mRNA Expression Levels Of HIF-1α Research Articles

The Suppression of Signal Transducer and Activator of Transcription-3 in A549human Lung Carcinoma Cells Induced by Marine Sponge Callyspongia aerizusa.

Lung cancer is recognized as a highly lethal disease, demanding swift and accurate solutions. Previous analysis showed the cytotoxic impact of Callyspongia aerizusa (C. aerizusa) extract containing ergost-22-en-3-one and ergost-7-en3-ol against A549 lung cancer cells, with an IC50 value of 9.38μg/mL. However, the extract did not have cytotoxicity towards Het-1A esophagus epithelial cells. Several reviews also validated the upregulation of pro-apoptotic molecules and the inhibition of anti-apoptotic molecules linked to the caspase-dependent signaling pathway. The objective of this research was to extend the understanding of the effects of C. aerizusa extract on A549 lung carcinoma, examining its influence on various signaling pathways, malignancy, migration, and invasion. PCR was used to measure mRNA expression, targeting PTEN, Akt, mTOR, STAT-3, IL-6, VEGF, and HIF1α. Additionally, Western Blot analysis was adopted to assess PTEN, p-Akt, Akt, p-mTOR, and p-STAT-3 protein expressions. Wound healing and invasion assays were performed to measure the migration and invasion capabilities of A549 cells post-treatment with C. aerizusa extract. The mRNA expression analysis showed an increase in Akt and m-TOR but a decrease in PTEN and STAT-3 after 24hours of treatment with C. aerizusa extract. At the protein level, there was a downregulation of p-Akt, Akt, p-mTOR, and p-STAT-3, while PTEN increased during 24-hour treatment. Wound healing and invasion assay results showed a weakened ability of A549 cells after a 24-hour treatment with C. aerizusa extract. Moreover, IL-6 and HIF-1α mRNA expression levels decreased during 24hours, while VEGF mRNA had a slight decrease compared to untreated cells. In conclusion, the ergosteroids present in marine sponge C. aerizusa extract signified a remarkable reduction in malignancy, migration, and invasion capabilities in A549 lung carcinoma cells. These results suggested their promising candidacy for future anti-angiogenesis in anticancer therapy.

Taurine reduces glycolysis of pig skeletal muscle by inhibiting HIF-1α signaling.

The aim of this study was to investigate the effect of taurine on skeletal muscle glycolysis in pigs. The results showed that dietary supplementation of taurine significantly reduced the activities of hexokinase (HK), phosphofructose kinase (PFK), and pyruvate kinase (PK) in finishing pigs. Meanwhile, taurine reduced the protein and mRNA expression levels of hypoxia inducible factor 1α (HIF-1α) and the mRNA expression of glycolytic enzyme related genes (such as HK type II, HK Ⅱ; pyruvate kinase M2, PKM2; lactate dehydrogenase A, LDHA). In addition, taurine reduced the expression of HIF-1α, lactate content, and the expression of glycolysis related genes in porcine myotubes. These results suggest that taurine may regulate glycolysis in skeletal muscle of finishing pigs through the HIF-1α signaling pathway. To further investigate the mechanism by which taurine affects skeletal glycolysis, HIF-1α activator dimethyloxalyl glycine (DMOG) was used to treat porcine myotubes, our results showed that DMOG significantly increased the protein and mRNA expression levels of HIF-1α, lactate content, and glycolytic enzyme (HK, PFK, PK, and LDH) activity, but taurine treatment significantly inhibited this effect. Taken together, these results of in vivo and in vitro experiments revealed that taurine reduces skeletal muscle glycolysis by inhibiting HIF-1α signaling.

Sex Differences in the Expression of Cardiac Remodeling and Inflammatory Cytokines in Patients with Obstructive Sleep Apnea and Atrial Fibrillation.

