Prevention and treatment of hypoxia-induced colorectal cancer damage in albino Wister rats

Background:In the early metastasis of colon cancer,cancer cells detach,migrate,and infiltrate surrounding tissues,including lymph vessels and blood vessels.Tumor heterogeneity arises from both tumor cells and distinct microenvironments.Maldistribution of blood vessels,creates hypoxic regions within the tumors,fostering cancer stem cell-like properties due to reduced oxygen and nutrient supply.Under hypoxia,tumor cells shift to a glycolytic pathway,producing more lactic acid that acidifies the microenvironment and leads to unstable heart rate variability(HRV)factors,weight disparity,and a higher incidence of aberrant crypt foci(ACF).These hypoxic-induced parameters promote cancer cell invasion,increase radiation resistance,and facilitate cancer cell migration.Methods:In this study,we induced hypoxia-preneoplastic colon damage in albino Wister rats by administrating 1,2-dimethyl hydrazine(DMH).After successfully creating a hypoxic environment in albino Wister rats,resulting in preneoplastic colon damage,we randomly allocated Wistar albino rats into seven groups,each containing 8 animals,and conducted a 6-week study.Group 1-Normal control(administered 1 mM EDTA+saline,2 ml/kg/day,p.o.);group 2-Toxic control(administered DMH,30 mg/kg/week,s.c.);group 3-Standard treatment(DMH,30 mg/kg/week,s.c.for 6 weeks),followed by 5-fluorouracil and Leucovorin(25 mg/kg each on 1^(st),3^(rd),7^(th),and 10^(th) days,i.p.after 6 weeks administration of DMH);group 4-Low dose of P1(DMH,30 mg/kg/week,s.c.+P1,2 mg/kg,i.v.,weekly for 3 weeks);group 5-High dose P1(DMH,30 mg/kg/week,s.c.+P1,4 mg/kg,i.v.,weekly for 3 weeks),group 6-Low dose of P2(DMH,30 mg/kg/week,s.c.,+P2,2 mg/kg,i.v.,weekly for 3 weeks),group 7-High dose of P2(DMH,30 mg/kg/week,s.c.,+P2,4 mg/kg,i.v.weekly for 3 weeks).Results:DMH-treated rats exhibited alterations in HRV factors,weight disparity,elevated gastric pH,increased total acidity,a higher incidence of ACF,and changes in antioxidant markers(TBARs,SOD,catalase,GSH).Brightfield microscopy at 40x magnification revealed the presence of large crypts within aberrant crypt foci in the toxic control group.Conclusion:Treatment groups P1 and P2 containing triazine derivatives initiated proteasomal degradation of Hypoxia Inducible Factor-1α(HIF-1α)by activating Prolyl Hydroxylase(PHDs)pathways.HIF-1αunder a hypoxic environment is responsible for activating a multitude of genes involved in angiogenesis,metastasis,invasiveness,pH changes,metabolic reprogramming,stem cell maintenance,resistance to radiation,and downstream regulation of the immune system.Treatment with P1 and P2 groups helped minimize the ACF count and restored HRV factors,weight disparity,pH levels,total acidity,and oxidative balance.Our findings emphasize the potential role of 1,2,4-triazine derivatives in suppressing hypoxia-induced colon carcinogenesis.

Role of ROS/Kv/HIF Axis in the Development of Hypoxia-Induced Pulmonary Hypertension

Hypoxic pulmonary hypertension (HPH) is a common complication in patients with chronic obstructive pulmonary disease (COPD), sleep-disordered breathing, or dwellers in high altitude. The exact mechanisms underlying the development of HPH still remain unclear. Reactive oxygen species (ROS),hypoxia inducible factors (HIF), and potassium channels (KV) are believed as the main factors during the development of HPH. We propose that the “ROS/Kv/HIF axis” may play an important initiating role in the development of HPH. Being formed under a hypoxic condition, ROS affects the expression and function of HIFs or KV, and consequently triggers multiple downstream signaling pathways and genes expression that participate in promoting pulmonary vasoconstriction and arterial remodeling. Thus, further study determining the initiating role of “ROS/Kv/HIF axis” in the development of HPH could provide theoretic evidences to better understand the underlying mechanisms of HPH, and help identify new potential targets in the treatment of HPH.

