The roles of hypoxia-inducible Factor-1 and iron regulatory protein 1 in iron uptake induced by acute hypoxia.

BACKGROUND INFORMATION: Divalent metal transporter 1 (DMT1) and transferrin receptor (TfR1) are vital proteins for cellular iron uptake. These proteins have hypoxia-responsive elements (HREs) in their 5'-regulatory region, and they are regulated by hypoxia-inducible factor 1α (HIF-1α) transcriptionally under hypoxic condition. Besides, iron regulatory protein 1 (IRP1) regulates DMT1 and TfR1 by binding to iron-responsive elements (IREs) present in their mRNAs to control cellular iron homeostasis. RESULTS: Here, we explored the effect of acute hypoxia on iron uptake. Ferrous iron uptake was elevated by DMT1(+IRE) and TfR1 under acute hypoxia. The luciferase activity analysis revealed that the functional HREs of DMT1 and TfR1 were increased. However, their IREs-dependent luciferase activities were reduced simultaneously. The mRNA stability of TfR1 and DMT1(+IRE) was suppressed under acute hypoxia. The mRNA levels of TfR1 and DMT1(+IRE) were restrain by silencing IRP1. In sharp contrast, HIF-1α overexpression enhanced the mRNA levels of TfR1 and DMT1(+IRE), which reversed the inhibition of IRP1 on both. HIF-1α konckdown suppressed the hypoxia-induced increase expression of TfR1 and DMT1(+IRE), whereas both proteins had little change when further decreased the IRP1 expression under hypoxia. Hypoxia upregulated the protein expression of Ferrtin-L in a time-dependent manner, yet there was no different when IRP1 silencing or overexperssion under hypoxia. The lactate dehydrogenase (LDH) release induced by hypoxia was increased by TfR1 siRNA silence. CONCLUSIONS: We propose that HIF-1/HRE system might play a principal part in hypoxia induced iron uptake.

Iron Regulatory Protein 1 Suppresses Hypoxia-Induced Iron Uptake Proteins Expression and Decreases Iron Levels in HepG2 Cells.

Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1-IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia.

Hypoxia regulates the ferrous iron uptake and reactive oxygen species level via divalent metal transporter 1 (DMT1) Exon1B by hypoxia-inducible factor-1.

Hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including ceruloplasmin, transferrin, and transferring receptor. Divalent metal transporter 1 (DMT1) is a transmembrane protein that is important in divalent metal ion transport, in particular iron. Although previous studies have provided that DMT1 exon1A is regulated by hypoxia, little is known about the relationship between DMT1 exon1B and hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor which is stabilized when mammalian cells are subjected to hypoxia. In this study, we have identified a functional hypoxia-response element (HRE) at position of -327 to -323 (-ACGTG-) in DMT1 exon1B promoter using a combination of bioinformatics and biological approaches. Both the total cellular iron and ferrous uptake increased after hypoxia, decreased after DMT1 RNA interference. Reactive oxygen species (ROS) were also changed by +iron responsive element (IRE) DMT1 exon1B overexpression. These findings implicated DMT1 exon1B was a target gene for HIF-1. Hypoxia might affect cellular iron uptake through regulating the expression of DMT1. When iron was present in excess, cells might be damaged by the ROS production.

Sex Difference of Ribosome in Stroke-Induced Peripheral Immunosuppression by Integrated Bioinformatics Analysis.

Ischemic stroke (IS) greatly threatens human health resulting in high mortality and substantial loss of function. Recent studies have shown that the outcome of IS has sex specific, but its mechanism is still unclear. This study is aimed at identifying the sexually dimorphic to peripheral immune response in IS progression, predicting potential prognostic biomarkers that can lead to sex-specific outcome, and revealing potential treatment targets. Gene expression dataset GSE37587, including 68 peripheral whole blood samples which were collected within 24 hours from known onset of symptom and again at 24-48 hours after onset (20 women and 14 men), was downloaded from the Gene Expression Omnibus (GEO) datasets. First, using Bioconductor R package, two kinds of differentially expressed genes (DEGs) (nonsex-specific- and sex-specific-DEGs) were screened by follow-up (24-48 hours) vs. baseline (24 hours). 30 nonsex-specific DEGs (1 upregulated and 29 downregulated), 79 female-specific DEGs (25 upregulated and 54 downregulated), and none of male-specific DEGs were obtained finally. Second, bioinformatics analysis of female-specific DEGs was performed. Gene Ontology (GO) functional annotation analysis shows that DEGs were mainly enriched in translational initiation, cytosolic ribosome, and structural constituent of ribosome. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis shows that the top 6 enrichment pathways are ribosome, nuclear factor--kappa B (NF-kappa B) signaling pathway, apoptosis, mineral absorption, nonalcoholic fatty liver disease, and pertussis. Three functional modules were clustered in the protein-protein interaction (PPI) network of DEGs. The top 10 key genes of the PPI network constructed were selected, including RPS14, RPS15A, RPS24, FAU, RPL27, RPL31, RPL34, RPL35A, RSL24D1, and EEF1B2. Sex difference of ribosome in stroke-induced peripheral immunosuppression may be the potential mechanism of sex disparities in outcome after IS, and women are more likely to have stroke-induced immunosuppression. RPS14, RPS15A, RPS24, FAU, RPL27, RPL31, RPL34, RPL35A, RSL24D1, and EEF1B2 may be novel prognostic biomarkers and potential therapeutic targets for IS.

