HIF-1α Protein Research Articles

HIF-1α Regulates the Progression of Cervical Cancer by Targeting YAP/TAZ.

Cervical carcinoma is one of the serious pernicious cancers that influence women's health. Invasion and metastasis are the chief reason of poor prognosis of cervical carcinoma. Hypoxia-inducible factor-1α (HIF-1α) is a significant regulatory factor of intracellular oxygen supersession, and its expression or increased activity is closely related to the arise and expansion of various human tumors. However, the relationship between HIF-1α (hypoxia-inducible factor 1) and Hippo pathway target gene Yes-related protein (YAP) and transcriptional coactivator (TAZ) in cervical carcinoma remains unclear. Here, we studied the clinical correlation of HIF-1α and YAP/TAZ expression in normal tissues, cervical intraepithelial neoplasia (CIN), and cervical squamous cell carcinoma (CSCC). In order to analyze the role of HIF-1α in CCSC in vitro, SiHa cells with high expression of HIF-1α and C33a cells with low expression of HIF-1α were screened by detection. After transfection with lentivirus, HIF-1α levels were downregulated in SiHa cells and upregulated in C33a Cells, respectively. Then, the expression of HIF-1α in transfected cervical cancer cells Siha and C33a was detected by qRT-PCR and Western blot, and the expression of YAP/TAZ was detected in cervical squamous cell carcinoma cells after HIF-1α expression was altered. To explore HIF-1α role in cell proliferation, invasion, and metastasis, we examined the changes of cell function in cervical cancer cells with HIF-1α overexpression and inhibition by MTT assay, wound healing assay, Transwell test, and other cell function tests. At the same time, HIF-1α overexpression and HIF-1α inhibition cervical cancer cells were transplanted into nude mice, and tumors were isolated from the nude mice, and tumor volume and weight were observed. In conclusion, HIF-1α significantly promotes the proliferation, invasion, and migration of cervical carcinoma cells by upregulating YAP/TAZ. In addition, YAP/TAZ, the target gene of Hippo pathway, plays an important role in CCSC cells, pointing out that HIF-1α is provided with treatment potential for the treatment of CCSC.

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells.

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.

Exploring the biomolecular mechanism of resveratrol in the treatment of nephrotic syndrome based on network pharmacology

ObjectiveTo investigate the molecular mechanism underlying the therapeutic effect of resveratrol for nephrotic syndrome using various online network pharmacology databases. MethodsGenes related to resveratrol and nephrotic syndrome were downloaded from TCMSP, PubChem, and SwissTargetPrediction databases, and Venny2.1.0 was used to obtain the intersected genes. The protein interaction (PPI) network of the intersected genes was generated using the STRING database. The R package clusterprofiler was used to perform GO and KEGG enrichment analysis of the intersected genes. Cytoscape 3.7.2 software was used to construct compound-target-pathway-disease network models. The effect of resveratrol on HIF-1α protein expression in hypoxic 24 h mpc5 cells was detected by Western blot. ResultsThe intersection of resveratrol and nephrotic syndrome-related genes yielded Seventeen genes. The PPI network indicated that the intersected genes were correlated. Functional enrichment analysis yielded 371 biological processes, 69 molecular functions, 9 cellular components, and 112 related pathways, among which the HIF-1 pathway was the most significantly enriched. Western blot showed that resveratrol significantly reduced HIF-1α protein expression in mpc5 cells after 24 h of hypoxia. Conclusion Resveratrol may act as a therapeutic agent for nephrotic syndrome via the HIF-1 signaling pathway mediated by targeting HIF-1α.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTIONWhat are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies?SUMMARY ANSWERThere are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness).WHAT IS KNOWN ALREADYPatient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues.STUDY DESIGN, SIZE, DURATIONA prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy.PARTICIPANTS/MATERIALS, SETTING, METHODSThe changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21).MAIN RESULTS AND THE ROLE OF CHANCEAn increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities.WIDER IMPLICATIONS OF THE FINDINGSCareful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data.STUDY FUNDING/COMPETING INTEREST(S)The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women’s Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

Explore the Effect of Asthma Regulating HIF-1 Pathway on Sperm Quality Based on Rat Model.

