Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

Mechanism of Xinguanzhongqizhenliao formula (XGZQZLF) in treatment of COVID-19 by network pharmacology / 中草药

Objective: To research the mechanism of Xinguanzhongqizhenliao formula (XGZQZLF) for COVID-19 treatment by network pharmacology. Methods: “Drug-active ingredient-target-disease” network was constructed by Cytoscape based on TCMSP and GeneCards databases. Mechanism of XGZQZLF in treatment of COVID-19 was implemented by GO and KEGG analysis. Results: A total of 80 active ingredients of XGZQZLF were screened out. They could regulate 208 targets of COVID-19, including AKT1, IL-6, MAPK3, VEGFA, CASP3 and so on, which exerted therapeutic effect on COVID-19 through regulating multiple signaling pathways such as TNF signaling pathway, IL-17 signaling pathway, Th17 cell differentiation, HIF-1 signaling pathway and so on. Conclusion: XGZQZLF plays an important role in the treatment of COVID-19 through multi-component, multi-target and multi-pathway, which can provide reference for the synergistic theory of TCM compound prescription.

Effect of Metabolic Reprogramming on Toxin Eliminating Therapeutics in Targeting Lung Cancer Stem Cells / 中国实验方剂学杂志

Objective::To observe the effect of realgar nanoparticles (a representative drug in toxin eliminating therapeutics) targeting hypoxia-inducible factors (HIF), which act as effector molecules on metabolic reprogramming of lung cancer stem cells, and to explore the effect mechanism of lung cancer stem cells and metabolic reprogramming in the process of lung cancer metastasis, so as to verify the effectiveness of toxin eliminating therapeutics in the prevention and treatment of lung cancer metastasis. Method::Lung cancer A549 cells were cultured in vitro, and lung cancer stem cells were then identified and selected. The stem cells were divided into blank control group, cisplatin group (5 mg·L-1), realgar nanoparticles low, medium and high dose groups (100, 200, 400 mg·L-1). After intervention, glucose oxidase method was used to detect the effect of realgar nanoparticles on the glucose metabolism of lung cancer stem cells, real-time polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression levels of hypoxia-inducible factors-1α (HIF-1α), C-myc and p53, while Western blot was used to detect the expression of related proteins HIF-1α, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mammalian target of rapamycin (mTOR), and enzyme linked immunosorbent assay (ELISA) was used to detect the glucose transporter 1 (GLUT1), pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase M (PKM), phosphofructokinase(PFK), pyruvate dehydrogenase (PDH) and lactic dehydrogenase (LDH) expression. Result::As compared with the blank control group, realgar nanoparticles can reduce the glucose consumption of lung cancer stem cells, and the glucose consumption was reduced with the increase of dose in a time-and dose-dependent manner (P<0.01). Realgar nanoparticles can inhibit the mRNA expression of HIF-1α, a key factor in metabolic reprogramming of lung cancer stem cells (P<0.05, P<0.01), down-regulated C-myc mRNA and up-regulated the p53 mRNA expression (P<0.05, P<0.01), down-regulated protein expressions of PI3K, Akt, mTOR(P<0.05, P<0.01), and inhibited the expression of related enzymes GLUT1, PDK1, PFK, PKM, PDH, and LDH levels (P<0.05, P<0.01). With the increase of dose, the regulation and control ability of realgar nanoparticles gradually increased. Conclusion::Toxin eliminating therapeutics can drive the metabolic reprogramming of lung cancer stem cells by targeting HIF effector molecule, and then inhibit the invasion and metastasis of lung cancer.