Effect of NADPH Oxidase Inhibitor Apocynin on the Expression of Hypoxia-induced Factor-1α and Endothelin-1 in Rat Carotid Body Exposed to Chronic Intermittent Hypoxia

The effects of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase inhibitor apocynin on the enhanced hypoxia induced factor-let （HIF-lct） and endothelin-1 （ET-1） expression, elevated systolic blood pressure under chronic intermittent hypoxia （CIH） condition and its action mechanism were investigated. Thirty healthy 8-week old Sprague-Dawley （SD） male rats were randomly divided into three groups （n=10 each）： sham group, CIH group, and apocynin-treated CIH group. Tail artery systolic blood pressure was measured by tail-cuff method. Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the mRNA expression of HIF-lu and ET-1 in the carotid body, and the HIF-1a protein expression was examined by using Western blotting. The levels of malondialdehyde （MDA） and superoxide dismutase （SOD） were determined by using colorimetric method. In addition, the plasma ET-1 and HIF-1a levels were measured by using enzyme-linked immunosorbent assay. It was found that CIH exposure was associated with increased MDA levels, and apo- cynin-treated CIH animals showed reduction in MDA levels. Apocynin treatment prevented CIH-induced hypertension as well as CIH-induced decrease in SOD. The increases of HIF-1a and ET-1 mRNA along with HIF-la protein expression in the carotid body, and elevated circulating HIF-1a and ET-1 levels were observed in CIH-exposed animals. Treatment with apocynin significantly decreased the ET-1 mRNA, HIF-lct protein expression and circulating HIF-la level in CIH-exposed animals, and there was no statistically significant difference in the HIF-lu mRNA expression between CIH group and apo- cynin-treated group. These results indicated that apocynin alleviated CIH-induced hypertension by inhibiting NADPH oxidase, further leading to the reduced vasoconstrictor ET-1 level and oxidative stress. HIF-1a/ET-1 system signal pathway may interact with CIH-induced NADPH oxidase-dependent oxidative stress. Inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for prevention and treatment of obstructive sleep apnea hypopnea syndrome-induced hypertension.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Expressions of G2A and OGR1 in peripheral blood cells of patients with hypoxia-induced pulmonary hypertension

Objective: To detect the expression changes of proton-sensing receptor G protein-coupled receptor 2A (G2A) and ovarian cancer G protein-coupled receptors 1 (OGR1) in human peripheral blood cells of patients with hypoxia-induced pulmonary hypertension (HPH). Methods: Thirty-one patients with HPH were enrolled for IPH group, 16 males and 15 females, aged (65.19 ± 5.86) years;and 30 healthy people were enrolled for control group (NC group), 15 males and 15 females, aged (63.47 ± 6.16) years. The peripheral blood samples were collected and the mRNA expressions of G2A and OGR1 were determined by using real-time fluorescent quantitative PCR. The pulmonary arterial pressure (PAP) of HPH group was detected with echocardiography for the analysis of blood gas and pulmonary function testing. Human peripheral blood was collected to detect the mRNA levels of G2A, OGR1 and the serum levels of tumor necrosis factor-α (TNF-α). Results: PaCO2 was increased significantly in HPH group than that in NC group (p < .05). The percentage of forced expiratory volume in 1 s in predicted value (FEV1 pro%) and the ratio of FEV1/forced vital capacity (FVC) in HPH group were significant lower than those in NC group (p < .05). The expressions of peripheral blood G2A mRNA and TNF-α in HPH group were increased dramatically than those in NC group (p < .05). The expressions of OGR1 mRNA in peripheral blood had no difference between HPH group and NC group. The expressions of G2A mRNA and TNF-α in HPH group were positively related to pulmonary artery pressure significantly. Conclusions: The expression of proton-sensing receptor G2A and the level of TNF-α were increased in peripheral blood cells of patients with pulmonary hypertension. The expressions of TNF-α and G2A had positive correlations with pulmonary artery pressure.

NDRG1 promotes endothelial dysfunction and hypoxia-induced pulmonary hypertension by targeting TAF15

