Danlou Tablet Improves Chronic Intermittent Hypoxia-Induced Dyslipidemia and Arteriosclerosis by HIF-1α-Angptl4 mRNA Signaling Pathway / 中国结合医学杂志

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 from 3T3-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 1,000 µg/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 1,000 µg/mL DLT submicron powder solution). Groups 1-4 used mature adipocytes and groups 5-8 used HIF-1 α-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-1 α and 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 expression 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-1 α, with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1 α -transfected cells. DLT inhibited HIF-1 α and Angptl4 mRNA expression (P<0.05 or P<0.01) and reduced HIF-1 α and Angptl4 protein expressions with DMOG in mature adipocytes (all P<0.01), as the effect on HIF-1 α protein also existed in the presence of siHIF-1 α (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 reduce 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-1 α-Angptl4 mRNA signaling pathway.

Combination effect of doxorubicin and HIF inhibitor on MCF-7 CD44+/CD24- subpopulation cells in hypoxic condition

Abstract Hypoxia-inducible factors (HIFs) and cancer stem cells (CSCs) are two challenging causes of radiotherapy and chemotherapy resistance, leading to most cases of failure and recurrence in breast cancer therapy. This study was conducted to investigated the inhibitory effect of combination therapy with doxorubicin (an anthracycline) and FM19G11 (an HIF inhibitor) on MCF-7 cells and their CSC-like cells (CSC-LCs). MCF-7 CSC-LCs with a CD44+/CD24- phenotype were sorted and characterized by flow cytometry. A combination of doxorubicin and FM19G11 caused more cytotoxic effects on MCF-7 and CSC-LCs compared to doxorubicin monotherapy. The largest synergistic effect was observed in CSC-LCs under hypoxic conditions; however, MCF-7 cells showed no synergism in normoxic conditions. The administration of doxorubicin and FM19G11 induced late apoptotic and necrotic cell death in MCF-7 and CSC-LCs. Additionally, G2 phase arrest was observed in both cells. Our results demonstrated that co-administration of FM19G11 and doxorubicin had a synergistic effect in hypoxia and improved drug resistance in breast cancer stem cells.

Hypoxia preconditioning promotes bone marrow mesenchymal stem cells survival and vascularization through the activation of hif-1α/malat1/vegfa pathway / 中国组织工程研究

BACKGROUND: Previous study demonstrated that hypoxia preconditioning promoted mesenchymal stem cells survival and their therapeutic efficacy, and this effect was mediated by hypoxia induced factor-1α (HIF-1α). However, specific downstream mechanism remained unclear. OBJECTIVE: To observe the influence of hypoxia preconditioning on the survival and vascularization potential of bone marrow mesenchymal stem cells in vitro and explore the regulatory mechanism of HIF-1α/MALAT1/VEGFA pathway. METHODS: Bone marrow mesenchymal stem cells were obtained and cultured in vitro. Cells were divided into hypoxia (1% O2) and normoxia control groups (20% O2), and cultured for 24 hours. Cells proliferation, apoptosis and vascularization were evaluated. The expression of HIF-1α, MALAT1, and VEGFA was detected. HIF-1α and MALAT1 were inhibited by their siRNAs separately. HIF-1α siRNA scramble and MALAT1 siRNA scramble were used as negative controls before hypoxia preconditioning. Alterations of the molecules were examined and compared in different groups. RESULTS AND CONCLUSION: (1) Compared with the normoxia control group, cell viability was significantly enhanced; and cell apoptosis percentage was significantly declined in the hypoxia group; vascular lumen like structure was also increased significantly in the hypoxia group (P < 0.01); expression of HIF-1α, MALAT1, and VEGFA was significantly increased in the hypoxia group (P < 0.01). (2) After the inhibition of HIF-1α and hypoxia preconditioning, both MALAT1 and VEGFA expression levels were significantly reduced (P < 0.01). The expression of VEGFA was also significantly suppressed after the blockage of MALAT1 (P < 0.01). (3) This study suggested that hypoxia preconditioning effectively promoted bone marrow mesenchymal stem cell survival and vascularization through the activation of HIF-1α/MALAT1/VEGFA pathway.

