Expression Levels Of HIF-1α Research Articles

Effects of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens: A systematic quantitative assessment

IntroductionBlood samples are the most common biospecimen in biobanks, and RNA from such blood samples is an important material for biomedical research. High-quality RNA is essential for consistent, reliable results. Preanalytical environmental conditions can affect the quality of blood RNA. Here, we carried out a quantitative assessment of the influence of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens in biobanks. MethodsBefore RNA purification, blood samples from volunteers were exposed to 4 °C for 2, 6, 12, 24, or 48 h, 3 days, or 1 week, or exposed to room temperature (22–30 °C) for 1, 2, 6, 12, or 24 h. Hemolyzed samples were collected from laboratory department and some of them were prepared using the freeze-thaw method. After exposure to different preanalytical environmental conditions, the RNA simple Total RNA Kit was used to purify the RNA, following which a NanoDrop™ One and Qsep 100 Bio-Fragment Analyzer were used to assess RNA concentration, purity, and integrity. In addition, a part of the RNA was immediately reverse transcribed into cDNA when it was purified, then the relative expression levels of 18S, ACTB, HIF1α, HMOX1, and MKI67 were determined by real-time quantitative PCR. Finally, 30 blood samples were collected from the surplus samples in our laboratory department to assess their RNA quality without knowledge of their storage conditions (duration/temperature). ResultsFor blood samples stored at 4 °C, there was a significant difference between the RNA integrity after 1 week compared to after 2 h. For blood samples stored at room temperature (22–30 °C), the RNA integrity was also significantly different at 6 h and 0 h. Hemolysis caused by freeze-thawing severely affected RNA quality, whereas clinical hemolysis generally produced no significant effects. Moreover, expression of 18S, ACTB, HIF1α, HMOX1, and MKI67 in whole blood stored under different conditions showed irregular changes, suggesting that preservation conditions are also important for gene expression. ConclusionRNA integrity was qualified for blood samples stored at 4 °C for up to 72 h or at room temperature (22–30 °C) for up to 2 h. Hemolysis usually does not affect the RNA quality of blood samples unless the hemolysis method damages leukocytes.

Study on the mechanism of early pancreatic exocrine function changes in severely scalded rats

Study on the mechanism of early pancreatic exocrine function changes in severely scalded rats

Prolonged stretching of rat uteri causes hypoxia and inhibits contractility via potassium channel TREK1.

During pregnancy, uterine kept quiescence along with uterine overdistention before labor. Prolonged stretching induced uterus myometrial hypoxia, increased TREK1 expression, and relaxed the myometrium, which may contribute to uterine quiescence and atony during pregnancy. The mechanisms underlying pre-labor uterine quiescence and uterine atony during overdistention are unclear. TREK1 (a two-pore domain potassium channel) and hypoxia-inducible factor-1α (HIF-1α) are activated by mechanical stretch, and their expression is upregulated by decreased uterine contractility. HIF-1α is a nuclear factor which regulates numerous target proteins, but whether it regulates TREK1 during the uterine stretch to cause uterine quiescence and/or atony is unclear. We investigated uterine contractility at different gestational stages in rats, as well as in non-pregnant uteri, which were induced by prolonged stretching and hypoxia. We also assessed the effects of incubating the uteri with or without echinomycin or l-methionine. Moreover, we analyzed HIF-1α and TREK1 expression levels in each group, as well as at various gestational stages of pregnant human uteri. We found that contractility was significantly decreased in pregnant uteri when compared with non-pregnant uteri, and this decrease was associated with increases in HIF-1α and TREK1 expression levels. HIF-1α and TREK1 expression levels in human uteri increased with the gestational length. Decreased uterine contractility and increased HIF-1α and TREK1 expression levels were also observed in non-pregnant rat uteri under 8 g of stretching tension or hypoxia. Inhibition of hypoxia with echinomycin restored normal uterine contractility, while HIF-1α and TREK1 protein expression remained reduced. TREK1 inhibition with l-methionine also restored uterine contractility under tension or hypoxia. In conclusion, we demonstrated that prolonged stretching induces myometrial hypoxia, increases TREK1 expression, and relaxes the myometrium, which may contribute to uterine quiescence and atony.