This study is to verify the effect of asthma on sperm quality and explore its potential underlying mechanism. We randomly categorized the Sprague-Dawley (SD) rats into control (Group C) and asthma model (Group M) groups. Rats in the asthma model group were induced allergic asthma by intraperitoneal injection of ovalbumin solution. We evaluated the sperm motility and sperm concentration. The expression of the Interleukin-6 (IL6), phosphorylation-signal transducer and activator of transcription 3 (p-Stat3), and hypoxia-inducible factor-1α (HIF-1α) proteins and mRNAs in the testicular tissue was detected by western blotting and RT-qPCR. Compared with group C, sperm concentration and sperm motility in group M rats were significantly decreased (P < 0.05). Meanwhile, compared with group C, the expression levels of IL6, Stat3, and HIF-1α proteins and mRNAs in group M rats were significantly increased (P < 0.05). Asthma can regulate the HIF-1 signaling pathway, promoting the expression of IL6, Stat3, and HIF-1α protein and mRNAs, so as to promote sperm apoptosis and ultimately causing male infertility.

Network Pharmacology and Transcriptomic Sequencing Analyses Reveal the Molecular Mechanism of Sanguisorba officinalis Against Colorectal Cancer.

BackgroundColorectal cancer (CRC) is the most common malignant cancer worldwide. Sanguisorba officinalis has been shown to have anti-inflammatory, anti-bacterial, antioxidant, and anti-tumor effects, while its molecular mechanism against CRC remains unclear. The aim of this study is to explore the underlying mechanism of S. officinalis against CRC cell lines using network pharmacology and transcriptomic sequencing methods.MethodFirstly, the active ingredients and potential targets of S. officinalis against CRC were screened from databases. Secondly, the networks of ingredient–target, ingredient–target–CRC and protein–protein interaction were constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of network pharmacology and transcriptomic sequencing were performed. Finally, the effect of S. officinalis against CRC was verified by in vitro experiments.ResultsIn total, 14 active ingredients and 273 potential targets against CRC were identified in S. officinalis by network pharmacology. PI3K–Akt, HIF-1, and MAPK signaling pathways related to cell proliferation were regulated by S. officinalis in enrichment analyses and transcriptomic sequencing. In vitro, S. officinalis inhibited the proliferation and migration of CRC cells and arrested the cell cycle at the G0–G1 phase. The western blot showed that S. officinalis downregulated the expression of p-PI3K, p-Akt, HIF-1A, VEGFA, cyclin D1, c-Myc, and p-MAPK proteins in CRC cells.ConclusionIn conclusion, network pharmacology and transcriptomic sequencing analyses, in combination with in vitro studies, have been successfully applied to study the underlying mechanism of S. officinalis against CRC cells. Our results demonstrate that S. officinalis suppresses the proliferation, survival, and migration of CRC cells through regulating the PI3K–Akt, HIF-1, and MAPK signaling pathways.

Treatment of malignant pleural effusion in non-small cell lung cancer with VEGF-directed therapy

Background Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion. Methods In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by 18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry. Results The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted in vivo apoptosis and decreased microvessel density. Conclusions Mechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE. KEY MESSAGES Combined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVmax of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.

Endothelial PRMT5 plays a crucial role in angiogenesis after acute ischemic injury.