Inhibition of the hypoxia-induced factor-1α and vascular endothelial growth factor expression through ginsenoside Rg3 in human gastric cancer cells

Objective: The aim of this study is to probe in the inhibitory effects of ginsenoside Rg3 on the expression of hypoxia-induced factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in human gastric cancer cells. Materials and Methods: Human gastric cancer BGC823 cells were divided into the control group and experiment group, and expression levels of HIF-1α and VEGF were detected by immunocytochemistry and Western blot after cells were cultured under hypoxia for different durations. Results: Under hypoxia, expression of HIF-1α and VEGF in human gastric cancer BGC823 cells showed an increasing trend, and that was remarkably lower in experiment group than in the control group after applying Rg3, which was obvious at 12 and 24 h (P < 0.05). Conclusion: Rg3 can inhibit expression of HIF-1α and VEGF in human gastric cancer cells and may influence abdominal implantation metastasis of gastric cancer through inhibiting its expression

Effect of dexmedetomidine on expression of hypoxia-inducible factor-1α during endotoxin-caused apoptosis in macrophages of mice / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine on expression of hypoxia-inducible factor-1α (HIF-1α) during endotoxin-caused apoptosis in macrophages of mice.Methods Mouse macrophage cell line RAW264.7 cultured in vitro were seeded in 6-well or 96-well plates and divided into 4 groups (n=16 each) when cell confluence reached 60％-70％ using a random number table method:control group (group Con),dexmedetomidine group (group Dex),lipopolysaccharide (LPS) group,and LPS plus dexmedetomidine group (group LPS+Dex).Phosphate buffer solution was added in group Con.Dexmedetomidine 1 μmol/L was added in group Dex.LPS 1 μg/ml was added in LPS and LPS+Dex groups.Dexmedetomidine 1 μmol/L was added immediately after adding LPS in group LPS+Dex.Cells were then cultured for 24 h in each group.Cell apoptosis was measured using TUNEL,mitochondrial membrane potential using JC-1,reactive oxygen species (ROS) content by ROS kit,and ATP content by ATP kit.The apoptosis rate was calculated.The expression of HIF-1α,cytochrome C (Cyt-c),caspase-9 and cleaved caspase-3 was detected by Western blot.Results Compared with group Con,the apoptosis rate and ROS content were significantly increased,ATP content and mitochondrial membrane potential were decreased,the expression of HIF-1α,Cyt-c,caspase-9 and cleaved caspase-3 was up-regulated in group LPS (P＜ 0.05),and no significant change was found in the parameters mentioned above in group Dex (P＞0.05).Compared with group LPS,the apoptosis rate and ROS content were significantly decreased,ATP content and mitochondrial membrane potential were increased,the expression of HIF-1α was up-regulated,and the expression of Cyt-c,caspase-9 and cleaved caspase-3 was down-regulated in group LPS + Dex (P＜0.05).Conclusion Dexmedetomidine can reduce endotoxin-caused oxidative stress injury to macrophages,improve mitochondrial function and inhibit mitochondrial apoptosis,and the mechanism may be related to upregulating the expression of HIF-1α in mice.

The expression and clinical significance of hypoxia-induced factor-1 α in gallbladder carcinoma tissues and its role on metformin-suppressed metastasis in GBC-SD cells / 中华肝胆外科杂志