Decellularized Mouse Liver as a Small-Scale Scaffold for the Creation of a Miniaturized Human Liver

The liver is the main organ responsible for drug metabolism; hepatocyte-based culture models play a fundamental role in understanding liver physiology. While different types of two-dimensional and three-dimensional liver models have been developed, the failure to accurately mimic native liver, including extracellular matrix (ECM) composition, complex structure, and rich vascular network as well as shortage of liver donors, impede their clinical translation. Herein, we have realized a “miniature human liver” based on the infusion of primary human hepatocytes into a decellularized liver template (DC-liver) made from the right liver lobe of mouse. We performed detergent-based decellularization of the mouse liver sections via portal vein (PV) perfusion and confirmed the successful removal of cell content, and the preservation of the vascular network. Subsequently, the DC-liver templates were recellularized at varying cell densities, including 3 × 106 and 1 × 107 cells/mL, and liver specific gene expression was assessed. Overall, hepatocytes in the recellularized liver constructs (RC-liver) expressed liver-specific mRNA expression markers (Hnf4α, Alb) at a level comparable to the unseeded, freshly isolated hepatocytes and to hepatocytes seeded inside collagen gels. However, the RC-liver with the highest cell density exhibited poor cell distribution and blockage of the PV, in general, hepatocytes showed elevated levels of Cyp1a1. Further analysis hinted at hypoxic conditions inside the constructs, showing higher mRNA expression levels of hypoxia-related genes (Hif-1α, Casp3, Zo-1). Fortunately, oxygen supplementation appeared to alleviate hypoxia, which markedly reduced expression levels of Hif-1α and Cyp1α1. As a proof-of-concept, primary human hepatocytes were also seeded into the DC-liver templates, and we could confirm liver specific mRNA expression (HNF4A, ALB, CYP3A4). Altogether, the above results indicate a profound potential in the use of DC-liver tissue for the fabrication of a miniature human liver, which may have potential application prospects for regenerative medicine, tissue engineering (TE) and other related disciplines.

KDM3A, a Novel Blood-Based Biomarker in Colorectal Carcinogenesis.

Colorectal cancer (CRC) is one of the leading causes of cancer-linked deaths globally. The determination of biomarkers is important in the prognosis and treatment of CRC. Previous studies emphasized the relationship between hypoxia and CRC in humans, and there is strong evidence that this process is strongly related to HIF-1. KDM3A is a histone demethylase that could directly bind to HIF-1α, a subunit of HIF-1. This study aimed to reveal whether the expression level of the KDM3A gene could be used as a predictor of CRC. The expression levels of HIF-1α, KDM3A, and Epithelial-Mesenchymal Transition (EMT) genes were evaluated by qRT-PCR in leukocyte samples of 50 CRC patients in different stages and 50 healthy controls. HIF-1α and KDM3A expression levels were significantly higher in the CRC group, compared to the controls. Slug and ZEB-1 genes, the mesenchymal markers, showed the same significance pattern between groups. We acquired 0.664 AUC with 54% sensitivity and 85.4% specificity for separating controls from CRC patients by using the KDM3A expression levels in ROC analysis. This data support that KDM3A could be a novel supplementary biomarker in diagnosis of CRC, which could be noninvasively detected in circulation.

Eriocitrin attenuates sepsis-induced acute lung injury in mice by regulating MKP1/MAPK pathway mediated-glycolysis.

Metabolic reprogramming has been shown to aggravate sepsis-induced acute lung injury. In particular, enhanced glycolysis is closely associated with inflammation and oxidative stress. Eriocitrin (ERI) is a natural flavonoid found in citrus fruit that exhibits various pharmacological activities, with antioxidant, anti-inflammatory, anti-diabetic, and anti-tumor properties. However, the role of ERI in lung injury is not well understood. We established a septic mouse model of acute lung injury (ALI) using lipopolysaccharide (LPS) for induction. Primary peritoneal macrophages were isolated to verify the relevant molecular mechanism. Tissues were assessed for lung pathology, pro-inflammatory cytokines, markers of oxidative stress, and protein and mRNA expression levels. In vivo experiments showed that ERI effectively alleviated LPS-induced pathological injury, suppress the inflammatory response (TNF-α, IL-1β, IL-6 levels) and decreased oxidative stress (MDA, ROS) in murine lung tissue. In vitro, ERI increased the resistance of LPS-treated cells to excessive inflammation and oxidative stress by inhibiting the enhancement of glycolysis (indicated by expression levels of HIF-1α, HK2, LDHA, PFKFB3, and PKM2). Specifically, the beneficial effects of ERI following LPS-induced lung injury occurred through promoting the expression of MKP1, which mediates the inactivation of the MAPK pathway to inhibit enhanced glycolysis. These results demonstrate that ERI has a protective effect on sepsis-induced ALI by regulating MKP1/MAPK pathway mediated-glycolysis. Hence, ERI is a promising candidate against ALI via inhibiting glycolysis.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Poly-D,L-Lactic Acid Stimulates Angiogenesis and Collagen Synthesis in Aged Animal Skin.