Although there is a link between obstructive sleep apnea (OSA) and atrial fibrillation (AF) and numerous investigations have examined the mechanism of AF development in OSA patients, which includes cardiac remodeling, inflammation, and gap junction-related conduction disorder, there is limited information regarding the differences between the sexes. This study analyzes the impact of sex differences on the expression of cardiac remodeling, inflammatory cytokines, and gap junctions in patients with OSA and AF. A total of 154 individuals diagnosed with sleep-related breathing disorders (SRBDs) were enrolled in the study and underwent polysomnography and echocardiography. Significant OSA was defined as an apnea-hypopnea index (AHI) of ≥15 per hour. Exosomes were purified from the plasma of all SRBD patients and incubated in HL-1 cells to investigate their effects on inflammatory cytokines and GJA1 expression. The differences in cardiac remodeling and expression of these biomarkers in both sexes were analyzed. Of the 154 enrolled patients, 110 patients were male and 44 patients were female. The LA sizes and E/e' ratios of male OSA patients with concomitant AF were greater than those of control participants and those without AF (all p < 0.05). Meanwhile, female OSA patients with AF had a lower left ventricular ejection fraction than those OSA patients without AF and control subjects (p < 0.05). Regarding the expression of inflammatory cytokines and GJA1, the mRNA expression levels of GJA1 were lower and those of IL-1β were higher in those male OSA patients with AF than in those male OSA patients without AF and control subjects (p < 0.05). By contrast, mRNA expression levels of HIF-1α were higher in those female OSA patients with and without AF than in control subjects (p < 0.05). In conclusion, our study revealed sex-specific differences in the risk factors and biomarkers associated with AF development in patients with OSA.

Acevaltrate promotes apoptosis and inhibits proliferation by suppressing HIF-1α accumulation in cancer cells

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.

Comparative Assessment of the Effectiveness of HSP70 / HIF-1α System Modulators after Prenatal Hypoxia

Prenatal hypoxia (PH) poses a significant threat to fetal development and may be responsible for neonatal mortality or neurodevelopmental abnormalities. The proteins HSP70 and HIF-1, which hold a distinct significance in the cellular reaction to PH, can be regarded as potential targets for pharmaceutical interventions aimed at mitigating the repercussions of chronic PH. This study aimed to identify a possible correlation between offspring survival and stages of expression of endogenous neuroprotective factors (HSP70 and HIF-1) after chronic prenatal hypoxia with course administration of potential HSP70 modulators (angiolin, piracetam, thiotriazoline, nicomex, cerebrocurin, tamoxifen, L-arginine, glutoredoxin, HSF-1, and mildronate). In the rat offspring after PH we determined the plasma concentrations of HSP70 and HIF-1 by solid-phase ELISA immunoassay, and the expression of HIF-1 mRNA and HSP70 mRNA by real-time PCR. For the first time, we found a positive correlation between offspring survival after PH and the expression of HIF-1 and HSP70, both in groups without experimental therapy and in groups receiving pharmacological agents. The course administration of HSP70/HIF-1α modulators, especially angiolin (50 mg/kg), cerebrocurin (150 mg/kg), and HSF-1 (50 mg/kg), to rats that underwent PH reduces postnatal lethality, increases blood plasma concentrations of HSP70 and HIF-1α, and positively affects the expression level of HIF-1α mRNA in the rat brain. These drugs can be considered as the most promising drug candidates for new therapeutic strategies of pharmacological correction of the consequences of chronic PH.

Impaired nitric oxide mechanisms underlying lower urinary tract dysfunction in aging rats.

The study aimed to investigate the bladder and urethral activity and nitric oxide (NO)-related molecular changes in aging rats. Rats were divided into two groups: Group Y (young rats; 12 wk) and Group A (aging rats; 15 mo). A 24-h voiding assay was performed, and the urodynamic parameters were evaluated using awake cystometry (CMG) and urethral perfusion pressure (UPP) recordings under urethane anesthesia. The mRNA expression levels of NO-, ischemia-, and inflammation-related markers in urethra and bladder tissues and cGMP levels in the urethra were assessed. Body weight was significantly higher in Group A than in Group Y. Voiding assay results (24 h) were insignificant. In the CMG, the number of non-voiding contractions per voiding cycle and post-void residual volume were significantly higher in Group A than in Group Y; voiding efficiency was significantly lower in Group A than in Group Y. In the UPP recordings, the urethral pressure reduction and high-frequency oscillation (HFO) amplitude were significantly lower in Group A than in Group Y. The mRNA expression levels of Hif-1α, Vegf-a, and Tgf-β1 in the bladder were significantly higher in Group A than in Group Y. The mRNA expression levels of Nos1 and Prkg1 and the cGMP concentrations in the urethra were significantly lower in Group A than in Group Y. Aging rats can be useful models for studying the natural progression of age-related lower urinary tract dysfunctions, for which impaired NO-mediated transmitter function is likely to be an important mechanism.NEW & NOTEWORTHY Aging rats can be useful models for studying the natural progression of age-related lower urinary tract dysfunctions, for which impaired nitric oxide-mediated transmitter function is likely to be an important mechanism.