Background:Pulmonary hypertension(PH)represents a threatening pathophysiologic state that can be induced by chronic hypoxia and is characterized by extensive vascular remodeling.However,the mechanism underlying hypoxia-induced vascular remodeling is not fully elucidated.Methods and Results:By using quantitative polymerase chain reactions,western blotting,and immunohistochemistry,we demon-strate that the expression of N-myc downstream regulated gene-1(NDRG1)is markedly increased in hypoxia-stimulated endothelial cells in a time-dependent manner as well as in human and rat endothelium lesions.To determine the role of NDRG1 in endothelial dysfunction,we performed loss-of-function studies using NDRG1 short hairpin RNAs and NDRG1 over-expression plasmids.In vitro,silencing NDRG1 attenuated proliferation,migration,and tube formation of human pulmonary artery endothelial cells(HPAECs)un-der hypoxia,while NDRG1 over-expression promoted these behaviors of HPAECs.Mechanistically,NDRG1 can directly interact with TATA-box binding protein associated factor 15(TAF15)and promote its nuclear localization.Knockdown of TAF15 abrogated the effect of NDRG1 on the proliferation,migration and tube formation capacity of HPAECs.Bioinformatics studies found that TAF15 was involved in regulating PI3K-Akt,p53,and hypoxia-inducible factor 1(HIF-1)signaling pathways,which have been proved to be PH-related pathways.In addition,vascular remodeling and right ventricular hypertrophy induced by hypoxia were markedly alleviated in NDRG1 knock-down rats compared with their wild-type littermates.Conclusions:Taken together,our results indicate that hypoxia-induced upregulation of NDRG1 contributes to endothelial dysfunction through targeting TAF15,which ultimately contributes to the development of hypoxia-induced PH.

Hypoxia-induced ROS aggravate tumor progression through HIF-1α-SERPINE1 signaling in glioblastoma

Hypoxia,as an important hallmark of the tumor microenvironment,is a major cause of oxidative stress and plays a central role in various malignant tumors,including glioblastoma.Elevated reactive oxygen species(ROS)in a hypoxic microenvironment promote glioblastoma progression;however,the underlying mechanism has not been clarified.Herein,we found that hypoxia promoted ROS production,and the proliferation,migration,and invasion of glioblastoma cells,while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine(NAC)and diphenyleneiodonium chloride(DPI).Hypoxia-induced ROS activated hypoxia-inducible factor-1α(HIF-1α)signaling,which enhanced cell migration and invasion by epithelial-mesenchymal transition(EMT).Furthermore,the induction of serine protease inhibitor family E member 1(SERPINE1)was ROS-dependent under hypoxia,and HIF-1αmediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region,thereby facilitating glioblastoma migration and invasion.Taken together,our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway,and that targeting ROS may be a promising therapeutic strategy for glioblastoma.

Thymoquinone affects hypoxia-inducible factor-1αexpression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

BACKGROUND Pancreatic cancer(PC)is associated with some of the worst prognoses of all major cancers.Thymoquinone(TQ)has a long history in traditional medical practice and is known for its anti-cancer,anti-inflammatory,anti-fibrosis and antioxidant pharmacological activities.Recent studies on hypoxia-inducible factor-1α(HIF-1α)and PC have shown that HIF-1αaffects the occurrence and development of PC in many aspects.In addition,TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α.Therefore,we speculate whether TQ affects HIF-1αexpression in PC cells and explore the mechanism.AIM To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1αexpression.METHODS Cell counting kit-8 assay,Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity,migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial(hTERTHPNE)cells.Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1αmRNA and protein in PC cells.The effects of TQ on the HIF-1αprotein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation.RESULTS TQ significantly inhibited proliferative activity,migration,and invasion ability and promoted apoptosis of PANC-1 cells;however,no significant effects on hTERT-HPNE cells were observed.TQ significantly reduced the mRNA and protein expression levels of HIF-1αin PANC-1,AsPC-1,and BxPC-3 cells.TQ significantly inhibited the expression of the HIF-1αinitial expression pathway(PI3K/AKT/mTOR)related proteins,and promoted the ubiquitination degradation of the HIF-1αprotein in PANC-1 cells.TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1αprotein but affected the stability of the HIF-1αprotein by inhibiting the interaction between HIF-1αand HSP90,thus promoting its ubiquitination degradation.CONCLUSION The regulatory mechanism of TQ on HIF-1αprotein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1αprotein by inhibiting the interaction between HIF-1αand HSP90;Secondly,TQ reduced the initial expression of HIF-1αprotein by inhibiting the PI3K/AKT/mTOR pathway.