Angiogenesis promotes rejuvenation in multiple organs, including the skin. Heat shock protein 90 (HSP90), hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) are proangiogenic factors that stimulate the activities of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase 1/2 (ERK1/2). Poly-D,L-lactic acid (PDLLA), polynucleotide (PN), and calcium hydroxyapatite (CaHA) are dermal fillers that stimulate the synthesis of dermal collagen. However, it is not yet known whether these compounds promote angiogenesis, which leads to skin rejuvenation. Here, we evaluated whether PDLLA, PN, and CaHA stimulate angiogenesis and skin rejuvenation using H2O2-treated senescent macrophages and endothelial cells as an in vitro model for skin aging, and we used young and aged C57BL/6 mice as an in vivo model. Angiogenesis was evaluated via endothelial cell migration length, proliferation, and tube formation after conditioned media (CM) from senescent macrophages was treated with PDLLA, PN, or CaHA. Western blot showed decreased expression levels of HSP90, HIF-1α, and VEGF in senescent macrophages, but higher expression levels of these factors were found after treatment with PDLLA, PN, or CaHA. In addition, after exposure to CM from senescent macrophages treated with PDLLA, PN, or CaHA, senescent endothelial cells expressed higher levels of VEGF receptor 2 (VEGFR2), PI3K, phosphorylated AKT (pAKT), and phosphorylated ERK1/2 (pERK1/2) and demonstrated greater capacities for cell migration, cell proliferation, and tube formation. Based on the levels of 4-hydroxy-2-nonenal, the oxidative stress level was lower in the skin of aged mice injected with PDLLA, PN, or CaHA, while the tumor growth factor (TGF)-β1, TGF-β2, and TGF-β3 expression levels; the density of collagen fibers; and the skin elasticity were higher in the skin of aged mice injected with PDLLA, PN, or CaHA. These effects were greater in PDLLA than in PN or CaHA. In conclusion, our results are consistent with the hypothesis that PDLLA stimulates angiogenesis, leading to the rejuvenation of aged skin. Our study is the first to show that PDLLA, PN, or CaHA can result in angiogenesis in the aged skin, possibly by increasing the levels of HSP90, HIF-1α, and VEGF and increasing collagen synthesis.

Uncover DNA damage and repair-related gene signature and risk score model for glioma

Background Glioma is a common primary central nervous system tumor with complex pathogenesis. DNA damage and repair (DDR) is widely involved in regulating cell proliferation and tumorigenesis by correcting and repairing DNA damage mechanisms. Recent studies have reported the following properties in cancer cells in glioma, increased DNA damage and reduced DNA repair capacity. However, the relationship between glioma and DDR-related genes was unclear. Methods DDR-related risk score model was built. The validity of this model was validated in detail through the Kaplan-Meier survival analysis, tumor mutational burden (TMB) analysis, immune cell infiltration, sensitivity to treatment regimens. Moreover, the model’s adaptability was validated in different glioma data cohorts and different glioma subgroups. To further investigate the molecular mechanism of one of DDR-related gene (NUDT1) in glioma, U251 cell was used for the knockdown experiment, followed by MTT, wound healing and transwell analysis. Results Ten prognostic-related DDR-related signature genes were obtained, including EID3, MGMT, YWHAG, PMS1, SHPRH, HUS1, NUDT1, GADD45G, APEX1 and FAM175A. The RT-qPCR results suggested that the latter five genes were highly expressed in glioma patients. Interestingly, high TMB score had longer survival. In high-risk score groups, reduced immune cell infiltration in the tumor microenvironment lead to poorer patient outcomes. Sensitivity to treatment regimens analysis indicated that low-risk score groups were more sensitive to chemotherapeutics. Moreover, the risk score model had a good prediction effect on different glioma datasets and different glioma subgroups. In vitro mechanism study showed that knockdown of NUDT1 reduced tumorigenesis. Furthermore, knockdown of NUDT1 remarkably reduced the expression level of HIF-1α. Conclusion DDR-related risk score model built-in this work has good predictive performance for glioma. Key messages Ten prognostic-related DDR-related signature genes were obtained, including EID3, MGMT, YWHAG, PMS1, SHPRH, HUS1, NUDT1, GADD45G, APEX1 and FAM175A. In high-risk score groups, reduced immune cell infiltration in the tumor microenvironment leads to poorer patient outcomes. The risk score model had a good prediction effect on different glioma datasets and different glioma subgroups. Knockdown of NUDT1 reduced tumorigenesis of glioma and remarkably reduced the expression level of HIF-1α.

