBM tumor microenvironment (GBM-TME) remains elusive. Because pro-tumorigenic GBM-TME is primarily due to angiogenesis and immune tolerance, we evaluated whether the RBBP4/p300 complex may control these critical processes. To that end, we used shRNA to silence each RBBP4/p300 complex member in GBM43 cells and RNAseq to identify angiogenesis and immune genes regulated by this complex. Our data demonstrated that silencing RBBP4 or p300 suppressed the expression of HIF1α and several other hypoxia response genes, including VEGFA and DDIT4, suggesting that RBBP4/p300 complex may regulate angiogenesis. Consistent with a role in regulating angiogenesis, culture medium conditioned by GBM43 cells expressing the control non-targeting shRNA (shNT) significantly induced angiogenic tube formation by human umbilical vein endothelial cells (HUVEC). In contrast, this effect was blocked by the medium conditioned by the shRBBP4 or shp300 expressing cells. Both shRBBP4 and shp300 expressing cells failed to activate HIF1α accumulation and other signaling in response to hypoxia. Additionally, the control shNT-conditioned medium inhibited IFN-γ production while the medium conditioned by the shRBBP4 or shp300 expressing cells supported IFN-ɣ production from CD8+ T cells. Intriguingly, silencing RBBP4 or p300 also suppressed the expression of CCL2 and PD-L1 (CD274). CCL2 protein is a chemokine that promotes TME infiltration by myeloid-derived suppressor cells (MDSCs), while PD-L1 interacts with PD1 to suppress cytotoxic CD8+ T cell activation. Although this is an ongoing investigation, we conclude that RBBP4/p300 complex mediates the expression of hypoxia response genes, including HIF1α, and immunosuppressive genes, including PD-L1, and this activity may enhance angiogenic and immunosuppressed GBM-TME, making this complex a potential target for GBM therapy.

Low‒Dose Cyclophosphamide Enhances the Tumoricidal Effects of 5-Day Spacing Stereotactic Ablative Radiotherapy by Boosting Antitumor Immunity.

To investigate the potential role of low‒dose cyclophosphamide (Cy) as a radiosensitizer by evaluating its impact on the immune response and the abscopal effect of stereotactic ablative radiotherapy (SABR) through preclinical models. CT26 tumors (immunologically hot) and 4T1 tumors (immunologically cold), grown in immunocompetent BALB/c and immunodeficient BALB/c‒nude mice, were irradiated with 20 Gy in two fractions with 5‒day spacing followed by intraperitoneal injections of 9 mg/kg Cy every 3 days. Immunological changes in CT26 tumors caused by the treatments were assessed using flow cytometry. Changes in the expression of HIF‒1α in tumors were also assessed. Splenocytes and bone marrow‒derived dendritic cells (DCs) were exposed to various concentrations of Cy to assess T cell proliferation and DC differentiation. The combination of Cy with RT (RT+Cy) significantly suppressed tumor growth compared to RT alone in immunocompetent mice, while that effect was not observed in immunodeficient mice. Additionally, RT+Cy effectively induced abscopal effects in hot and cold tumors, with increased CD8+ T cells in blood and tumors. Significantly higher expression levels of Granzyme B, IFN‒γ, and TNF‒α were observed in RT+Cy group compared to the RT alone group. In vitro data indicated that low‒dose Cy promotes DC differentiation. Low‒dose Cy suppressed the radiation‒induced upregulation of HIF‒1α in the tumors. Low‒dose Cy enhances tumoricidal effects of 5‒day spacing high‒dose RT by increasing anti-tumor immune responses.

Salidroside Ameliorates Macrophages Lipid Accumulation and Atherosclerotic Plaque by Inhibiting Hif-1α-Induced Pyroptosis