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 ...

Mechanism of active components of "Notoginseng Radix et Rhizoma-Drynariae Rhizoma" in treatment of osteoporosis based on network pharmacology and in vitro cell experiment

The present study aimed to explore the main active components and potential mechanisms of Panax notoginseng saponins(PNS) and osteopractic total flavone(OTF) in the treatment of osteoporosis(OP) through network pharmacology, molecular docking and in vitro cell experiments, which was expected to provide a theoretical basis for clinical applications. The blood-entering components of PNS and OTF were obtained from literature search and online database, and their potential targets were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The OP targets were obtained by means of searching Online Mendelian Inheritance in Man(OMIM) and GeneCards. The common targets of the drug and disease were screened by Venn. Cytoscape was used to construct a &quot;drug-component-target-disease&quot; network, and the core components were screened according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened according to the node degree. GO and KEGG enrichment analysis of potential therapeutic targets were carried out by R language. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock Vina. Finally, HIF-1 signaling pathway was selected for in vitro experimental verification according to the results of KEGG pathway analysis. Network pharmacology showed that there were 45 active components such as leachianone A, kurarinone, 20(R)-protopanaxatriol, 20(S)-protopanaxatriol, and kaempferol, and 103 therapeutic targets such as IL6, AKT1, TNF, VEGFA and MAPK3 involved. PI3K-AKT, HIF-1, TNF and other signaling pathways were enriched. Molecular docking revealed that the core components had good binding ability to the core targets. In vitro experiments found that PNS-OTF could up-regulate the mRNA expression levels of HIF-1α, VEGFA and Runx2, indicating that the mechanism of PNS-OTF in treating OP may be related to the activation of HIF-1 signaling pathway, and thus PNS-OTF played a role in promoting angiogenesis and osteogenic differentiation. In conclusion, this study predicted the core targets and pathways of PNS-OTF in treating OP based on network pharmacology and carried out in vitro experimental verification, which reflected the characteristics of multi-component, multi-target and multi-pathway synergy of PNS-OTF, and provided new ideas for the future clinical treatment of OP.

125I seed implantation enhances arsenic trioxide-induced apoptosis and anti-angiogenesis in lung cancer xenograft mice.

Arsenic trioxide (ATO) exerts anticancer effects on lung cancer. However, the clinical use of ATO is limited due to its systemic toxicity and resistance of lung cancer cells. The present study aimed to investigate the effects of ATO, alone and in combination with 125I seed implantation on tumor growth and proliferation in lung cancer xenograft mice, and investigate the possible molecular mechanisms. The transmission electron microscope observed the tumor ultrastructure of lung cancer xenograft mice. The proliferation index of Ki-67 and the number and morphology of tumor microvessels were detected with immunohistochemical staining. The protein and mRNA expression were examined by western blot and real-time PCR assay. The in vivo results demonstrated that ATO combined with 125I seed significantly inhibited tumor growth and proliferation, as well as promoted apoptosis, and decreased the Ki-67 index and microvessel density in lung cancer xenograft mice. Moreover, ATO combined with 125I seed decreased the protein and mRNA expression levels of HIF-1α, VEGF, and BCL-2, and increased those of BAX and P53. ATO combined with 125I seed significantly inhibited tumor growth and proliferation in lung cancer, which may be accomplished by inhibiting tumor angiogenesis and inducing apoptosis.