Ligustilide prevents cognitive impairment and attenuates neurotoxicity in D-galactose induced aging mice brain.

Ligustilide (LIG) is a principal active ingredient of traditional Chinese medicine, Radix Angelica sinensis, which has versatile pharmacological activities including neuroprotection. Previous studies have demonstrated that LIG has beneficial effects on cognition deficits associated with cerebral damage or neurodegenerative disorders. In present study, we investigated the neuroprotective effect of LIG on cognitive impairment and neurotoxicity in the brain of aging mouse induced by d-galactose (d-gal). The aging model mice were induced by subcutaneous (S.C.) injection of d-gal once daily for 8 weeks and LIG (80 mg/kg) was simultaneously administered orally. The Morris water maze (MWM) test was used to assess the spatial learning and memory abilities. The activity of Na(+)-K(+)-ATPase and the content of lipid peroxidation product malondialdehyde (MDA) in brain were examined. The levels of glial fibrillary acidic protein (GFAP), growth-associated protein GAP-43, and cleaved caspase-3 in brain were also determined by immunohistochemistry. The MWM test showed that LIG administration markedly improved behavioral performance of d-gal treated mice. This action could be partly explained by the results that LIG reduced the level of MDA as well as increased the activity of Na(+)-K(+)-ATPase in the brain of d-gal induced aging mice. Moreover, LIG significantly raised the expression of GAP-43 and reduced cleaved caspase-3 and GFAP levels in the brain of d-gal treated mice. These results demonstrated that LIG improves d-gal-induced cognitive dysfunction and brain toxicity, which suggests that LIG may be developed as a new medicine for the treatment of aged-related conditions.

Meta-analysis on the relationship between HLA-DRBl gene polymorphism and cervical cancer in Chinese population.

AIM: To determine the association between HLA-DRB1 haplotypes and risk of cervical cancer in unselected and samples from Chinese ethnicities. METHODS: A comprehensive search for articles from their inception to April 1st, 2013 was conducted from PubMed, Medline, Elsevier Science, Springer Link, Cochrane Library database, China biology medical literature database (CBM),China National Knowledge Infrastructure (CNKI),VIP,and Chinese literature database(Wang fang). A total of 1596 patients with cervical cancer and 2048 controls from the 12 studies on the relationship between gene polymorphism of HLA-DRB l and cervical cancer were performed and data were analyzed and processed using Review Manager 5.0 and Stata 11.0. RESULTS: Among the 13 family alleles, two (DRB1*03 and DRB1*08) were found to be negatively associated with cervical cancer in all studies or in Uighur subgroups, and two (DRB1*10 and DRB1*15) were positively associated with in all studies or in Uighur subgroups. Among the 25 specific alleles, six (DRB1*0301, *0403,*0404, *0803, *1312 and *1502) were associated with an increased risk cervical cancer in all studies. No significant association was established for other HLA-DRB1 family alleles and specific alleles. Ethnicity partially explained the race influence of DRB1*12, DRB1*14, DRB1*0301, DRB1*0403, DRB1*0404, DRB1*0803, DRB1*1312 and DRB1*1502 phenotypes. CONCLUSION: Our results support the hypothesis that the HLA-DRB1 family alleles and specific alleles might influence the susceptibility or resistance to cervical cancer, suggesting that immune regulation may play a key role in this disease, although further investigations are still needed.