Identification and clinical validation of hypoxia-inducible factor -1α protein as the potential biomarker in patients with sepsis.

Sepsis is a complex disease characterized by an inflammatory response and tissue hypoxia. Hypoxia-inducible factor-1α (HIF-1α) expression level is regulated by hypoxia and inflammation. This study aimed to explore the correlation between HIF-1α expression level and sepsis by bioinformatics analysis and clinical investigation. Bioinformatics tools were used to identify differentially expressed genes (DEGs) between sepsis and non-sepsis groups using the Gene Expression Omnibus (GEO) dataset. A clinical investigation was carried out to validate HIF-1α protein level in 54 non-septic patients and 173 septic patients who were followed up for 28 days. Bioinformatics analysis revealed that HIF-1α mRNA level was significantly different between septic and non-septic patients (P < 0.05). Consistent with the study hypothesis, higher HIF-1α levels in plasma were found in septic patients compared with those in non-septic patients. The diagnostic accuracy for sepsis, as quantified by the area under the curve, was 0.926 (0.885-0.968) for HIF-1α expression level combined with oxygen saturation to fraction of inspired oxygen (SpO2/ FiO2), white blood cell (WBC) and blood urea nitrogen (BUN). The HIF-1α expression level was also significantly correlated with the severity of the disease. The results of the restricted cubic splines (RCS) model indicated a U-shaped relationship between HIF-1α expression level and intensive care unit (ICU) mortality. Univariate and multivariate linear regression analyses indicated that septic patients with the elevated HIF-1α expression levels had shorter length of ICU stay versus those with the lower HIF-1α expression levels. HIF-1α expression level can be used for diagnosing disease, assessing severity, and predicting length of ICU stay in septic patients.

Effect of diosgenin on mTOR/FASN/HIF-1α/VEGFA expression in rats with non-alcoholic fatty liver disease

The present study aimed to investigate the effect of diosgenin on mammalian target of rapamycin(mTOR), fatty acid synthase(FASN), hypoxia inducible factor-1α(HIF-1α), and vascular endothelial growth factor A(VEGFA) expression in liver tissues of rats with non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin on lipogenesis and inflammation in NAFLD. Forty male SD rats were divided into a normal group(n=8) fed on the normal diet and an experimental group(n=32) fed on the high-fat diet(HFD) for the induction of the NAFLD model. After modeling, the rats in the experimental group were randomly divided into an HFD group, a low-dose diosgenin group(150 mg·kg~(-1)·d~(-1)), a high-dose diosgenin group(300 mg·kg~(-1)·d~(-1)), and a simvastatin group(4 mg·kg~(-1)·d~(-1)), with eight rats in each group. The drugs were continuously given by gavage for eight weeks. The levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were detected by the biochemical method. The content of TG and TC in the liver was detected by the enzyme method. Enzyme-linked immunosorbent assay(ELISA) was used to measure interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the serum. Lipid accumulation in the liver was detected by oil red O staining. Pathological changes of liver tissues were detected by hematoxylin-eosin(HE) staining. The mRNA and protein expression levels of mTOR, FASN, HIF-1α, and VEGFA in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction(PCR) and Western blot, respectively. Compared with the normal group, the HFD group showed elevated body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P&lt;0.01), increased lipid accumulation in the liver(P&lt;0.01), obvious liver steatosis, up-regulated mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.01), and increased protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.01). Compared with the HFD group, the groups with drug treatment showed lowered body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P&lt;0.05, P&lt;0.01), reduced lipid accumulation in the liver(P&lt;0.01), improved liver steatosis, decreased mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.05, P&lt;0.01), and declining protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.01). The therapeutic effect of the high-dose diosgenin group was superior to that of the low-dose diosgenin group and the simvastatin group. Diosgenin may reduce liver lipid synthesis and inflammation and potentiate by down-regulating the mTOR, FASN, HIF-1α, and VEGFA expression, playing an active role in preventing and treating NAFLD.