Hypoxia-inducible factor-1αin myocardial infarction

Hypoxia-inducible factor 1(HIF1)has a crucial function in the regulation of oxygen levels in mammalian cells,especially under hypoxic conditions.Its importance in cardiovascular diseases,particularly in cardiac ischemia,is because of its ability to alleviate cardiac dysfunction.The oxygen-responsive subunit,HIF1α,plays a crucial role in this process,as it has been shown to have cardioprotective effects in myocardial infarction through regulating the expression of genes affecting cellular survival,angiogenesis,and metabolism.Furthermore,HIF1αexpression induced reperfusion in the ischemic skeletal muscle,and hypoxic skin wounds in diabetic animal models showed reduced HIF1αexpression.Increased expression of HIF1αhas been shown to reduce apoptosis and oxidative stress in cardiomyocytes during acute myocardial infarction.Genetic variations in HIF1αhave also been found to correlate with altered responses to ischemic cardiovascular disease.In addition,a link has been established between the circadian rhythm and hypoxic molecular signaling pathways,with HIF1αfunctioning as an oxygen sensor and circadian genes such as period circadian regulator 2 responding to changes in light.This editorial analyzes the relationship between HIF1αand the circadian rhythm and highlights its significance in myocardial adaptation to hypoxia.Understanding the changes in molecular signaling pathways associated with diseases,specifically cardiovascular diseases,provides the opportunity for innovative therapeutic interventions,especially in low-oxygen environments such as myocardial infarction.

Hypoxia-inducible factor 1alpha and vascular endothelial growth factor in Glioblastoma Multiforme:a systematic review going beyond pathologic implications

Glioblastoma multiforme(GBM)is an aggressive primary brain tumor characterized by extensive heterogeneity and vascular proliferation.Hypoxic conditions in the tissue microenvironment are considered a pivotal player leading tumor progression.Specifically,hypoxia is known to activate inducible factors,such as hypoxia-inducible factor 1alpha(HIF-1α),which in turn can stimulate tumor neo-angiogenesis through activation of various downward mediators,such as the vascular endothelial growth factor(VEGF).Here,we aimed to explore the role of HIF-1α/VEGF immunophenotypes alone and in combination with other prognostic markers or clinical and image analysis data,as potential biomarkers of GBM prognosis and treatment efficacy.We performed a systematic review(Medline/Embase,and Pubmed database search was completed by 16th of April 2024 by two independent teams;PRISMA 2020).We evaluated methods of immunoassays,cell viability,or animal or patient survival methods of the retrieved studies to assess unbiased data.We used inclusion criteria,such as the evaluation of GBM prognosis based on HIF-1α/VEGF expression,other biomarkers or clinical and imaging manifestations in GBM related to HIF-1α/VEGF expression,application of immunoassays for protein expression,and evaluation of the effectiveness of GBM therapeutic strategies based on HIF-1α/VEGF expression.We used exclusion criteria,such as data not reporting both HIF-1αand VEGF or prognosis.We included 50 studies investigating in total 1319 GBM human specimens,18 different cell lines or GBM-derived stem cells,and 6 different animal models,to identify the association of HIF-1α/VEGF immunophenotypes,and with other prognostic factors,clinical and macroscopic data in GBM prognosis and therapeutic approaches.We found that increased HIF-1α/VEGF expression in GBM correlates with oncogenic factors,such as miR-210-3p,Oct4,AKT,COX-2,PDGF-C,PLDO3,M2 polarization,or ALK,leading to unfavorable survival.Reduced HIF-1α/VEGF expression correlates with FIH-1,ADNP,or STAT1 upregulation,as well as with clinical manifestations,like epileptogenicity,and a favorable prognosis of GBM.Based on our data,HIF-1αor VEGF immunophenotypes may be a useful tool to clarify MRI-PET imaging data distinguishing between GBM tumor progression and pseudoprogression.Finally,HIF-1α/VEGF immunophenotypes can reflect GBM treatment efficacy,including combined first-line treatment with histone deacetylase inhibitors,thimerosal,or an active metabolite of irinotecan,as well as STAT3 inhibitors alone,and resulting in a favorable tumor prognosis and patient survival.These data were supported by a combination of variable methods used to evaluate HIF-1α/VEGF immunophenotypes.Data limitations may include the use of less sensitive detection methods in some cases.Overall,our data support HIF-1α/VEGF’s role as biomarkers of GBM prognosis and treatment efficacy.