Expression of HIF-1α and markers of angiogenesis and metabolic adaptation in molecular subtypes of breast cancer

Hypoxic zones exist in solid tumors, where oxygen levels are significantly lower than in normal tissues. Hypoxia makes chemo-radiation therapeutics less effective and renders the metastatic potential more favorable. Emerging research has found that the transcriptional expression of hypoxia-inducible factor-1alpha (HIF-1α) promotes the transcription of vascular endothelial growth factor A (VEGF-A) and Hexokinase-I (HK-I), which are associated to cellular growth, angiogenesis, and metastatic invasion in many malignancies. However, it is still unclear whether VEGFA and HK-I expression has any influence on survival based on the intrinsic subtypes of breast cancer. Their prognostic significance remains a debatable topic. In the present study, quantitative Real-time polymerase chain reaction (qRT-PCR) was employed to check the relative expression of HIF-1α, VEGF-A and HK-I. The hazard ratios (HR) of breast cancer-specific and overall mortality were calculated using Cox proportional hazards model, which were adjusted for demographic, clinicopathological, and associated molecular variables, as well as the diagnosis year. The relative mRNA expression levels of HIF-1α (p = 0.0010) and VEGFA (p = 0.0119) were significantly higher in tumor tissues. The expression of both HIF-1α (p = 0.0111) and VEGFA (p = 0.0078) was higher in the TNBC group of breast cancers, while HK-I (p = 0.0106) was higher in ER/PR-positive, HER2-negative group. HIF-1α and HK-I overexpression were associated with a higher likelihood of survival, while overexpression of VEGFA was associated with a low survival rate, although it was not statistically significant.

Effect of "nape seven needles" on expression of hypoxia inducible factor-1α and Notch1 in cervical intervertebral disc degeneration rats

To observe the effect of "nape seven needles" on the expressions of hypoxia inducible factor-1α (HIF-1α) and Notch1 in cervical intervertebral disc of cervical disc degeneration (CDD) rats, so as to reveal its underlying mecha-nisms in improving CDD. SD male rats were randomly divided into normal, model, non-acupoint and nape seven needles groups, with 10 rats in each group. Staticdynamic imbalance method was used to establish CDD model. Rats in the nape se-ven needles group were treated with acupuncture at "Fengfu"(GV16), and bilateral "Fengchi"(GB20), "Wangu"(GB12) and "Tianzhu"(BL10), and rats in the non-acupoint group received acupuncture at the sham acupoints at the caudal tip and armpit, both for 20 min, 6 days a week for 4 weeks. After intervention, tilted plane test and spiral CT were used to assess the neck movement function and cervical degeneration degree of rats; immunohistochemistry was used to observe the protein expression levels of HIF-1α and Notch1 in cervical discs tissue; Western blot and real-time quantitative PCR were used to detect the protein and mRNA expression levels of HIF-1α and Notch1 in cervical discs tissue, respectively. After modeling, the cervical curvature was straightened, with narrowed intervertebral space, rough and hardened articular surface, osteophytes, and blurred articular space and articular process, which was relatively milder in the nape seven needles group. Compared with the normal group, the angle of tilted plane was significantly reduced (P<0.05), while cervical scores, HIF-1α mRNA expression level in cervical intervertebral disc tissue were significantly increased (P<0.05) in the model group. Compared with the model and the non-acupoint groups, cervical scores were significantly reduced (P<0.05), while the angle of tilted plane, HIF-1α and Notch1 positive expressions, HIF-1α mRNA expression level, Notch1 protein and mRNA expression levels in cervical intervertebral disc tissue were significantly increased (P<0.05) in the nape seven needles group. Acupuncture of "nape seven needles" can reduce the degree of cervical degeneration in rats, which was possibly associated with its effects in up-regulating the expressions of HIF-1α and Notch1 in cervical intervertebral disc tissue, promoting the proliferation and recovery of endogenous cells in nucleus pulposus.

Paclitaxel Induces the Apoptosis of Prostate Cancer Cells via ROS-Mediated HIF-1α Expression.