Arginine methylation mediated by protein arginine methyltransferases (PRMTs) has been shown to be an important posttranslational mechanism involved in various biological processes. Herein, we sought to investigate whether PRMT5, a major type II enzyme, is involved in pathological angiogenesis and, if so, to elucidate the molecular mechanism involved. Our results show that PRMT5 expression is significantly upregulated in ischemic tissues and hypoxic endothelial cells (ECs). Endothelial-specific Prmt5-KO mice were generated to define the role of PRMT5 in hindlimb ischemia–induced angiogenesis. We found that these mice exhibited impaired recovery of blood perfusion and motor function of the lower limbs, an impairment that was accompanied by decreased vascular density and increased necrosis as compared with their WT littermates. Furthermore, both pharmacological and genetic inhibition of PRMT5 significantly attenuated EC proliferation, migration, tube formation, and aortic ring sprouting. Mechanistically, we showed that inhibition of PRMT5 markedly attenuated hypoxia-induced factor 1-α (HIF-1α) protein stability and vascular endothelial growth factor–induced (VEGF-induced) signaling pathways in ECs. Our results provide compelling evidence demonstrating a crucial role of PRMT5 in hypoxia-induced angiogenesis and suggest that inhibition of PRMT5 may provide novel therapeutic strategies for the treatment of abnormal angiogenesis-related diseases, such as cancer and diabetic retinopathy.

Apigenin sensitizes radiotherapy of mouse subcutaneous glioma through attenuations of cell stemness and DNA damage repair by inhibiting NF-κB/HIF-1α-mediated glycolysis

We used radioresistant SU3-5R stem cells-inoculated subcutaneous glioma model to investigate the radiosensitization effect of apigenin. After treatment of glioma with apigenin 20 mg/kg for 12 days, irradiation 8 Gray twice or their combination, the tumor volume and weight were decreased, especially in the combination group. Apigenin inhibited the activities of glycolytic enzymes and expressions of nuclear factor kappa B (NF-κB) p65, hypoxia inducible factor-lα (HIF-1α), glucose transporter (GLUT)-1/3 and pyruvate kinase isozyme type M2 (PKM2) proteins in tumor tissues. After treatment of SU3-5R cells with apigenin 7.5 µM, the fluorescence intensity of CD133 positive cells was decreased, the percentage of cells with comet tails was increased, and the expressions of lipopolysaccharide-induced NF-κB p65, HIF-1α, GLUT-3 and PKM2 proteins were reduced. These results demonstrate that apigenin can sensitize the radiotherapy of glioma via the attenuations of cell stemness and DNA damage repair by inhibiting NF-κB/HIF-1α-mediated glycolytic enzymes and protein expressions.

MO067: Role of ANT2 in the Development of Obesity-Induced Chronic Kidney Disease

Abstract BACKGROUND AND AIMS Obesity, a widespread condition with an ever-increasing prevalence, imposes a great medical concern since it serves as a risk factor for a wide range of conditions, including the development of chronic kidney disease (CKD). Lipid accumulation and impaired fatty acid β-oxidation in the renal proximal tubular cells (RPTCs) as well as an overall mitochondrial dysfunction have been shown to induce kidney injury, inflammation and fibrosis, leading to the development of CKD. However, the molecular mechanisms linking mitochondrial dysfunction in RPTCs to obesity-induced CKD are not fully understood and require further examination. Adenine nucleotide translocases (ANTs), which transport ADP and ATP through the mitochondrial inner membrane, play an essential role in energy metabolism of eukaryotic cells. ANT2 (Slc25a5) mediates fatty acid-induced uncoupled respiration, and its complete deletion is embryonic lethal. Here we explore its specific role in RPTCs in the development of obesity-induced CKD. METHOD ANT2 protein levels were measured using western blot and immunohistochemistry analyses. RPTC-ANT2-/- animals were generated using the Cre/loxP system. The null mice and their wild-type littermate controls were fed with either a high-fat diet (HFD) or a standard diet (STD) for 24 weeks. Kidney function and lipid content were assessed by biochemical analysis. Histological techniques were used to visualize the morphological changes in the kidney as well as fibrogenesis. Kidney injury and inflammatory markers were measured via qPCR and ELISAs. Mitochondrial function was tested in primary mouse RPTCs extracted from the null and WT animals by measuring oxygen consumption rate via Seahorse as well as reactive oxygen species (ROS) production by DCFDA staining. RESULTS As evident from the double-stained immunofluorescence staining of mouse kidney, high levels of ANT2 were found in RPTCs. Interestingly, its protein expression was downregulated in HFD-fed WT mice. Nullification of ANT2 specifically in RPTCs prevented the obesity-induced kidney dysfunction as reflected by lowered albumin-to-creatinine ratio. This was associated with the improved renal morphology in HFD-fed RPTC-ANT2-/- mice, evident by the reduction in obesity-related increased glomerular and Bowman's space areas and mesangial expansion. Compared with their littermate controls, obese RPTC-ANT2-/- mice showed reduced renal KIM-1 and NGAL protein levels and downregulation in the expression levels of Mcp1 and Lcn2, markers of renal injury and inflammation, respectively. The null mice were also protected from obesity-induced fibrosis, measured by trichrome staining, as well as accumulation of triglycerides and cholesterol in the kidney. In addition, preserved mitochondrial function was found in primary mouse RPTCs lacking ANT2, measured by increased spare respiratory capacity and decreased ROS production. Lastly, downregulated Hif1α gene and protein expression levels were found in RPTCs and kidney tissue lacking ANT2. CONCLUSION Our findings introduce ANT2 in the RPTCs as a key player in the development of obesity-induced CKD. Deletion of RPTC-ANT2 protects the kidney from the deleterious effects of lipotoxicity. Therefore, targeted manipulation of renal mitochondrial metabolism, particularly via inhibiting ANT2, may represent a novel approach for the treatment of obesity-induced CKD.