Objective To study the expression and the clinical significance of hypoxia-induced factor-1α (HIF-1α) in gallbladder cancer tissues,and the role and mechanism of HIF-1α in metformin-suppressed metastasis in gallbladder carcinoma GBC-SD cells.Methods 24 specimens of gallbladder cancer tissues and 5 specimens of chronic cholecystitis were collected from the First Affiliated Hospital of Zhengzhou University between June 2016 and February 2017.Immunohistochemistry and qPCR were used to detect the expression of HIF-1α in gallbladder cancer tissues,in adjacent non-cancer tissues and in chronic cholecystitis,and the clinical significance was analyzed.The model of metastasis was induced by hypoxia;the wound healing assay and the Transwell assay were used to detect the ability of cell metastasis;the expressions of HIF-1α and VEGF in gallbladder carcinoma GBC-SD cells were detected by western blotting assay and immunofiuorescence.Results The expression of HIF-1α in gallbladder cancer tissues was higher than the adjacent non-cancer tissues and in chronic cholecystitis.The expression of HIF-1α was correlated with lymph node metastasis and TNM staging in gallbladder cancer tissues (P ＜ 0.05).The wound healing rate after 48 h in the negative control group and in the treatment with hypoxia group (1％ O2) in GBC-SD cells were (46.5 ± 4.8) ％ and (67.3 ± 4.0) ％,respectively.The Transwell data showed that the numbers of metastasis after 24 h in the negative control group and in the treatment with hypoxia group GBC-SD cells were (147.4 ± 11.7) and (234.4 ± 17.7),respectively.When compared with the negative control group,treatment with hypoxia significantly increased the ability of metastasis and up-regulated the expression of HIF-1α and VEGF in GBC-SD cells (P ＜ 0.05).The wound healing rate after 48 h in the negative control group,the metformin group,the hypoxia group and the metformin and hypoxia group in GBC-SD cells were (40.6 ± 7.1) ％,(16.4 ± 9.4) ％,(69.5 ± 4.0) ％ and (22.4 ± 7.4) ％,respectively.The Transwell data showed that the numbers of metastasis after 24 h in the negative control group,the metformin group,the hypoxia group and the metformin and hypoxia group in GBC-SD cells were (148.4 ± 6.9),(90.0 ± 8.4),(185.8 ± 10.2) and (113.4± 8.6),respectively.When comparcd with the hypoxia group,treatment with metformin and hypoxia significantly decreased the ability of metastasis and down-regulated the expression of HIF-1α and VEGF in GBC-SD cells (P ＜ 0.05).The wound healing rate after 48 h in the negative control group,the 2MeoE2 group,the hypoxia group,the 2MeoE2 and hypoxia group in GBC-SD cells were (43.4 ±4.4)％,(25.9 ±9.0)％,(63.3 ±2.2)％,(46.2 ±4.5)％,respectively.The Transwell data showed that the numbers of metastasis after 24 h in the negative control group,the 2MeoE2 group,the hypoxia group,the 2MeoE2 and hypoxia group in GBC-SD cells were (144.2 ± 12.6),(80.2 ±7.7),(203.8 ±7.0),(124.0 ± 5.2),respectively.When compared with the hypoxia group,treatment with HIF-1α inhibitor 2MeoE2 and hypoxia significantly decreased the ability of metastasis and down-regulated the expression of HIF-1α and VEGF in GBC-SD cells (P ＜ 0.05).Conclusions The expression of HIF-1 α was correlated with lymph node metastasis and TNM staging in gallbladder cancer tissues.Treatment with hypoxia significantly increased the expression of HIF-1α and VEGF and promoted metastasis of GBC-SD cells,while treatment with metformin decreased the ability of metastasis induced by hypoxia via inhibiting the HIF-1o/VEGF pathway in GBC-SD cells.

The expression of hypoxia induced factor-1α signaling in nonacoholic fatty liver disease in rats / 中华内分泌代谢杂志

Objective To investigate the role of hypoxia induced factor-1 α (HIF-1 α) signaling pathway in development of nonacoholic fatty liver disease (NAFLD) by using a rat model.Methods Forty SD rats were randomly divided into 5 groups (normal control group,4 weeks model group,8 weeks model group,12 weeks model group,and 16 weeks model group according to random number table).NAFLD model was induced by high fat diet.Serum biochemical parameters and liver histological examinations were observed.The HIF-1α,peroxisome proliferator-activated receptor α (PPARα),and nuclear factor-κB (NF-κB) mRNA transcriptions of liver tissue were detected with realtime PCR.Results Compared with the control group,a remarkable rise of serum alanine amiotransferase,triglyceride,cholesterol,and reduction of high density lipoprotein-cholesterol were observed in rats treated with high fat diet after 12 weeks and 16 weeks (P＜0.05).Compared with model group,steatosis,grade of inflammation,and degree of fibrosis in the liver were also significantly aggravated.Fed with high fat diet for 8 weeks,the expression of HIF-1α was significantly higher than that in the control group;the expression of PPARα in 4 weeks group was significantly higher than that in the control group.The expression of PPARα was again significantly elevated in 16 weeks group (P＜0.05);NF-κB expression was increased gradually with high fat diet (P＜0.05).The expression of HIF-1 α mRNA was related with PPARα (r =0.82,P＜0.05) and NF-κB (r =0.67,P＜0.05),and PPARα mRNA was related with NF-κB (r =0.78,P＜0.05).Conclusion HIF-1 α signaling may regulate PPARα/NF-κB and seems to be related with development of NAFLD.