Unveiling the role of hypoxia-inducible factor 2alpha in osteoporosis:Implications for bone health

BACKGROUND Osteoporosis(OP)has become a major public health problem worldwide.Most OP treatments are based on the inhibition of bone resorption,and it is necessary to identify additional treatments aimed at enhancing osteogenesis.In the bone marrow(BM)niche,bone mesenchymal stem cells(BMSCs)are exposed to a hypoxic environment.Recently,a few studies have demonstrated that hypoxiainducible factor 2alpha(HIF-2α)is involved in BMSC osteogenic differentiation,but the molecular mechanism involved has not been determined.AIM To investigate the effect of HIF-2αon the osteogenic and adipogenic differentiation of BMSCs and the hematopoietic function of hematopoietic stem cells(HSCs)in the BM niche on the progression of OP.METHODS Mice with BMSC-specific HIF-2αknockout(Prx1-Cre;Hif-2αfl/fl mice)were used for in vivo experiments.Bone quantification was performed on mice of two genotypes with three interventions:Bilateral ovariectomy,semilethal irradiation,and dexamethasone treatment.Moreover,the hematopoietic function of HSCs in the BM niche was compared between the two mouse genotypes.In vitro,the HIF-2αagonist roxadustat and the HIF-2αinhibitor PT2399 were used to investigate the function of HIF-2αin BMSC osteogenic and adipogenic differentiation.Finally,we investigated the effect of HIF-2αon BMSCs via treatment with the mechanistic target of rapamycin(mTOR)agonist MHY1485 and the mTOR inhibitor rapamycin.RESULTS The quantitative index determined by microcomputed tomography indicated that the femoral bone density of Prx1-Cre;Hif-2αfl/fl mice was lower than that of Hif-2αfl/fl mice under the three intervention conditions.In vitro,Hif-2αfl/fl mouse BMSCs were cultured and treated with the HIF-2αagonist roxadustat,and after 7 d of BMSC adipogenic differentiation,the oil red O staining intensity and mRNA expression levels of adipogenesis-related genes in BMSCs treated with roxadustat were decreased;in addition,after 14 d of osteogenic differentiation,BMSCs treated with roxadustat exhibited increased expression of osteogenesis-related genes.The opposite effects were shown for mouse BMSCs treated with the HIF-2αinhibitor PT2399.The mTOR inhibitor rapamycin was used to confirm that HIF-2αregulated BMSC osteogenic and adipogenic differentiation by inhibiting the mTOR pathway.Consequently,there was no significant difference in the hematopoietic function of HSCs between Prx1-Cre;Hif-2αfl/fl and Hif-2αfl/fl mice.CONCLUSION Our study showed that inhibition of HIF-2αdecreases bone mass by inhibiting the osteogenic differentiation and increasing the adipogenic differentiation of BMSCs through inhibition of mTOR signaling in the BM niche.

Danlou Tablet Improves Chronic Intermittent Hypoxia-Induced Dyslipidemia and Arteriosclerosis by HIF-1α-Angptl4 mRNA Signaling Pathway

Objective:To detect whether Danlou Tablet(DLT)regulates the hypoxia-induced factor(HIF)-1α-angiopoietin-like 4(Angptl4)mRNA signaling pathway and explore the role of DLT in treating chronic intermittent hypoxia(CIH)-induced dyslipidemia and arteriosclerosis.Methods:The mature adipocytes were obtained from3 T3-L1 cell culturation and allocated into 8 groups including control groups(Groups 1 and 5,0.1 mL of cell culture grade water);DLT groups(Groups 2 and 6,0.1 mL of 1,000μg/mL DLT submicron powder solution);dimethyloxalylglycine(DMOG)groups(Groups 3 and 7,DMOG and 0.1 mL of cell culture grade water);DMOG plus DLT groups(Groups 4 and 8,DMOG and 0.1 mL of 1,000μg/mL DLT submicron powder solution).Groups1-4 used mature adipocytes and groups 5-8 used HIF-1α-siRNA lentivirus-transfected mature adipocytes.After 24-h treatment,real-time polymerase chain reaction and Western blot were employed to determine the mRNA and protein expression levels of HIF-1αand Angptl4.In animal experiments,the CIH model in ApoE^(-/-)mice was established.Sixteen mice were complete randomly divided into 4 groups including sham group,CIH model group[intermittent hypoxia and normal saline(2 mL/time)gavage once a day],Angptl4 Ab group[intermittent hypoxia and Angptl4 antibody(30 mg/kg)intraperitoneally injected every week],DLT group[intermittent hypoxia and DLT(250 mg/kg)once a day],4 mice in each group.After 4-week treatment,enzyme linked immunosorbent assay was used to detect the levels of serum total cholesterol(TC)and triglyceride(TG).Hematoxylin-eosin and CD68 staining were used to observe the morphological properties of arterial plaques.Results:Angptl4 expression was dependent on HIF-1α,with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1α-transfected cells.DLT inhibited HIF-1αand Angptl4 mRNA expression(P<0.05 or P<0.01)and reduced HIF-1αand Angptl4 protein expressions with DMOG in mature adipocytes(all P<0.01),as the effect on HIF-1αprotein also existed in the presence of siHIF-1α(P<0.01).ApoE^(-/-)mice treated with CIH had increased TG and TC levels(all P<0.01)and atherosclerotic plaque.Angptl4 antibody and DLT both reduced TG and TC levels(all P<0.01),as well as reducing atherosclerotic plaque areas,narrowing arterial wall thickness and alleviating atherosclerotic lesion symptoms to some extent.Conclusion:DLT had positive effects in improving dyslipidemia and arteriosclerosis by inhibiting Angptl4 protein level through HIF-1α-Angptl4 mRNA signaling pathway.