PD23-03 IMPAIRED NITRIC OXIDE MECHANISMS UNDERLYING LOWER URINARY TRACT DYSFUNCTION IN AGING RATS

You have accessJournal of UrologyCME1 Apr 2023PD23-03 IMPAIRED NITRIC OXIDE MECHANISMS UNDERLYING LOWER URINARY TRACT DYSFUNCTION IN AGING RATS Daisuke Gotoh, Kenta Onishi, Yosuke Morizawa, Shunta Hori, Yasushi Nakai, Makito Miyake, Kazumasa Torimoto, Fujimoto Kiyohide, and Naoki Yoshimura Daisuke GotohDaisuke Gotoh More articles by this author , Kenta OnishiKenta Onishi More articles by this author , Yosuke MorizawaYosuke Morizawa More articles by this author , Shunta HoriShunta Hori More articles by this author , Yasushi NakaiYasushi Nakai More articles by this author , Makito MiyakeMakito Miyake More articles by this author , Kazumasa TorimotoKazumasa Torimoto More articles by this author , Fujimoto KiyohideFujimoto Kiyohide More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003296.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Nitric oxide (NO) promotes the intracellular production of cyclic guanosine monophosphate (cGMP) in smooth muscles, reduces the intracellular calcium concentration, and enhances smooth muscle relaxation. However, it remains unknown whether changes in the expression of NO-related molecules are associated with the development of lower urinary tract dysfunction (LUTD) with age. Therefore, we examined changes in bladder and urethral activities, and in NO-related molecule expression using aging rats. METHODS: Fourteen Female Sprague–Dawley rats were divided into two age groups: (A) young rats (12 weeks old, n=8) and (B) aging rats (15 months old, n=6). In both groups, 24 h voiding assay was performed, and the urodynamic parameters were evaluated using cystometry (CMG) and urethral perfusion pressure (UPP) recordings. mRNA expression levels of NO-, ischemia-, and inflammation-related markers in bladder and urethra tissues were assessed, and the cGMP level in the urethra was measured. RESULTS: There was no significant difference in the 24-h voiding assay results between the groups. In CMG, the number of non-voiding contractions (NVCs) per voiding cycle and post-void residual (PVR) were significantly higher, and voiding efficiency (VE) was significantly lower in Group B than in Group A (Figure 1A, B). In the UPP recordings, urethral pressure (UP) reduction and high frequency oscillation (HFO) amplitudes were both significantly lower in Group B than in Group A (Figure 1C, D). In molecular studies, mRNA expression levels of HIF-1α, VEGF, and TGF-β1 in the bladder were significantly higher in Group B than in Group A (Figure 2A). mRNA expression levels of NOS1 and PKG were significantly lower in Group B than in Group A (Figure 2B). In addition, the concentration of cGMP in the urethra was significantly lower in Group B than in Group A (Figure 2C). CONCLUSIONS: Aging rats would be a useful model for the study of the natural progression of age-related LUTD, for which impaired NO-mediated transmitter function is likely to be an important mechanism. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e671 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daisuke Gotoh More articles by this author Kenta Onishi More articles by this author Yosuke Morizawa More articles by this author Shunta Hori More articles by this author Yasushi Nakai More articles by this author Makito Miyake More articles by this author Kazumasa Torimoto More articles by this author Fujimoto Kiyohide More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

A cell-permeable peptide inhibitor of p55PIK signaling alleviates suture-induced corneal neovascularization and inflammation