Effect of Estradiol and its Metabolite on Hypoxic Induced Factor-1αand Alkane Hydroxylase in Experimental Rats With Ovariectomy and Hypoxic Pulmonary Hypertension / 中国循环杂志

Objective: To explore the effects of 17 β-estrogen (E2) and 2-methoxyestradiol (2ME) on hypoxic induced factor-1α (HIF-1α) and alkane hydroxylase (AlkB) in experimental rats with ovariectomy and hypoxic pulmonary hypertension. Methods: A total of 60 healthy female SD rats with castrated surgery were randomly divided into 6 groups:①Routine oxygen group,②Routine oxygen + E2 group, the rats received subcutaneous injection of E2 (20 μg/kg?d),③Routine oxygen + 2ME group, the rats received 2ME (240 μg/kg?d) and④Hypoxia group,⑤Hypoxia + E2 group,⑥Hypoxia + 2ME group.n=10 in each group and all animals were treated for 8 weeks to establish the hypoxic pulmonary hypertension model. The mean pulmonary artery pressure (mPAP) was measured after bloodletting, right ventricle hypertrophy index (RVHI) was calculated and small pulmonary artery remodeling was observed by HE staining. The expression level of HIF-1α and AlkB were examined by RT-PCR and Western blot analysis. Results: Compared with Routine oxygen group, the rats in Hypoxia group had obviously thickened small pulmonary artery wall with narrowed lumen, increased mPAP and RVHI; the above changes in Hypoxia + E2 and Hypoxia + 2ME groups were relatively smaller, their mPAP and RVHI were higher than Routine oxygen group, while mPAP and RVHI were similar between Hypoxia + E2 and Hypoxia + 2ME groups. There were no real morphological changes in small pulmonary vessels in Routine oxygen + E2 and Routine oxygen + 2ME groups. The HIF-1α expression was obviously elevated in Hypoxia group than Routine oxygen group, while the elevation was less in Hypoxia + E2 and Hypoxia + 2ME groups. HIF-1α expression had no real changes in Routine oxygen+E2 and Routine oxygen + 2ME groups. The AlkB expression was obviously reduced in Hypoxia group than Routine oxygen group, while the reduction was less in Hypoxia + E2 and Hypoxia + 2ME groups. AlkB expression had no real changes in Routine oxygen + E2 and Routine oxygen + 2ME groups. Conclusion: Estradiol E2 and 2ME could remit pulmonary hypertension which might be via up-regulating AlkB expression and down-regulating HIF-1α expression in experimental rats with hypoxic pulmonary hypertension.

Mechanism of radiosensitization effect of berberine on human nasopharyngeal carcinoma in hypoxia / 中华放射医学与防护杂志

Objective To investigate the radiosensitiation effect of berberine on human nasopharyngeal carcinoma ( NPC) in hypoxia condition and explore the underlying mechanisms. Methods MTT assay, clonogenic assay and flow cytometry were performed to analyze cell proliferation, colony formation and apoptosis, respectively. Male nude mice inoculated subcutaneously with CNE-2 cells were used to examine the radiosensitization effect of berberine in vivo. The expressions of HIF-1α and VEGF were assessed by Western blot. Results Berberine efficiently inhibited the proliferation of CNE-2 cells in time-dependent and dose-dependent fashions with an IC50 of ( 14?9 ± 2?2 ) μmol/L. Clonogenic survival assay showed that berberine ( 5 μmol/L ) sensitized CNE-2 cells to ionizing radiation in hypoxia and its SERD0 was 1?27. Under hypoxic condition, berberine alone (5, 15 μmol/L) could induce apoptosis (t=5?01, 9?02,P<0?05) and it further promoted 8 Gy radiation-induced apoptosis (t =5?31, 9?91,P <0?05). Moreover, berberine significantly delayed the tumor growth in the combination group (berberine +irradiation) compared with the mice received irradiation alone or PBS (t =2?96, 14?52, P <0?05). Immunobloting assay showed that berberine inhibited the upregulation of HIF-1α and VEGF induced by hypoxia in CNE-2 cells. Conclusion Berberine confers radiosensitivity on hypoxic NPC in vitro and in vivo, which is probably associated with the downregulation of HIF-1α and VEGF expressions.