Prostate cancer (PCa) is the most common malignancy to endanger the health of male genitourinary system. Clinically, paclitaxel (PTX) (C47H51NO14), a diterpene alkaloid, is commonly used as an effective natural antineoplastic drug during the treatment of PCa. However, the mechanism and pathway involved in the function of PTX are poorly understood. In the current study, we employed the CCK-8 assay, revealing that PTX can inhibit the survival and induce the apoptosis of PC3M cells (a human prostate cancer cell line) in a concentration-dependent manner. Reactive oxygen species (ROS), as a metabolic intermediate produced by the mitochondrial respiratory chain, are highly accumulated under the PTX treatment, which results in a sharp decrease of the mitochondrial membrane potential in PC3M cells. Additionally, the migration and invasion of PC3M cells are weakened due to PTX treatment. Further analysis reveals that N-acetylcysteine (NAC), which functions as an antioxidant, not only rescues the decreased mitochondrial membrane potential induced by the abnormal ROS level, but also restores the migration and invasion of PC3M cells. In a subsequent exploration of the detailed mechanism, we found that hypoxia-inducible factor (HIF)-1α works as a downstream gene that can respond to the increased ROS in PC3M cells. Under PTX treatment, the expression levels of HIF-1α mRNA and protein are significantly increased, which stimulate the activation of JNK/caspase-3 signaling and promote the apoptosis of PC3M cells. In summary, we demonstrate that PTX regulates the expression of HIF-1α through increased ROS accumulation, thereby promoting the activation of JNK/caspase-3 pathway to induce the apoptosis of PCa cells. This study provides new insights into the mechanism of antineoplastic action of taxanes and unveils the clinical benefit of the ROS-HIF-1α signaling pathway, which may offer a potential therapeutic target to prevent the development of PCa.

Inhibitory effect of low‑intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF‑1α in a carrageenan‑induced knee osteoarthritis rat model.

Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF-1α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF-1α in IFP fibrosis using a carrageenan-induced KOA rat model and evaluated the potential of low-intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra-articular injection of 0.5% carrageenan (50 µl) using 8-week-old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra-articular injection. The mRNA expression levels of HIF-1α and fibrosis-related molecules, CTGF and VEGF, were analyzed using reverse transcription-quantitative PCR, and the DNA binding activity of HIF-1α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra-articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF-1α. Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF-1α and decreased the gene expression levels of HIF-1α, CTGF, and VEGF. The present study demonstrated that activation of HIF-1α promoted fibrosis of IFP in carrageenan-induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF-1α and inhibited fibrosis. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA, through its modulation of HIF-1α.

HIF1α: A Novel Biomarker with Potential Prognostic and Immunotherapy in Pan-cancer.

Cancer is a catastrophic disease that seriously affects human health. HIF1α plays an important role in cancer initiation, progression, and prognosis. However, little is known about the specific role of HIF1α in pan-cancer. Therefore, we systematically and comprehensively analyzed HIF1α using GEPIA, HPA, GeneMANIA, STRING, SMPDB, cBioPortal, UALCAN, and TISDB databases and also 33 cancer and normal tissues in TCGA downloaded from the Genome Data Commons (GDC) data portal. Data and statistical analysis were performed using R software v4.0.3. Our results found that there were differences in the mRNA expression levels of HIF1α in human pan-cancer and its corresponding normal tissues. The expression level of HIF1α correlated with tumor stage in LIHC and also significantly correlated with prognosis in LIHC, LUSC, STAD, OV, PAAD, PRAD, THCA, LUAD, MESO, and READ. The small molecule pathways involved in HIF1α include succinate signaling, fumarate, and succinate carcinogenesis-related pathways. The highest mutation frequency of the HIF1α gene in pan-cancer was head and neck cancer, and the HIF1α methylation level in most tumors is significantly reduced. HIF1α was not only associated with immune cell infiltration but also with immune checkpoint genes and immune regulators TMB and MSI. There were currently 5 small molecule drugs targeting HIF1α.

Periodontal ligament cells under mechanical force regulate local immune homeostasis by modulating Th17/Treg cell differentiation.