BackgroundHipoxia-inducible factor 1 alpha (Hif-1α) is a significant risk factor for atherosclerotic cardiovascular disease. Salidroside (SAL) has demonstrated anti-oxidative and anti-cardiovascular disease effects. Currently, there are no relevant studies investigating the interaction between SAL and Hif-1α in the progression of atherosclerosis . MethodsHif-1α was either knocked down or upregulated in Ana-1 macrophages-derived foam cells, and atherosclerosis ApoE−/− mice were treated with or without SAL. A Protein-protein network involving Hif-1α and pyroptosis-related genes was identified through bioinformatic analysis and validated in human vascular tissues. The Oil Red O and DiI staining were used to detect the intracellular ox-LDL accumulation. The HE and Oil Red O staining were employed to evaluate atherosclerotic plaque in vivo. The levels of relevant molecules were quantified using WB, qRT-PCR, ELISA, and immunohistochemistry. The target proteins of SAL were identified through Molecular docking and Cell Thermal Shift Assay (CESTA). ResultsBoth Hif-1α knockdown and SAL treatment markedly reduced lipid accumulation in macrophages-derived foam cells. Hif-1α was closely associated with Caspase1, Gsdmd, NRLP3, and IL-1β, and co-located in CD86+ macrophages-derived foam cells within atherosclerotic plaque. SAL inhibited Hif-1α-induced Caspase-1-dependent pyroptosis and lipid accumulation by directly bonding to Hif-1α. In vivo, SAL treatment decreased atherosclerotic plaque and improved plasma lipid profiles. Furthermore, SAL reduced M1 macrophages infiltration and the levels of Hif-1α, C-Caspase1, Gsdmd-N, NRLP3, IL-18, and IL-1β in atherosclerotic plaque. ConclusionSAL alleviated the lipid accumulation in macrophages and atherosclerotic plaques by inhibiting pyroptosis pathway via directly binding to Hif-1α, which may be a promising therapeutic strategy for AS treatment.

Metformin boosts doxorubicin efficacy and increases CD8 + T cell frequency in mouse breast cancer.

Breast cancer is a leading cause of cancer-related deaths among women worldwide. The presence and function of CD8 + T cells in the tumor microenvironment have been associated with better patient outcomes. Drug toxicity has posed a significant challenge to the clinical implementation of chemotherapy-based strategies in cancer treatment. In this study, we employed flow cytometry to investigate the potential synergistic immunomodulatory effects on CD8 + T cells in a mouse model of breast cancer by combining metformin, an anti-diabetic medication, with doxorubicin. Through flow cytometry analysis, we observed that the combination of the two drugs led to an increased percentage of CD8 + T cells compared to either drug alone. The modulation of HIF-1α and STAT3 gene expression in the combination group further confirmed the efficacy of this approach. Our findings suggest that combining metformin with doxorubicin can enhance the anticancer activity of doxorubicin and decrease its cytotoxicity in a 4T1 breast cancer mouse model.

Adolescent exposure to organophosphate insecticide malathion induces spermatogenesis dysfunction in mice by activating the HIF-1/MAPK/PI3K pathway

Chemical-caused reproductive dysfunction has emerged as a global public health concern. This study investigated the adverse effects of the organophosphorus pesticide malathion on reproductive function in adolescent male mice at environmentally relevant concentrations. The results indicated that eight-week malathion exposure reduced testis weight, caused sex and thyroid hormone disorders, and induced testicular spermatogenic epithelium damage and oxidative stress. Testicular RNA sequencing indicated that malathion significantly affected testicular energy metabolism, hypoxia-inducible factor 1 (HIF-1) signaling, and steroid hormone biosynthesis pathways. Malathion significantly increased the gene and protein expression of HIF-1α by upregulating key genes in the mitogen-activated protein kinase (MAPK) pathway (Map2k2, Mapk3, and Eif4e2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Pik3r2 and Akt1). Furthermore, malathion downregulated HIF-1α degradation-regulating genes while upregulating anaerobic metabolism and inflammation-related genes, thereby inhibiting normoxia and promoting hypoxia processes. Testicular hypoxia subsequently induced steroid hormone biosynthesis disorders and spermatogenesis dysfunction. Molecular docking verified that malathion interfered with HIF-1α and steroid hormone synthases (CYP11A1, CYP17A1 and CYP19A1) by forming hydrogen bonds and hydrophobic interactions with these proteins. This study presents the first evidence that malathion triggers spermatogenesis dysfunction in mice through activating the HIF-1/MAPK/PI3K pathway, providing a comprehensive understanding of the reproductive toxicity risks associated with organophosphorus pesticides.