Apelin-APJ Effects of Ginsenoside-Rb1 Depending on Hypoxia-Induced Factor 1α in Hypoxia Neonatal Cardiomyocytes

Objective： To investigate whether ginsenoside-Rbl （Gs-Rb1） inhibits the apoptosis of hypoxia cardiomyocytes by up-regulating apelin-APJ system and whether the system is affected by hypoxia-induced factor 1α （Hif-1α）. Methods： Neonatal rat cardiomyocytes were randomly divided into 6 groups： a control group, a simple COCl2 group, a simple Gs-Rbl group, a CoCl2 and Gs-Rbl hypoxia group, a CoCl2 and 3-（5＇-hydroxymethyl-2＇-furyl）-1-benzylindazole （YC-1） group, a CoCl2 and YC-1 group and a Gs-Rbl group, in which YC-1 inhibits the synthesis and accelerates the degradation of Hif-la. The concentration of COCI2, Gs-Rbl and YC-1 was 500 μmol/L, 200 μ mol/L and 5 μmol/L, respectively; the apoptosis ratio was analyzed with a flow cytometer; and apelin, APJ and Hif-1 c= were assayed with immunocytochemistry, Western blot assays and reverse transcription polymerase chain reaction （RT-PCR）. Results： （1） The anti-apoptosis effect of Gs-Rbl on hypoxia cardiomyocytes was significantly inhibited by YC-1; （2） Hypoxia significantly up-graded the expression of mRNA and protein of apelin; this effect was further reinforced by Gs-Rbl and significantly inhibited by YC-1; （3） Gs-Rb1 further strengthened the expression of APJ mRNA and APJ proteins once hypoxia occurred, which was significantly inhibited by YC-1; （4） Gs-Rb1 significantly increased the expression of Hif-1α, which was completely abolished by YC-1; （5） There was a negative relationship between AR and apelin （or APJ, including mRNA and protein）, a positive correlation between apelin （or APJ） protein and Hif-1a protein, in hypoxia cardiomyocytes. Conclusion： The apelin-APJ system plays an important role in the anti-apoptosis effect of Gs- Rb1 on hypoxia neonatal cardiomyocytes, which was partly adjusted by Hif-1α.

Protective effects of astragalus extract against intermittent hypoxia-induced hippocampal neurons impairment in rats

Background Intermittent hypoxia is the main pathophysiological cause of the obstructive sleep apnea syndrome. Astragalus shows improvement of spatial learning and memory abilities under intermittent hypoxia. Our study aimed to investigate the protective effect of astragalus against intermittent hypoxia induced-hippocampal neurons impairment in rats and lay the theoretical foundation for the sleep apnea improvement in cognitive function by astragalus. Methods Male Wistar rats were divided into 4 groups： blank control group, normoxia group, intermittent hypoxia group and astragalus treated intermittent hypoxia group. After 6-week treatment, apoptosis of neurons was evaluated by terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling （TUNEL） assay. Furthermore, the expression of HIF-la was detected by real-time reverse transcription polymerase chain reaction （RT-PCR） at the mRNA level as well as by immunohistochemistry （IHC） and Western blotting at the protein level. Results HPLC analysis indicated that astragaloside IV, astragaloside II and astragaloside I were the main compounds in astragals extract. Astragalus extract reduced the apoptosis of hippocampal neurons （P 〈0.05） and decreased the expression of HIF-la at both the mRNA and protein levels in hippocampus compared with non-treated groups （P 〈0.05）. Conclusion Astragalus protects aqainst intermittent hypoxia-induced hippocampal neurons impairment in rats.