Improper orthodontic force often causes root resorption or destructive bone resorption. There is evidence that T helper 17 (Th17) cells and regulatory T (Treg) cells may be actively involved in bone remodeling during tooth movement. In a combination of in vitro and in vivo studies, we investigated the effect of human periodontal ligament cells (hPDLCs) on Th17/Treg cells under different orthodontic forces and corticotomy. hPDLCs were cultured in vitro and subjected to different mechanical forces. The expression of interleukin (IL)-6 and transforming growth factor (TGF)-β in the supernatant and the mRNA levels of hypoxia inducible factor (HIF)-1α, Notch1, and TGF-β in hPDLCs were investigated. Supernatants were collected and co-cultured with activated CD4+T cells, and the differentiation of Th17/Treg cells was analyzed by flow cytometry. We also established an animal model of tooth movement with or without corticotomy. The tooth movement distance, alveolar bone height, and root resorption were analyzed using micro-computed tomography. Expression of interleukin (IL)-17A, forkhead Box P3 (Foxp3), and IL-6 were analyzed using immunohistochemistry, while osteoclasts were evaluated by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of IL-17A, IL-6, Foxp3, IL-10, HIF-1α, notch1, and C-X-C motif chemokine ligand 12 (CXCL12) in alveolar bone and gingiva were investigated. Heavy force repressed cell viability and increased the mortality rate of hPDLCs; it also improved the expression of IL-6, declined the expression of TGF-β, and promoted the mRNA expression level of HIF-1α. The expression of TGF-β and Notch1 mRNA decreased and then increased. The supernatant of hPDLCs under heavy force promotes the polarization of Th17 cells. The heavy force caused root resorption and decreased alveolar bone height and increased the positive area of IL-17A immunohistochemical staining and the expression of IL-17A, IL-6, HIF-1α, and Notch1 mRNA. Corticotomy accelerated tooth movement, increased the proportion of Foxp3-positive cells, and up-regulated the expression of Foxp3, IL-10, and CXCL12 mRNA. During orthodontic tooth movement, the heavy force causes root resorption and inflammatory bone destruction, which could be associated with increased expression of Th17 cells and IL-6. Corticotomy can accelerate tooth movement without causing root resorption and periodontal bone loss, which may be related to the increased expression of Treg cells. Altogether, this report provides a new perspective on the prevention of inflammatory injury via the regulation of Th17/Treg cells in orthodontics.

Pyruvate Kinase M2 Regulates Hif-1alpha Activity in Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia

Hypoxia-inducible factor 1 (HIF-1) is a nuclear protein with transcriptional activity. HIF-1 can be activated by immune cells exposed to hypoxia and regulate glycolysis. In this regulation, pyruvate kinase M2 (PKM2), a key enzyme in the cell glycolytic pathway, is an important regulatory site. Our previous studies suggest that PKM expression is increased in myeloid dendritic cells (mDCs) in SAA patients. Therefore, we investigated the expression level of HIF-1α in mDCs and its interaction with PKM2 in SAA patients. 1.The HIF-1α expression of mRNA and protein on mDCs in SAA untreated patients was significantly higher than that in SAA remission patients and normal controls.2.In the SAA patients, the HIF-1α expression on mDCs was positively correlated with the numbers of mDCs (p < 0.01), CD80+mDC/mDC (r = 0.689, p < 0.01), and CD86+mDC/mDC in PB (p < 0.05).3.In SAA patients, the HIF-1α expression on mDCs was negatively correlated with the CD4+T/CD8+T ratio in peripheral blood (PB) (p < 0.05), the percentage of granulocytoid and erythroid cells in bone marrow (p < 0.05), the WBC count in PB (p < 0.05), ANC in PB (p < 0.05), and the percentage of Rets in the PB (p < 0.05); was positively correlated with the percentage of lymphoid cells in bone marrow(p < 0.05); and was not statistically correlated with the megakaryocyte number in bone marrow, absolute PBL count, HGB in PB, absolute Ret count in PB, or PLT count in PB.4.In the correlation analysis we observed that there was a positively correlation between HIF-1α and PKM2 expression on mDCs in SAA patients (p < 0.001). To evaluate whether there is a mutual adjustment relationship between HIF-1α and PKM2, we successfully reduced PKM2 gene expression in this cell population via siRNA transfection. This process resulted in significantly lower levels of PKM2 protein expression relative to cells transfected with siControl which were evaluated by western blotting. We observed that the relative expression levels of HIF-1α mRNA in mDCs transfected with PKM2 siRNA was lower than siControl group（P< 0.01）.Conclusions 1.In this study, we found that untreated patients with SAA had higher HIF-1α expression on mDCs compared with recovering SAA patients and normal controls. The expression of HIF-1α was correlated with the number and function in mDCs and the severity of pancytopenia of SAA.2.The results indicated that the mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2.3.The mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The above experimental results confirmed that HIF-1α plays an important role in the abnormal immune response in patients with severe aplastic anemia, mainly by regulating the activity of PKM2 and thereby affecting the energy metabolism in immune cells. DisclosuresNo relevant conflicts of interest to declare.