Orexin A protects against cerebral ischemia-reperfusion injury by enhancing reperfusion in ischemic cortex via HIF-1α-ET-1/eNOS pathway

The purpose of this study was to investigate the protective effect and underlying mechanism of orexin A on cerebral ischemia-reperfusion injury, specifically through vasodilation mediated by the hypoxia inducible factor-1α (HIF-1α)-Endothelin-1(ET-1)/endothelial nitric oxide synthase (eNOS) pathway. A model of middle cerebral artery occlusion was established in both wild-type SD rats with exogenous orexin A intervention and in orexin A transgenic rats. Neurological deficit scores and cerebral infarction areas were assessed, and ischemic cortical blood flow was monitored. Gene and protein expression levels of HIF-1α, HIF-2α, ET-1, and three types of NOS were detected using real-time RT-qPCR and Western blot analysis, respectively. Additionally, nitric oxide (NO) levels in the cortex were analyzed through biochemical detection methods. Orexin A demonstrated a protective effect by reducing cerebral infarction and improving neurological deficits, which was achieved by increasing cortical blood flow during reperfusion. This protective mechanism was associated with upregulated HIF-1α expression, downregulated ET-1 expression, upregulated eNOS expression, and increased NO production. This study demonstrates the protective effect of orexin A on cerebral ischemia-reperfusion injury, achieved by regulating the release of vasomotor substances to enhance cortical blood flow during reperfusion. These findings suggest that orexin A may represent a potential therapeutic strategy for ischemic stroke.

Hypoxia Reduces Mouse Urine Output via HIF1α-Mediated Upregulation of Renal AQP1

Introduction: Patients with acute mountain sickness (AMS) due to hypoxia at high altitudes often exhibit abnormal water metabolism. Hypoxia-inducible factors (HIFs) are major regulators of adaptive responses to hypoxia. As transcription factors, HIFs are involved in the regulation of erythropoiesis, iron metabolism, angiogenesis, energy metabolism, and cell survival by promoting the transcriptional expression of hundreds of target genes. Roxadustat, a novel drug for the treatment of anemia associated with chronic kidney disease (CKD), acts by inhibiting the degradation of HIFs to increase their protein levels. However, the clinical use of roxadustat is frequently associated with peripheral edema, suggesting the involvement of HIFs in regulating the body’s water balance possibly by modulating water reabsorption in the kidney. Methods: We first evaluated the effect of hypoxia (8% O2) on mouse urine output. We then performed in vitro experiments using hypoxia (1% O2) and roxadustat on mouse primary proximal tubular cells (mPTCs). The quantitative polymerase chain reaction, Western blot, and immunofluorescence were used to assess AQP1 mRNA and protein expression levels. Luciferase, Chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) were used to investigate the transcriptional regulation of AQP1 by HIF1α. Results: We found that mice exposed to hypoxia (8% O2) had significantly reduced urine volume compared to mice exposed to normoxia (21% O2). Hypoxia significantly elevated AQP1 expression at both mRNA and protein levels. In vitro experiments using mouse primary cultured proximal tubular cells (mPTCs) revealed that both hypoxia and roxadustat increased AQP1 expression. Mechanistically, overexpression of HIF1α, but not HIF2α, markedly increased AQP1 protein expression. Furthermore, the upregulation of AQP1 by hypoxia and roxadustat can be blocked by the HIF1α inhibitor PX-478 in mPTCs. Finally, we found that the AQP1 gene promoter contains a putative hypoxia response element and confirmed that AQP1 is a target gene of HIF1α using Luciferase reporter, ChIP, and EMSA assays. Conclusion: This study demonstrates that hypoxia can reduce the urine volume of mice via upregulating AQP1 expression by HIF1α in the proximal tubular epithelial cells. Our findings also suggest a potential mechanism involved in water metabolism disorders in patients with AMS and in patients with CKD receiving roxadustat treatment.

Hypoxia-inducible Factor-1α Pathway in Cerebral Ischemia: From Molecular Mechanisms to Therapeutic Targets.

Ischemic injury to the brain can result in a variety of life-threatening conditions, mortality, or varying degrees of disability. Hypoxia-inducible factor 1α (HIF 1α), an oxygen- sensitive transcription factor that controls the adaptive metabolic response to hypoxia, is a critical constituent of cerebral ischemia. It participates in numerous processes, such as metabolism, proliferation, and angiogenesis, and plays a major role in cerebral ischemia. Through the use of a number of different search engines like Scopus, PubMed, Bentham, and Elsevier databases, a literature review was carried out for investigating the pharmacological modulation of HIF-1α pathways for the treatment of cerebral ischemia. Various signalling pathways, such as Mitogen-activated protein kinase (MAPK), Janus kinase/ signal transducers and activators (JAK/STAT), Phosphoinositide-3-kinase (PI3-K), and cAMPresponse element binding protein (CREB) play a vital role in modulation of HIF-1α pathway, which helps in preventing the pathogenesis of cerebral ischemia. The pharmacological modulation of the HIF-1α pathway via various molecular signalling pathways, such as PI3-K, MAPK, CREB, and JAK/STAT activators, offer a promising prospect for future interventions and treatment for cerebral ischemia.