To prepare an ophthalmic solution with a cell-permeable TAT peptide (TAT-N24) as the main cell-permeable peptide inhibitor of p55PIK signaling and observe its therapeutic effect on suture-induced corneal neovascularization (CNV) in rats. Sprague-Dawley rats were used to establish a corneal suture (CS) model of CNV. The vehicle and 0.9% TAT-N24 ophthalmic solution was topically administered. CNV induction was assessed on the basis of the clinical performance of each group. Hematoxylin-eosin staining was used to observe pathological changes, and immunohistochemical staining and confocal immunofluorescence were used to determine the localization of factors associated with corneal tissue. The mRNA expression levels of hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF-A), nuclear transcription factor κB (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin (IL)-6 were determined using real-time quantitative polymerase chain reaction. Western blotting was performed to detect the protein expression levels of HIF-1α and NF-κB p65. TAT-N24 slowed CNV production and reduced the expression of HIF-1α and inflammatory factors in CS models. The mRNA levels of HIF-1α, VEGF-A, NF-kB, TNF-α, IL-1β, and IL-6 significantly decreased. Moreover, the protein levels of HIF-1α and NF-κB p65 were significantly decreased. TAT-N24 can treat CNV and ocular inflammation by inhibiting the HIF-1α/NF-κB signaling pathway in CS. In the early treatment of corneal foreign body trauma, topical application of TAT-N24 can not only reduce the inflammatory response but also inhibit corneal neovascularization.

Expression of HIF1α, BNIP3, and beclin-1 in the brain of newborn and adult yaks (Bos grunniens).

As a main consumer of energy, the brain is particularly susceptible to the effects of hypoxia. However, during long-term evolution, the brain of the plateau yak developed adaptive mechanisms enabling it to maintain normal physiological conditions. A total of 20 male yaks belonging to two age groups [newborns (1-6 days old; n = 10) and adults (3-5 years old; n = 10)] were obtained, and the brain tissue was fixed and processed by standard methods. RT-qPCR, ELISA and IHC assays were used to investigate the expression and localization of HIF1α, BNIP3 and beclin-1 in the hippocampus, cerebral cortex, thalamus, medulla oblongata and cerebellum of newborn and adult yak brains and to explore their potential neuroprotective role. We found that the expression levels of HIF1α, BNIP3 and beclin-1 at the mRNA and protein levels varied in the different regions of yak brain, with the highest expression observed in the hippocampus, followed by the cerebral cortex, thalamus, medulla oblongata and the cerebellum. Moreover, the HIF1α, BNIP3 and beclin-1 expression were significantly higher in the newborn yaks' brains than in the adult yak. The IHC results showed that HIF1α, BNIP3 and beclin-1 were mainly distributed in the neurons of the cerebral cortex, hippocampus, thalamus, medulla oblongata and cerebellum. In particular, HIF1α accumulated in the nucleus and cytoplasm. Furthermore, the immunoreactivity of BNIP3 and beclin-1 was concentrated in the cytoplasm. The results indicate that the yak hippocampus and cerebral cortex may be more resistant to hypoxia than thalamus, medulla oblongata and cerebellum, and the expression of BNIP3 and beclin-1 may be regulated by HIF1α to serve a neuroprotective role in the yak's brain to adaptation to hypoxia. Additionally, the brain of adult yaks may have a higher tolerance to hypoxia than the brain of newborn yaks.

Hypoxia-induced degradation of PICK1 by RBCK1 promotes the proliferation of nasopharyngeal carcinoma cells

AimsHypoxia is an important feature of nasopharyngeal carcinoma (NPC). “Protein interacting with PRKCA 1” (PICK1) is commonly downregulated in human malignancies and is functionally related to poor prognosis. However, there is a limited understanding of the upstream mechanisms regulating PICK1 currently. Main methodsPICK1 and HIF-1α expression levels were analyzed by Immunohistochemistry (IHC), western blotting, and quantitative real-time PCR assay. Protein stability and ubiquitin assays were used to investigate PICK1 protein degradation. Immunofluorescence and co-immunoprecipitation assays were used to demonstrate the interaction between RBCK1 and PICK1. Gene knockdown by siRNA transfection was used to investigate the role of HIF-1α and RBCK1 in hypoxia-induced PICK1 degradation. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of RBCK1 and PICK1 in NPC cell proliferation. Key findingsPICK1 expression in NPC tissue was negatively relative to that of HIF-1α. HIF-1α downregulated PICK1 expression by facilitating its ubiquitination by the E3 ligases RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1), thereby enhancing proteasome-mediated PICK1 degradation. RBCK1 knockdown inhibited NPC cell proliferation, which was ameliorated by double knockdown of RBCK1/PICK1. SignificanceThese data provide evidence for an NPC cell adaptation mechanism to hypoxia, where HIF-1α regulates RBCK1, which targets PICK1 for degradation to promote cell proliferation.

Potent anti-inflammatory responses: Role of hydrogen in IL-1α dominated early phase systemic inflammation.