Digitoxin promotes apoptosis and inhibits proliferation and migration by reducing HIF-1α and STAT3 in KRAS mutant human colon cancer cells

Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor. New treatment options are needed to improve survival in patients with KRAS mutated colorectal cancer. Digitoxin is a cardiotonic drug, which has been demonstrated to exhibit anticancer effects in a number of cancers. However, the anticancer mechanisms of digitoxin in KRAS mutant human colon cancer cells remain elusive. Our result demonstrated that digitoxin but not cetuximab markedly decreased the expression of hypoxia-inducible factor-1α (HIF-1α), signal transducer and activator of transcription 3 (STAT3) and p-STAT3 protein in KRAS mutant colon cancer cells. Further analysis revealed that digitoxin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. The phosphorylation levels of ribosomal protein S6 kinase (p70S6K) and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by digitoxin. Digitoxin inhibited the expression and activation of STAT3 through upregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN), SHP1 and protein inhibitors of activated STAT3 (PIAS3) and direct binding to STAT3. Meanwhile, digitoxin inhibited HIF-1α in STAT3-independent manner in KRAS mutant colon cancer cells. Moreover, digitoxin promoted apoptosis and inhibited proliferation and migration, which was potentially mediated by suppression of HIF-1α and STAT3. We also found that digitoxin administration inhibited tumor growth in a mouse xenograft model. Taken together, our findings highlight the therapeutic potential of digitoxin for the treatment of cetuximab-resistant human colon cancer.

Experimental Brain Injury Induced by Acute Hypobaric Hypoxia Stimulates Changes in mRNA Expression and Stress Marker Status

The present study was proposed to investigate the brain injury under acute hypobaric hypoxia following alteration in mRNA expression and stress markers in a time-dependent manner. SD clean graded male rats were randomly divided into four groups for this experimental brain injury, the control group at Xining (altitude, 2270m) and hypoxia treatment groups with different time exposure day1, day2, and day3 at (altitude, 7000m) in a hypobaric chamber. After day3 exposure, the brain tissues were collected. The level of mRNA expression of VEGF and HIF1-α was assessed using qRT-PCR. The oxidative stress level of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined with commercial kits. AHH with time duration significantly increased the MDA level and decreased in the activity of SOD was seen in all hypoxia treated groups as compared to the control (P&lt; 0.001). The mRNA expression level of HIF1-α and VEGF in day1, day2, and day3 AHH groups was markedly raised when it is compared to control (P&lt; 0.05). Ultimately, in conclusion, such results indicate that AHH stimulates oxidative stress induces brain damage in rats. Keywords: Acute hypobaric hypoxia, Brain injury, HIF-1α, Oxidative stress.

Metabolic Consequences of Neuronal HIF1α-Deficiency in Mediobasal Hypothalamus in Mice.