MO282: Sigma-1 Receptor Agonists are Protective in Renal Ischemia/Reperfusion Injury

Abstract BACKGROUND AND AIMS Renal ischemia/reperfusion injury (IRI)-induced acute kidney injury is associated with high mortality and morbidity and effective therapies are lacking. We previously showed that Sigma-1 receptor (S1R) agonist fluvoxamine treatment is protective against renal IRI (Hosszu et al, 2017). Here in a rat model, we studied the renoprotective effect of specific, high affinity S1R agonists focusing on the S1R-Akt-eNOS signaling pathway and renal vasoregulation. METHOD 8-week old male Wistar rats (n = 8/group) were subjected to 50 min unilateral ischemia with contralateral nephrectomy, kidney tissue was harvested 24 h after reperfusion. 30 min prior to the ischemic insult rats were treated i.p. as follows: (i) isotonic saline as vehicle; (ii) highly specific, high affinity S1R agonist SA-4503; or (iii) SA-4503 + S1R antagonist NE100. Sham-operated rats were used as controls. Renal functional parameters (BUN, serum creatinine, serum aspartate aminotransferase, kidney injury molecule 1 (Kim1), neutrophil gelatinase-associated lipocalin (Ngal) were evaluated. Structural damage was assessed on PAS-stained kidney sections. Renal S1R, phospho-Akt and phospho-eNOS, Hif-1α protein levels and serum nitric oxide (NO) concentration were measured. In vitro experiments were performed on HK2 human proximal tubular epithelial cells treated with a 10 µM dose of S1R agonist SA-4503 or PRE-084. RESULTS Impaired kidney function and structural damage after I/R were ameliorated by SA-4503. Expressions of early and sensitive tubular injury markers Kim1 and Ngal were markedly less elevated in SA-4503-treated rats. This recovery was blocked with the addition of the S1R antagonist NE100. S1R, phospho-Akt and phospho-eNOS protein levels were significantly elevated in the kidneys of SA-4503 treated rats. Vasodilator nitric oxide concentration was reduced in the kidney after IRI, but returned to control levels in SA-4503-treated rats. Treatment with S1R agonists SA-4503 and PRE-084 increased the NO production of HK-2 cells. CONCLUSION The specific and high affinity S1R agonist SA-4503 acts directly on proximal tubular cells by activating the S1R-NOS system. Thereby SA-4503 is renoprotective by increasing vasodilative NO production and thus improving post-ischemic renal perfusion. Based on our data activation of S1R and downstream signaling pathways could provide a novel therapeutic option in renal IRI. Founding OTKA PD-131 637; FK-124 491; 2020–4.1.1.-TKP2020-6 183 069 269; 2020–4.1.1.-TKP2020-6 183 169 273; KDP-2020/1 019 145