Hydroxypropyl-sulfobutyl-ß-cyclodextrin improves the oral bioavailability of edaravone by modulating drug efflux pump of enterocytes.

The objective of the study was to evaluate the effect of hydroxypropyl-sulfobutyl-ß-cyclodextrin (HP-SBE-ßCD) on the bioavailability and intestinal absorption of edaravone, and identify its mechanism of action. We devised HP-SBE-ßCD as a carrier and modulator of P-glycoprotein (Pgp) efflux pump, and edaravone as a model drug, and prepared edaravone/HP-SBE-ßCD inclusion complex. HP-SBE-ßCD improved the water solubility and enhanced the bioavailability of edaravone by 10.3-fold in rats. Then, in situ single-pass intestinal perfusion showed that HP-SBE-ßCD had an effect of improving the permeability and inhibiting the efflux of edaravone. Furthermore, the effects of HP-SBE-ßCD on Pgp were achieved through interfering with the lipid raft and depleting the cholesterol of enterocytes membrane. From the results, we presented the novel mechanisms. First, edaravone/HP-SBE-ßCD had a lower release from the inclusion compound to protect edaravone from the low pH of the stomach. Then, HP-SBE-ßCD modulated the membrane microenvironment of intestinal absorption epithelial cells. At last, the result was that HP-SBE-ßCD enhanced the absorption of edaravone by interfering with Pgp. In conclusion, HP-SBE-ßCD improves the bioavailability of drug not only because of its enhancing water solubility of the drug, but also because it modulates the Pgp-mediated efflux from enterocytes.

Z-ligustilide activates the Nrf2/HO-1 pathway and protects against cerebral ischemia-reperfusion injury in vivo and in vitro.

Z-ligustilide (LIG), the main lipophilic component of Radix Angelica sinensis, has been shown to protect against brain ischemic damage in rodents by oral and intra-peritoneal treatments. The present study aimed to confirm the therapeutic effect of LIG administered intravenously on 2h middle cerebral artery occlusion (MCAO) and 22 h reperfusion injury in rats since oral administration has low bioavailability, slow absorption and distribution. Moreover, whether LIG activated the NF-E2-related factor 2/ heme oxygenase-1 (Nrf2/HO-1) pathway was also investigated in vivo and in vitro to further elucidate the precise protective mechanisms. In vivo, rats treated intravenously with LIG immediately after the surgery was finished had less neurological dysfunction and smaller infarct volume than that of the vehicle-treated rats. Additionally, LIG promoted Nrf2 nuclear translocation, and further remarkably increased Nrf2 and HO-1 protein expression. In vitro, LIG induced Nrf2 nuclear translocation and up-regulated HO-1 expression in a time-dependent and concentration-dependent manner. Furthermore, LIG treatment reduced cell death induced by OGD, however, the protective action was abolished while Nrf2/HO-1 expression was knockdown by RNA interference. These results noted that intravenous post-treatment with LIG exhibits noticeable neuroprotective properties against brain damage by ischemia-reperfusion and the ability of LIG to activate Nrf2/HO-1 pathway may be partly responsible for it.

Neuroprotective effect of astaxanthin on H(2)O(2)-induced neurotoxicity in vitro and on focal cerebral ischemia in vivo.

Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. Much experimental evidence has proved that AST has the function of eliminating oxygen free radicals and can protect organisms from oxidative damage. The present study was carried out to further investigate the neuroprotective effect of AST on oxidative stress induced toxicity in primary culture of cortical neurons and on focal cerebral ischemia-reperfusion induced brain damage in rats. AST, over a concentration range of 250-1000nM, attenuated 50µM H(2)O(2)-induced cell viability loss. 500nM AST pretreatment significantly inhibited H(2)O(2)-induced apoptosis measured by Hoechst 33342 staining and restored the mitochondrial membrane potential (MMP) measured by a fluorescent dye, Rhodamine 123. In vivo, AST prevented cerebral ischemic injury induced by 2h middle cerebral artery occlusion (MCAO) and 24h reperfusion in rats. Pretreatment of AST intragastrically twice at 5h and 1h prior to ischemia dramatically diminished infarct volume and improved neurological deficit in a dose-dependent manner. Nissl staining showed that the neuronal injury was significantly improved by pretreatment of AST at 80mg/kg. Taken together, these results suggest that pretreatment with AST exhibits noticeable neuroprotection against brain damage induced by ischemia-reperfusion and the antioxidant activity of AST maybe partly responsible for it.