The impact of inflammatory and oxidative stress biomarkers on the sympathetic nervous system in severe coronary atherosclerosis.

The present study aimed at evaluating the association between sympathetic nervous system activation (SNS) and the severity of coronary artery disease (CAD). In addition, we tested the hypothesis that inflammation and oxidative stress influence the SNS activation. Adult patients with severe CAD scheduled for coronary artery bypass graft (CABG) surgery were enrolled. SYNTAX I score was calculated based on coronary angiography. Systemic activation of the SNS was estimated through circulating levels of norepinephrine (NE). Plasma levels of pro-inflammatory cytokines (IL 1β, IL 6 and HIF 1α) and oxidative stress molecules (SOD-1 and LOX-1) were obtained prior to surgery. Circulating NE levels were significantly correlated with the severity of CAD, as assessed by the SYNTAX I score (p 0.002; r 0.329). Elevated levels of circulating pro-inflammatory markers were significantly correlated with increased NE concentrations (for IL-1β: p < 0.001, r = 0.49; for IL-6 and NE: p = 0.003, r = 0.32; for HIF-1α and NE: p = 0.049, r = 0.21). Additionally, oxidative stress molecules were associated with circulating NE levels (for SOD-1 and NE: p = 0.016, r = 0.26; for LOX-1 and NE: p = 0.004, r = 0.31). In patients with CAD referred for CABG, SNS activation, indicated by plasma NE levels, was correlated with disease severity as assessed by the SYNTAX I score, as well as with markers of inflammation and oxidative stress. This suggests that inflammation, oxidative stress, and SNS activation form an interconnected network, with each component influencing the others. It might be of interest to develop a scoring system including inflammation and oxidative stress markers to identify patients that require a more aggressive approach to lower inflammation, oxidative stress and modulate the sympathetic nervous system. This could be of use especially in the setting of a scheduled intervention -such as CABG surgery.

Resveratrol enhances sensitivity of renal cell carcinoma to tivozanib: An in-vitro study

BackgroundSince tivozanib has many side effects in the treatment of kidney cancer, we decided to use resveratrol as a bioactive molecule with anticancer and antioxidant properties to make tivozanib more effective and also reduce its side effects in kidney cancer cell line. MethodIn this in vitro study, we evaluated the effect of tivozanib, resveratrol and tivozanib- resveratrol combination therapy in ACHN cell line as representatives of human kidney cancer. The assessment includes Hoechst dye staining, scratch-wound assay, 3D spheroid, 2D colony formation assay, flow cytometric analysis of apoptosis and DNA cell cycle, real-time PCR (BAX/BCL2, E-cadherin, Snail, HIF1α, VEGFC and KLK3 genes). ResultTo determine IC50 levels, ACHN cells was exposed to different concentration of tivozanib and resveratrol. Our data indicated that IC50 values for tivozanib (0.5 μM) and resveratrol (30 μM) with MTT in a dose and time-dependent manner. Due to the efficacy of resveratrol in combination with tivozanib, we used 20 μM resveratrol, and 0.25 μM tivozanib instead of 30 μM and 0.25 μM respectively. This data was approved by flow cytometry for ACHN cell line with 38.39, 14.74 and 66.06 percent apoptosis and 8.25, 5.12 and 15.6 percent subG1 for tivozanib, resveratrol and tivozanib-resveratrol combination respectively which was as a consequence of cell cycle arrest at G1/S phase. The treatment also reduced cells’ migration, fragmented nuclei, 3D spheroid and colony formation potentials in analyses. Evaluation of gene expression presented that the effect of the tivozanib and resveratrol combination in ACHN cell lines is completely different during the evaluation of apoptosis genes, BAX, P53 genes and E-Cadherin had significantly increased expression compared to single treatment groups (P < 0.01). Meanwhile, a significant decrease was observed in the expression of VEGFC and HIF1α genes in the combination group compared to the monotherapy groups (P < 0.001). ConclusionConsidering that resveratrol can increase the apoptosis of cancer cells alone and in combination with tivozanib and prevent the proliferation of cancer cells and also reduce the side effects of tivozanib, we suggest that resveratrol as a potential bioactive molecule can be used in treatment of kidney cancer should be used in combination with tivozanib.