Simultaneously targeting hyperinflammation and hyperglycemia following respiratory viral infection

Abstract Individuals with hyperglycemia are at great risk of developing exaggerated pulmonary inflammation following infection with respiratory viruses including SARS-CoV-2. Conversely, SARS-CoV-2 infection has been linked to the development of new onset of hyperglycemia and diabetes. Despite global vaccination efforts, SARS-CoV-2 infection continues to be a great public health challenge, and corticosteroids targeting hyper-inflammation remain the mainstay treatment for severe COVID-19. However, steroidal medicine arises the risk of hyperglycemia, limiting its usage. Here, we showed that a second-generation thiazolidines drug MSDC-0602K (MSDC), a mitochondrial pyruvate carrier inhibitor, simultaneously decreased excessive pulmonary inflammation and hyperglycemia in normal and obese hosts during influenza infection, as such, reducing host morbidity and mortality. scRNA-seq analysis revealed that MSDC treatment reduced lung macrophage inflammation, and myeloid-specific ablation of MPC2 led to dampened lung inflammation. Further, MSDC inhibited human lung macrophage inflammatory responses following SARS-CoV-2 infection, and dampened inflammation in lung autopsies of COVID-19 patients. Mechanistically, MSDC treatment destabilized HIF-1a protein, a transcription factor promoting inflammatory responses of lung macrophages, by repressing cellular Acetyl-CoA levels and HIF-1a acetylation. Our results suggest that MSDC has the great potential to treat severe COVID-19 or other respiratory viral infection, particularly in patients with underlying metabolic conditions, by simultaneously repressing hyper-cytokinemia and hyperglycemia. Supported by NIH AG069264, NIH AI150599

Role of Lysine-Specific Demethylase 1 in Metabolically Integrating Osteoclast Differentiation and Inflammatory Bone Resorption Through Hypoxia-Inducible Factor 1α and E2F1.

Hypoxia occurs in tumors, infections, and sites of inflammation, such as in the affected joints of patients with rheumatoid arthritis (RA). It alleviates inflammatory responses and increases bone resorption in inflammatory arthritis by enhancing osteoclastogenesis. The mechanism by which the hypoxia response is linked to osteoclastogenesis and inflammatory bone resorption is unclear. This study was undertaken to evaluate whether the protein lysine-specific demethylase 1 (LSD1) metabolically integrates inflammatory osteoclastogenesis and bone resorption in a state of inflammatory arthritis. LSD1-specific inhibitors and gene silencing with small interfering RNAs were used to inhibit the expression of LSD1 in human osteoclast precursor cells derived from CD14-positive monocytes, with subsequent assessment by RNA-sequencing analysis. In experimental mouse models of arthritis, inflammatory osteolysis, or osteoporosis, features of accelerated bone loss and inflammatory osteolysis were analyzed. Furthermore, in blood samples from patients with RA, cis-acting expression quantitative trait loci (cis-eQTL) were analyzed for association with the expression of hypoxia-inducible factor 1α (HIF-1α), and associations between HIF-1α allelic variants and extent of bone erosion were evaluated. In human osteoclast precursor cells, RANKL induced the expression of LSD1 in a mechanistic target of rapamycin-dependent manner. Expression of LSD1 was higher in synovium from RA patients than in synovium from osteoarthritis patients. Inhibition of LSD1 in human osteoclast precursors suppressed osteoclast differentiation. Results of transcriptome analysis identified several LSD1-mediated hypoxia and cell-cycle pathways as key genetic pathways involved in human osteoclastogenesis. Furthermore, HIF-1α protein, which is rapidly degraded by the proteasome in a normoxic environment, was found to be expressed in RANKL-stimulated osteoclast precursor cells. Induction of LSD1 by RANKL stabilized the expression of HIF-1α protein, thereby promoting glycolysis, in conjunction with up-regulation of the transcription factor E2F1. Analyses of cis-eQTL revealed that higher HIF-1α expression was associated with increased bone erosion in patients with RA. Inhibition of LSD1 decreased pathologic bone resorption in mice, both in models of accelerated osteoporosis and models of arthritis and inflammatory osteolysis. LSD1 metabolically regulates osteoclastogenesis in an energy-demanding inflammatory environment. These findings provide potential new therapeutic strategies targeting osteoclasts in the management of inflammatory arthritis, including in patients with RA.

Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1\u03b2

BackgroundThe main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer.MethodsThis project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed.ResultsBaP can enhance the binding ability of HIF-1α protein to HIF-1β/ARNT in a dose-dependent manner without CoCl2. However, the binding ability of AhR protein to HIF-1β/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl2.ConclusionIt is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.

Protective Effect of Brassica rapa Polysaccharide against Acute High-Altitude Hypoxia-Induced Brain Injury and Its Metabolomics.

Brassica rapa L., a traditional Tibetan medicine, has been wildly used for treating plateau disease. Polysaccharide is an important chemical component in B. rapa. The present study aimed to evaluate the effect of B. rapa polysaccharide (BRP) against acute high-altitude hypoxia (AHH) induced brain injury and its metabolic mechanism. The rats were randomly divided into six groups: control group, AHH group, Hongjingtian oral liquid group, and three BRP groups (38, 75, and 150 mg/kg/d). Serum levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and lactate dehydrogenase (LDH) were detected by commercial biochemical kits. Hippocampus and cortex histopathological changes were observed by H&E staining and Nissl staining. Neuronal apoptosis was observed by TUNEL staining. The protein and gene expression of Caspase-3, Bax, Bcl-2, p-PI3K, PI3K, p-Akt, Akt, HIF-1α, microRNA 210, ISCU1/2, and COX10 were detected by western blotting and qRT-PCR. Then, a brain metabolomics method based on UPLC-Q-Exactive-MS was performed to discover potential biomarkers and analyze metabolic pathways. It was found that BRP decreased levels of MDA, LDH, and GSSG, increased GSH and SOD, reduced the pathological changes, inhibited apoptosis, and activated the PI3K/Akt/HIF-1α signaling pathway as evidenced by increased phosphorylation of PI3K and Akt, enhanced protein expression of HIF-1α and gene levels of microRNA210, ISCU1/2, and COX10. Furthermore, 15 endogenous potential biomarkers were identified in the brain through metabolomics analysis. BRP can regulate 7 potential biomarkers and the corresponding metabolic pathways were mainly associated with pyruvate metabolism and glycolysis/gluconeogenesis. Collectively, BRP has a clear protective effect on AHH-induced brain injury and its mechanisms may be related to ameliorate oxidative stress injury, inhibit apoptosis by activating PI3K/Akt/HIF-1α signaling pathway, and reverse metabolic pathway disturbances.

Life-stage dependent histological characterization of liver HIF-1α expression in the Indonesian shortfin eel (Anguilla bicolor bicolor McClelland, 1884)