The role of hypoxia-induced factor 1α in breast cancer

Breast cancer usually grows very quickly,becoming insensitive to blood flow in nearby veins;because of that,inside solid tumors it's possible to find a hypoxic environment,in other words,an environment where oxygen is less available.Another feature of cancer is its angiogenesis rate,because of the high energy demand,new blood vessels must be produced to take nutrients inside the solid tumor mass.Even with normal blood flow bringing the cancer oxygen and nutrients,its cells favor hypoxia,in an event known as Warburg Effect.According to the Warburg Effect,cells,even with normal oxygen rates,prefer to use fermentation instead of the citric acid cycle to produce ATP.For the cancer to operate normally in hypoxia,a transcription factor family is activated,known as hypoxia-induced factors(HIF),composed of a HIF-1βand a HIF-1αsubunits.As HIF-1αis expressed during hypoxia,it is a great target for treatments and a breast cancer biomarker.Because of the role of HIF-1αin cancer and the high incidence of breast cancer worlwide,this review was performed in order to bring the most recent results concerning the role HIF-1αcan exert in breast cancer development and progression.

Hypoxia-inducible factor-1 alpha regulates the role of vascular endothelial growth factor on pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

Background Hypoxia-inducible factor-1α (HIF-1α) is one of the pivotal mediators in the response of lungs to decreased oxygen availability, and increasingly has been implicated in the pathogenesis of pulmonary hypertension. Vascular endothelial growth factor (VEGF), a downstream target gene of HIF-1α, plays an important role in the pathogenesis of hypoxic pulmonary hypertension and hypoxic pulmonary artery remodelling. In this study, we investigated the dynamic expression of HIF-1α and VEGF in pulmonary artery of rats with hypoxia-induced pulmonary hypertension. Methods Forty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were estimated. Lungs were inflated and fixed for in situ hybridisation and immunohistochemistry. Results mPAP values were significantly higher than the control values after 7days of hypoxia [(18.4±0.4) mmHg, P<0.05]. RVHI developed significantly after 14 days of hypoxia. Expression of HIF-1α protein increased in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterial tunica media, HIF-1α protein was markedly increased by day 3 (0.20±0.02, P<0.05), reached the peak by day 7, then declined after day 14 of hypoxia. HIF-1α mRNA increased significantly after day 14 of hypoxia (0.20±0.02, P<0.05). VEGF protein began to increase markedly after day 7 of hypoxia, reaching its peak around day 14 of hypoxia (0.15±0.02, P<0.05). VEGF mRNA began to increase after day 7 of hypoxia, then remained more or less stable from day 7 onwards. VEGF mRNA is located mainly in tunica intima and tunica media, whereas VEGF protein is located predominantly in tunica intima. Linear analysis showed that HIF-1α mRNA, VEGF and mPAP were correlated with hypoxic pulmonary artery remodelling. HIF-1α mRNA was positively correlated with VEGF mRNA and protein (P<0.01). Conclusion HIF-1α and VEGF are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.

Efficacy of Shouzhangshen(Rhizoma Gymnadeniae Crassinervidis)extract against acute high altitude hypoxia-induced brain injury in mice

OBJECTIVE:To evaluate the protective effect of Shouzhangshen(Rhizoma Gymnadeniae Crassinervidis)extract against acute high altitude hypoxia-induced brain injury in mice.METHODS:Sixty C57BL/6J mice were selected and assigned to six groups(n=10):normal control group,low-pressure hypoxia group,positive control group(dexamethasone 500 mg/kg),and three groups treated with Shouzhangshen extract(250,500,and 750 mg/kg,respectively).The Morris water maze test was performed to evaluate alterations in spatial learning and memory deficits.Nissl staining was performed to detect Nissl bodies and neuron damage.Hypoxia-inducible factor(HIF)-1α,interleukin(IL)-1β,tumor necrosis factor(TNF)-α,vascular endothelial growth factor(VEGF),and malondialdehyde(MDA)expression in brain tissue and serum,as well as superoxide dismutase(SOD)and glutathione(GSH)activity in brain tissues were measured by enzyme-linked immunosorbent assays,quantitative real-time-polymerase chain reaction and western blots.RESULTS:The Morris water maze test results showed that Shouzhangshen extract can significantly reduce the latency and swimming distance to escape onto a visible platform,increase neuron density and hierarchy and the number of pyramidal neurons,and decrease the expression of HIF-1α,IL-1β,TNF-α,and VEGF mRNAs and proteins in both brain tissue and serum(P<0.05).Furthermore,significantly lower MDA expression and higher GSH activity were detected in the three groups treated with Shouzhangshen compared with the low-pressure hypoxia group(P<0.05).However,no significant alteration was observed for SOD activity(P>0.05).CONCLUSION:Our findings suggest that Shouzhangshen extract may have a significant effect on acute high altitude hypoxia-induced brain injury in mice.