Introduction: It has been proven that hydrogen has obvious anti-inflammatory effects in animal experiments and clinical practice. However, the early dynamic process of the inflammatory response caused by lipopolysaccharide (LPS) and the anti-inflammatory effect of hydrogen has not been definitively reported. Methods: Inflammation in male C57/BL6J mice or RAW264.7 cells was induced with LPS, for which hydrogen was immediately administered until samples were taken. Pathological changes in lung tissue were assessed using hematoxylin and eosin (HE) staining. Levels of inflammatory factors in serum were determined using liquid protein chip. The mRNA levels of chemotactic factors in lung tissues, leukocytes, and peritoneal macrophages were measured by qRT-PCR. The expression levels of IL-1α and HIF-1α were measured by immunocytochemistry. Results: Hydrogen alleviated LPS-induced inflammatory infiltration in the lung tissues of mice. Among the 23 inflammatory factors screened, LPS-induced upregulation of IL-1α etc. was significantly inhibited by hydrogen within 1 hour. The mRNA expression of MCP-1, MIP-1α, G-CSF, and RANTES was inhibited obviously by hydrogen at 0.5 and 1 h in mouse peritoneal macrophages. In addition, hydrogen significantly blocked LPS or H2O2-induced upregulation of HIF-1α, and IL-1α in 0.5 h in RAW264.7 cells. Discussion: The results suggested that hydrogen is potentially inhibitive against inflammation by inhibiting HIF-1α and IL-1α release at early occurrence. The target of the inhibitive LPS-induced-inflammatory action of hydrogen is chemokines in macrophages in the peritoneal cavity. This study provides direct experimental evidence for quickly controlling inflammation with the translational application of a hydrogen-assisted protocol.

Expression of HIF-1α/PKM2 axis correlates to biological and clinical significance in papillary thyroid carcinoma.

hypoxia inducible factor-1α (HIF-1α) and pyruvate kinase M2 (PKM2) are 2 key metabolic regulatory proteins, they could engage in a positive feedback loop and drive cancer growth by enhancing glycolysis. This study aimed to investigate the expression of HIF-1α and PKM2 in papillary thyroid carcinoma (PTC) and its correlation with the patients clinicopathological features and with tumor invasion and metastasis. Surgically resected PTC specimens from 60 patients were collected. The expression levels of HIF-1α and PKM2 in PTC tissues were examined by immunohistochemical staining. The full clinical records of all patients were collected to analyze the relevance between HIF-1α and PKM2 expressions and the clinical pathological features of PTC. The results showed that the positive expressions of HIF-1α, PKM2, and HIF-1α/PKM2 axis (HIF-1α+/PKM2+) were all significantly higher in PTC than those in normal thyroid follicular epithelium, and a positive correlation was found between HIF-1α and PKM2 in PTC. Further analysis showed that in PTC, the positive expression of HIF-1α and HIF-1α/PKM2 axis (HIF-1α+/PKM2+) were significantly associated with bigger tumor size, moreover, the positive expressions of HIF-1α, PKM2 and HIF-1α/PKM2 axis (HIF-1α+/PKM2+) were all correlated with capsular invasion and lymph node metastasis, while they were all not correlated with gender, sex and multicentricity of tumor. This study identified HIF-1a/PKM2 axis as potential molecular marker for predicting the invasion and progression of papillary thyroid carcinoma.

MSC-EXO and tempol ameliorate bronchopulmonary dysplasia in newborn rats by activating HIF-1α.