The Indonesian shortfin eel (Anguilla bicolor bicolor McClelland, 1884) is catadromous fish. The developmental phase of the fish is influenced by the availability and type of feed, and dissolved oxygen levels (DO). Oxygen level fluctuations during migration from sea to fresh waters can cause hypoxia and trigger activation of the Hypoxia-inducible factor 1 alpha gene (HIF-1α). Therefore, undertanding HIF-1α expression is very important in understanding the life cycle and physiology of this organism. This study aimed to perform a histological comparison between the liver structure of eel in the elver eel phase to silver eel and compared the expression of HIF-1α in the liver. The eels were collected from Kulon Progo Regency, Segara Anakan in Cilacap, and Cangkringan in Sleman, respectively. The eel livers were prepared with standard paraffin method. The samples were stained with Hematoxylin-Eosin, Mallory Acid Fuchsin and Periodic Acid-Schiff Alcian Blue for histological observation. The HIF-1α protein expression was detected using immunohistochemistry. The results showed that the structure of liver elver, yellow, and silver eel shared similar tubular hepatocytes. Moreover, there was no significant differences in the parenchymal structure of liver of all stage of eel. Mucin levels were different in each life cycle phase. HIF-1α expression varied by eel phase, with the elver eel showing weak expression, yellow stage showing moderate expression, and silver eels displaying the highest levels of expression. We conclude that the highest expression of HIF-1α was found in the silver eel, which resides in freshwater environments and is found in freshwater while returning to the sea for reproduction.

Wogonoside alleviates high glucose-induced dysfunction of retinal microvascular endothelial cells and diabetic retinopathy in rats by up-regulating SIRT1

To investigate the effects of wogonoside on high glucose-induced dysfunction of human retinal microvascular endothelial cells (hRMECs) and streptozotocin (STZ)-induced diabetic retinopathy in rats and explore the underlying molecular mechanism. HRMECs in routine culture were treated with 25 mmol/L mannitol or exposed to high glucose (30 mmol/L glucose) and treatment with 10, 20, 30, 40 μmol/L wogonoside. CCK-8 assay and Transwell assay were used to examine cell proliferation and migration, and the changes in tube formation and monolayer cell membrane permeability were tested. ROS, NO and GSH-ST kits were used to evaluate oxidative stress levels in the cells. The expressions of IL-1β and IL-6 in the cells were examined with qRT-PCR and ELISA, and the protein expressions of VEGF, HIF-1α and SIRT1 were detected using Western blotting. We also tested the effect of wogonoside on retinal injury and expressions of HIF-1α, ROS, VEGF, TNF-α, IL-1β, IL-6 and SIRT1 proteins in rat models of STZ-induced diabetic retinopathy. High glucose exposure caused abnormal proliferation and migration, promoted angiogenesis, increased membrane permeability (P < 0.05), and induced inflammation and oxidative stress in hRMECs (P < 0.05). Wogonoside treatment concentration-dependently inhibited high glucose-induced changes in hRMECs. High glucose exposure significantly lowered the expression of SIRT1 in hRMECs, which was partially reversed by wogonoside (30 μmol/L) treatment; interference of SIRT1 obviously attenuated the inhibitory effects of wogonoside against high glucose-induced changes in proliferation, migration, angiogenesis, membrane permeability, inflammation and oxidative stress in hRMECs. In rat models of STZ-induced diabetic retinopathy, wogonoside effectively suppressed retinal thickening (P < 0.05), alleviated STZ-induced retinal injury, and increased the expression of SIRT1 in the retinal tissues (P < 0.001). Wogonoside alleviates retinal damage caused by diabetic retinopathy by up-regulating SIRT1 expression.

An Enhanced Photothermal Therapeutic Iridium Hybrid Platform Reversing the Tumor Hypoxic Microenvironment.

As hypoxia is closely associated with tumor progression, proliferation, invasion, metastasis, and strong resistance to therapy, regulating and overcoming the hypoxia tumor microenvironment are two increasingly important aspects of tumor treatment. Herein, we report a phototherapeutic platform that uses the organic photosensitizer diketopyrrolopyrrole (DPP) derivative and inorganic iridium salts (IrCl3) with photothermal activity and the capacity to decompose H2O2 efficiently. The characterization of their photophysical properties proved that DPP-Ir nanoparticles are capable of remarkable near-infrared (NIR) absorption, and compared to DPP nanoparticles, the photothermal conversion efficiency (PCE) increases from 42.1% in DPP nanoparticles to 67.0% in DPP-Ir nanoparticles. The hybrid nanoparticles utilize the catalytic decomposition of endogenous H2O2 to produce oxygen for the downregulation of the hypoxia-inducible factor 1 subunit alpha (HIF-1α) protein, which could reverse the tumor hypoxic microenvironment. Benefiting from the excellent optical properties and good biocompatibility, the hybrid platform exhibits efficient photothermal therapeutic effects as well as good biological safety. In conclusion, such a hybrid platform could improve photothermal therapy against cancer.