The effects of exogenous hydrogen sulfide on injury of rat hippocampus neurons induced by oxygen-glucose deprivation and restoration / 中华急诊医学杂志

Objective To investigate the effects of exogenous hydrogen sulfide (H2S) on injury of rat hippocampus neurons induced by oxygen-glucose deprivation and restoration (OGD/R) and explore its mechanism.Methods Hippocampus neurons were isolated from embryonic day 16-18 (E16-18) rat embryos.Hippocampus was immediately removed and digested with 0.25％ trypsin.The neurons were isolated and cultured at 37 ℃ for 7 days and neuron-specific enolase (NSE) was detected by immunohistochemical staining method to identify neurons.At 8th day,the neurons were placed in deoxygenated glucose-free medium and exposed to 95％ N2-5％ CO2 in an air tight chamber for 1 hour,and then replaced the glucose-free medium with original medium,and returned the cultures to a standard incubator in 5％ CO2 at 37 ℃ and incubated for another 24 h.The neurons were divided into 3 groups:group Ⅰ control; group Ⅱ OGD/R,and group ⅢOGD + NaHS,the latter was further divided into 5 subgroups:groups Ⅲ1-5 with 25,50,100,200,400 μmol/L NaHS added,respectively.Then cell viability was quantified by MTT method,the level of lactate dehydrogenase (LDH) were detected,apoptosis was measured by Annexin V FITC/PI Apoptosis Kits,and RT-PCR was used to assay HIF-1 α mRNA in neurons in all groups.Results Compared with control group,the LDH level,apoptosis and expression of HIF-1α mRNA in group Ⅱ were significantly increased,the cell viability was significantly decreased (P ＜ 0.01).There were no significant differences in the LDH level,apoptosis and expression of HIF-1 α mRNA and the cell viability between group Ⅱ and group Ⅲ1 (P ＞ 0.05).Compared with group Ⅱ,the LDH level,apoptosis and expression of HIF-1α mRNA in group Ⅲ2-4 were significantly increased,the cell viability was significantly increased (P ＜ 0.01).Compared with group Ⅱ,the LDH level,apoptosis and expression of HIF-1 α mRNA in the group Ⅲ 5 were significantly decreased,the cell viability was significantly decreased (P ＜ 0.01).Conclusions H2S of low concentration has no significant effects on injury of rat hippocampus neurons induced by OGD/R.H2S of moderate doses can protect rat hippocampus neurons from OGD/R injury and H2S of high concentration can aggravate injury.The expression of HIF-1α mRNA in rat hippocampus neurons was increased after OGD/R,and the protective role of H2S is associated with increase in the expression of HIF-1α mRNA.

Update on apoptosis signal pathway of retinal ganglion cell / 中华实验眼科杂志

Retinal ganglion cells (RGCs) apoptosis is the ultimate pathway of many disorders involved in the retina and optic nerve,and contributes to irreversible visual loss.RGCs are vulnerable to various stimulates including ocular trauma,intraocular hypertension,endoophthalmitis and neurotoxin,and eventually undergo apoptosis.Those external stimulates can activate multiple intracellular signal pathways,and regulate apoptosis associated genes.This paper reviewed the recent findings regarding external stimulates,intracellular signaling pathways and gene regulation involved in the pathological retinal ganglion cells death,in order to provide potential targets for the development of new specific therapies for disorders associate with RGCs degeneration.

A Study of Predicting Postpartum Depression and the Recovery Factor from Prepartum Depression / 대한주산의학회잡지

OBJECTIVE: To discover the risk factor of postpartum depression and whether this is different from the induced factor of prepartum depression and to clarify what is the recovery factor from prepartum depression. METHODS: In the first test stage, 310 pregnant women were examined and with their postpartum follow-up survey, materials from 85 people in total were retrieved. In order to predict the postpartum depression and find out the recovery factor from prepartum depression, longitudinal study was carried out. For the statistical analysis hierarchical regression analysis and MANOVA were used. RESULTS: Postpartum depression (Beck Depression Inventory; BDI score of 16 or greater) was prevalent amongst 22.4% of pregnant women and prepartum depression was experienced by 10.5% in pregnant women. There were no significant on psychological variable factor of prepartum and postpartum depression. Only preatum depression redicted 33% for postaprtum depression. In the case of depressed during pregnancy but not depressed after pregnancy, recovery factor is related to increase in self-esteem, husband support and improvement in marital satisfaction. Postpartum depression showed twice higher depression ration than prepartum depression and it was serious in terms of degree of depression. CONCLUSION: Postpartum depression is related with lack of husband support that is recovery factor from prepartum depression. Also, unstable attachment is vulnerable to depression but even people with unstable attachment can be recovered from depression with better marriage relationship. Even people without unstable attachment if husband support is reduced then it is suggested that can be subject to vulnerability in depression.