Bronchopulmonary dysplasia (BPD) is a major complication of premature infants and an important cause of morbidity and mortality. This study investigates the effect of the combination of mesenchymal stem cells-derived exosomes (MSC-EXO) and tempol on BPD and analyzes its mechanism. MSC-EXO was extracted by centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis, and western blot analysis(WB). Tidal volume (TV), minute ventilation (MV), peak inspiratory flow (PIF), and dynamic pulmonary compliance (Cdyn) of rats were measured by BuxCo pulmonary function experimental platform. Hematoxylin-eosin staining was performed to observe the lung morphology and radical alveolar count (RAC)and mean linear intercept (MLI) were assessed. Immunofluorescence (IF) was conducted to detect the expression of CD31 and α-SMA in pulmonary blood vessels. The kits were used to calculate malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (TAOC) concentration in lung tissue. Enzyme linked immunosorbent assaywas applied to detect the levels of IL-1β, IL-17, IL-6, and IFN-γ in bronchoalveolar lavage fluid. In addition, the expressions of HIF-1α, vascular endothelial growth factor(VEGF), p-PI3K, and p-AKT were analyzed by WB and IF. We successfully extracted and identified MSC-EXO. In BPD rats, TV, MV, PIF, and Cdyn decreased, alveoli were simplified, and the number of interalveoli small vessels, blood vessel density decreased. Moreover, RAC, CD31, TAOC, and SOD decreased, and MLI, α-SMA, MDA, IL-1β, IL-17, IL-6, and IFN-γ increased, which was reversed by the combination of MSC-EXO and tempol treatment after combined treatment. In addition, the expression levels of HIF-1α, VEGF, p-PI3K, and p-AKT were increased after combined treatment. Combined treatment could improve lung tissue injury, promote pulmonary vascular remodeling, restore lung function, and inhibit oxidative stress in BPD rats. These effects were achieved through activation of HIF-1α.

FtMt reduces oxidative stress-induced trophoblast cell dysfunction via the HIF-1α/VEGF signaling pathway

BackgroundPreeclampsia (PE) is a complication of pregnancy that causes long-term adverse outcomes for the mother and fetus and may even lead to death. Oxidative stress caused by the imbalance of oxidants and antioxidants in the placenta has been considered as one of the key mechanisms of preeclampsia (together with inflammation, etc.), in which the placental mitochondria play an important role. The expression of hypoxia-inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) is known to be increased in patients with PE. Mitochondrial ferritin (FtMt) is known to protect the mitochondria from oxidative stress, although its specific role in PE remains unclear.MethodsWe used qRT-PCR and western blotting to detect the expression levels of FtMt, HIF-1α, and VEGF in placental tissues from patients with PE. Human chorionic trophoblast cells were also administered with hypoxia treatment, followed by the detection of cell proliferation, invasion and angiogenic capacity by CCK8, Transwell, and endothelial cell angiogenesis assays; we also detected the expression of HIF-1α and VEGF in these cells. Finally, overexpression or inhibitory FtMt lentiviral vectors, along with negative control vectors, were constructed and transfected into hypoxia-treated human chorionic trophoblast cells; this was followed by analyses of cell function.ResultsThe expression levels of FtMt, HIF-1α and VEGF in the PE group were higher than those in the control group (P < 0.05). Following hypoxia, there was an increase in the expression levels of HIF-1α and VEGF protein in trophoblast cells. There was also an increase in invasion ability and vascular formation ability along with a reduction in cell proliferation ability. These effects were reversed by transfecting cells with the knockout FtMt lentivirus vector. The differences were statistically significant.ConclusionAnalyses showed that FtMt plays a key role in the vascular regulation of PE trophoblast cells after hypoxia possibly acting via the HIF-1α/VEGF signaling pathway. These results provide us an enhanced understanding of the pathogenesis of PE and suggest that the HIF-1α/VEGF signaling pathway represents a new target for the treatment of PE.

Role of hypoxia-inducible factor-1α and survivin in breast cancer recurrence and prognosis

ObjectiveTo analyze the expression of hypoxia-inducible factor-1α (HIF-1α) and survivin in breast cancer, and different molecular subtypes of breast cancer and to assess their relationship with recurrence and prognosis. MethodsThe expression levels of HIF-1α and survivin genes in breast cancer were investigated using bioinformatics. Their protein expression levels were then verified through immunohistochemistry (IHC), and their relationship with recurrence and prognosis was assessed. ResultsExpression levels of HIF-1α and survivin genes and proteins were increased in breast cancer tissues compared with normal tissues. Both were associated with clinical features of breast cancer and differentially expressed in different molecular subtypes of breast cancer, and both are related to the signal pathway of breast cancer growth and invasion. HIF-1α and survivin gene and protein expression levels were correlated, and both were associated with breast cancer recurrence (R = 0.380, P < 0.05; R = 0.673, P < 0.05, respectively). According to The Cancer Genome Atlas (TCGA) database, HIF1A and BIRC5 gene were not associated with breast cancer prognosis (P ≥ 0.05); however, HIF-1α and survivin protein were associated with recurrence patient's overall survival (OS) (P < 0.05). ConclusionHIF-1α and survivin are highly expressed in breast cancer and can be used as potential biomarkers to predict recurrence and assess prognosis.