TLR4 Agonist and Hypoxia Synergistically Promote the Formation of TLR4/NF-κB/HIF-1α Loop in Human Epithelial Ovarian Cancer.

Inflammation and hypoxia are involved in numerous cancer progressions. Reportedly, the toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathway and hypoxia-inducible factor-1α (HIF-1α) are activated and closely related to the chemoresistance and poor prognosis of epithelial ovarian cancer (EOC). However, the potential correlation between TLR4/NF-κB and HIF-1α remains largely unknown in EOC. In our study, the possible positive correlation among TLR4, NF-κB, and HIF-1α proteins was investigated in the EOC tissues. Our in vitro results demonstrated that LPS can induce and activate HIF-1α through the TLR4/NF-κB signaling in A2780 and SKOV3 cells. Moreover, hypoxia-induced TLR4 expression and the downstream transcriptional activity of NF-κB were HIF-1α-dependent. The cross talk between the TLR4/NF-κB signaling pathway and HIF-1α was also confirmed in the nude mice xenograft model. Therefore, we first proposed the formation of a TLR4/NF-κB/HIF-1α loop in EOC. The positive feedback loop enhanced the susceptibility and responsiveness to inflammation and hypoxia, which synergistically promote the initiation and progression of EOC. The novel mechanism may act as a future therapeutic candidate for the treatment of EOC.

Naoluo Xintong Decoction Ameliorates Cerebral Ischemia-Reperfusion Injury by Promoting Angiogenesis through Activating the HIF-1α/VEGF Signaling Pathway in Rats.

Background Naoluo Xintong decoction (NLXTD) is a traditional Chinese medicine (TCM) formula which has been used to improve neuronal functional recovery after cerebral ischemic stroke. However, the molecular mechanism underlying NLXTD's amelioration of ischemic stroke remains unclear. The present study was designed to explore the effect and mechanism of NLXTD on brain angiogenesis in a rat model with cerebral ischemia-reperfusion (I/R) injury targeting the hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway. Materials and Methods Cerebral I/R model was established by the classical middle cerebral artery occlusion (MCAO) method. Sprague-Dawley (SD) male rats (n = 80) were randomly divided into the sham-operation group, the model group, the HIF-1α inhibitor 2-methoxyestradiol (2ME2) group, the 2ME2 with NLXTD group, and the NLXTD group. Neurological deficit test, TTC staining, H&E staining, TUNEL staining, immunohistochemistry (IH), immunofluorescence (IF), western blot, and quantitative RT-PCR were performed to evaluate the effect of NLXTD after MCAO. Results Administration of NLXTD significantly decreased neuron deficiency scores, reduced brain infarct volume, and lowered damaged and apoptotic cells after brain I/R injury in rats. Meanwhile, NLXTD had a protective effect on angiogenesis by increasing the MVD and the expressions of BrdU and CD34, which enhanced the number of endothelial cells in the ischemic penumbra brain. NLXTD treatment significantly raised the protein and mRNA levels of HIF-1α, VEGF, VEGFR2, and Notch1 compared with the model treatment. In contrast, a specific HIF-1α inhibitor, 2ME2, inhibited the improvement of neurological function and angiogenesis in NLXTD-induced rats with cerebral I/R injury, suggesting that NLXTD played a positive role in ischemic brain injury by activating the HIF-1α/VEGF signaling pathway. Conclusions NLXTD exerts neuroprotection targeting angiogenesis by upregulating the HIF-1α/VEGF signaling pathway on cerebral I/R injury rats.