ObjectiveThis study aims to investigate whether hypoxia-inducible factor 1α (HIF1α) in the neurons of the mediobasal hypothalamus is involved in the regulation of body weight, glucose, and lipid metabolism in mice and to explore the underlying molecular mechanisms.MethodsHIF1α flox/flox mice were used. The adeno-associated virus that contained either cre, GFP and syn, or GFP and syn (controls) was injected into the mediobasal hypothalamus to selectively knock out HIF1α in the neurons of the mediobasal hypothalamus. The body weight and food intake were weighed daily. The levels of blood glucose, insulin, total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), high-density lipoprotein (HDL), and low-density lipoprotein (LDL)were tested. Intraperitoneal glucose tolerance test (IPGTT) was performed. The insulin-stimulated Akt phosphorylation in the liver, epididymal fat, and skeletal muscle were examined. Also, the mRNA expression levels of HIF1α, proopiomelanocortin (POMC), neuropeptide Y (NPY), and glucose transporter protein 4 (Glut4) in the hypothalamus were checked.ResultsAfter selectively knocking out HIF1α in the neurons of the mediobasal hypothalamus (HIF1αKOMBH), the body weights and food intake of mice increased significantly compared with the control mice (p < 0.001 at 4 weeks). Compared with that of the control group, the insulin level of HIF1αKOMBH mice was 3.5 times higher (p < 0.01). The results of the IPGTT showed that the blood glucose level of the HIF1αKOMBH group at 20–120 min was significantly higher than that of the control group (p < 0.05). The serum TC, FFA, HDL, and LDL content of the HIF1αKOMBH group was significantly higher than those of the control group (p < 0.05). Western blot results showed that compared with those in the control group, insulin-induced AKT phosphorylation levels in liver, epididymal fat, and skeletal muscle in the HIF1αKOMBH group were not as significantly elevated as in the control group. Reverse transcription-polymerase chain reaction (RT-PCR) results in the whole hypothalamus showed a significant decrease in Glut4 mRNA expression. And the mRNA expression levels of HIF1α, POMC, and NPY of the HIF1αKOMBH group decreased significantly in ventral hypothalamus.ConclusionsThe hypothalamic neuronal HIF1α plays an important role in the regulation of body weight balance in mice under normoxic condition. In the absence of hypothalamic neuronal HIF1α, the mice gained weight with increased appetite, accompanied with abnormal glucose and lipid metabolism. POMC and Glut4 may be responsible for this effect of HIF1α.

Profiles of messenger RNAs and MicroRNAs in hypoxia-induced hepatic stellate cells.

BackgroundMicroRNA (miRNA) plays an important role in hepatic stellate cell (HSCs) activation and liver fibrosis. The purpose of this study is to explore the effect of hypoxia on the differential expression of mRNAs and miRNAs in rat HSCs.MethodsHSC-T6 cells were treated with cobalt chloride (CoCl2), and the activity of HSC-T6 cells was measured by the CCK-8 assay. The mRNA expression levels of hypoxia inducible factor-1α (HIF-1α), collagen type I, transforming growth factor-β1 (TGF-β1), and Smad7 were measured by RT-qPCR. The protein expression levels of HIF-1α, Bax, Bcl-2, and caspase-3 were assayed by western blot. We used basal medium and 400 µmol/L CoCl2 medium to treat HSC-T6 cells for 48 h. Cells were harvested after 48 h to extract RNA. Transcriptome sequencing was performed to investigate differentially expressed miRNAs and mRNAs (fold change >2; P<0.05). Bioinformatics analysis was performed to predict the functions of differentially expressed miRNAs and mRNAs. Further, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ResultsWith the increase of CoCl2 concentration, the activity of HSC-T6 cells decreased (P<0.05). The mRNA expression levels of HIF-1α, collagen I, TGF-β1, and Smad7, and the protein expressions levels of HIF-1α, Bax, caspase-3, and the Bcl-2/Bax ratio were increased compared with the control group (P<0.05), while the expression of Bcl-2 decreased. A total of 54 miRNAs (20 upregulated and 34 downregulated) and 1,423 mRNAs (685 upregulated and 738 downregulated) were differentially expressed in the 400 µmol/L CoCl2 medium group compared to the control basal medium group. Further bioinformatics analysis demonstrated that the differentially expressed mRNAs and miRNAs were mainly enriched in the synthesis of extracellular matrix. In addition, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ConclusionsOur results presented the profiles of mRNAs and miRNAs in hypoxia-induced HSC-T6 cells in rats, the signaling pathways, and co-expression networks. These findings may suggest novel insights for the early diagnosis and treatment of HSC activation and liver fibrosis.