Mitochondrial dysfunction affects hepatic immune and metabolic remodeling in patients with hepatitis B virus-related acute-onchronic liver failure

BACKGROUND Immune dysregulation and metabolic derangement have been recognized as key factors that contribute to the progression of hepatitis B virus(HBV)-related acute-on-chronic liver failure(ACLF).However,the mechanisms underlying immune and metabolic derangement in patients with advanced HBV-ACLF are unclear.AIM To identify the bioenergetic alterations in the liver of patients with HBV-ACLF causing hepatic immune dysregulation and metabolic disorders.METHODS Liver samples were collected from 16 healthy donors(HDs)and 17 advanced HBV-ACLF patients who were eligible for liver transplantation.The mitochondrial ultrastructure,metabolic characteristics,and immune microenvironment of the liver were assessed.More focus was given to organic acid metabolism as well as the function and subpopulations of macrophages in patients with HBV-ACLF.RESULTS Compared with HDs,there was extensive hepatocyte necrosis,immune cell infiltration,and ductular reaction in patients with ACLF.In patients,the liver suffered severe hypoxia,as evidenced by increased expression of hypoxia-inducible factor-1α.Swollen mitochondria and cristae were observed in the liver of patients.The number,length,width,and area of mitochondria were adaptively increased in hepatocytes.Targeted metabolomics analysis revealed that mitochondrial oxidative phosphorylation decreased,while anaerobic glycolysis was enhanced in patients with HBV-ACLF.These findings suggested that,to a greater extent,hepa-tocytes used the extra-mitochondrial glycolytic pathway as an energy source.Patients with HBV-ACLF had elevated levels of chemokine C-C motif ligand 2 in the liver homogenate,which stimulates peripheral monocyte infiltration into the liver.Characterization and functional analysis of macrophage subsets revealed that patients with ACLF had a high abundance of CD68^(+)HLA-DR^(+)macrophages and elevated levels of both interleukin-1βand transforming growth factor-β1 in their livers.The abundance of CD206^(+)CD163^(+)macrophages and expression of interleukin-10 decreased.The correlation analysis revealed that hepatic organic acid metabolites were closely associated with macrophage-derived cytokines/chemokines.CONCLUSION The results indicated that bioenergetic alteration driven by hypoxia and mitochondrial dysfunction affects hepatic immune and metabolic remodeling,leading to advanced HBV-ACLF.These findings highlight a new therapeutic target for improving the treatment of HBV-ACLF.

Reveal more mechanisms of precondition mesenchymal stem cells inhibiting inflammation

Hypoxia can get more ability to inhibit inflammation.But how it impact on survival time of mesenchymal stem cells(MSCs)is confusing and how preconditioned MSCs inhibiting inflammation are partially known.Those issues decided the value of preconditioned MSCs by hypoxia.

HIFs in hypoxic regulation of the extracellular matrix:focus on little-known player HIF-3

The structural and associated molecules of the extracellular matrix(ECM)complex is an important component of the local milieu of cells,both for maintaining their functions and homeostasis.It is a dynamic structure that is finely tuned to changes in the microenvironment.One of these factors is hypoxia,which can arise in tissues due to physiological or pathological effects.As a result of the hypoxic effect,the properties of the ECM are significantly modified,stiffness increases,the balance between degradation and synthesis of structural proteins shifts,and the deposition of biologically active mediators’changes.Hypoxia-inducible factors(HIFs)contribute significantly to the modification of the properties of the ECM under hypoxia.Among the three HIF isoforms,HIF-1 is the most studied,with numerous identified target genes encoding proteins that participate in all matrisome compartments.Much less is known about HIF-2 and HIF-3.In the context of the effects of hypoxia on the matrisome,HIF-3 isoform is of particular interest.Unlike the first two isoforms,transiently expressed during the first hours of hypoxia,HIF-3 is activated after around 24 h of exposure to hypoxia and persists for a longer period.In addition,its transcription is more pronounced under hypoxia than are HIFs 1 and 2.HIF-3,rather than the first two isoforms,is possibly responsible for the changes that occur in matrisome during prolonged hypoxic conditions.This review attempts to summarize the available data on the involvement of HIF-3 in the matrisome modification under hypoxia.