Tibial dyschondroplasia is closely related to suppression of expression of hypoxia-inducible factors 1α, 2α, and 3α in chickens

Tibial dyschondroplasia (TD) cases has not been reported in Tibetan chickens (TBCs), but it is commonly seen in commercial broilers characterized by lameness. The underlying mechanism remains unclear. Hypoxia-inducible factors (HIFs) are important regulators of cellular adaptation to hypoxic conditions. In this study, we investigated the role of HIF-1α,

The expression and significance of hypoxia-inducible factors-1α and pyruvate kinase type M2 in gastric carcinoma patients with Hp infection / 中华普通外科杂志

Objective To study the expression of hypoxia-inducible factors-1 α and pyruvate kinase type M2 in Hp infected gastric carcinoma and their clinical significances.Methods The expression levels of hypoxia-inducible factors-1α and pyruvate kinase type M2 in 85 cases of gastric carcinoma tissues were detected by using RT-PCR and immunohistochemical method.The Virulence level of CagA gene were detected in Hp infected gastric tissue.Results As the degree of tumor differentiation,invasion depth and clinical stage of tumor increased,the positive expression rate of HIF-1 α and PKM2 increased,and the expression level of the two was in positive correlation.The positive expression rate of HIF-1α (73％)was related to lymph node metastasis (x2 =4.204,P =0.041).The positive expression rate of PKM2 (85 ％)was related to tumor diameter.The positive expression rate of HIF-1α and PKM2 (75％ and 85％,respectively)significantly increased in Hp infected gastric carcinoma tissue (x2 =6.486,P =0.010;x2 =7.341,P =0.009,respectively) and related to CagA + type.Conclusion HIF-1α and PKM2 may be closely correlated to development and metastasis of gastric carcinoma with Hp infection.

Hypoxia inducible factor in postmenopausal osteoporosis: Recent progress / 第二军医大学学报

Hypoxia inducible factor (HIF) plays a key role in the cellular response to hypoxia, and is involved in a variety of pathological and physiological processes such as glucose metabolism, vascular remodeling and erythropoiesis. Growing research suggests that HIF has an important effect on osteogenesis, bone resorption and angiogenesis; while the deterioration of bone vascular structures and functions is closely related to the development and progression of postmenopausal osteoporosis. In this review, we summarized the relationship between HIF and postmenopausal osteoporosis based on the roles of HIF in osteogenesis, bone resorption and angiogenesis, hoping to evaluate its therapeutic prospect on postmenopausal osteoporosis.

Mechanisms of tumor invasion metastasis caused by long-term anti-angiogenic therapy / 中国肿瘤临床

Hypoxia results from long-term anti-angiogenic therapy and can stimulate hypoxia-inducible factors (HIFs). HIF-induced hy-poxia signaling is involved in various steps in tumor invasive-metastatic cascade. On the one hand, HIFs regulate epithelial-mesenchy-mal transition. On the other hand, the characteristics of pericytes around vessels and the links among endothelial cells can change;thus, tumor cells can more easily intravasate into blood vessels, survive in peripheral blood, and then reach specific organs, ultimately resulting in metastasis. This review discusses the emerging mechanisms of long-term anti-angiogenic therapy and the occurrence of metastasis.

Role of hypoxia-inducible factors in ischemia/reperfusion injury: An update / 第二军医大学学报

Ischemia/reperfusion injury (IRI) is a common clinical pathophysiological process characterized by ischemia and hypoxia. Hypoxia-inducible factors (HIFs) are a group of transcription factors vital to cell responding and adapting to hypoxia environment. Hypoxia can activate HIF, thus enhancing the tolerance of cells to hypoxia. The role of HIF in IRI has become a research focus of many scientists. Elucidating the related mechanism can not only help to reduce IRI, but also lay a basis for further studying the role of HIF in other pathophysiological processes. In this paper, we reviewed the structure, function of HIF and the specific role and related mechanisms of HIF in IRI.

Role of Hypoxia Inducible Factors in Obesity Pathogenesis.

The prevalence of obesity, metabolic syndrome and diabetes has been increasing rapidly worldwide. These are a group of metabolic disorders characterized by a chronic hyperglycaemic condition resulting from defects in insulin secretion, insulin action or both. The control of body weight and blood glucose concentrations depends on the exquisite coordination of the function of several cells, organs and tissues. Underlying mechanisms of obesity and insulin resistance remain uncertain. Adipose tissue is composed of heterogeneous cell types. Immune cells within adipose tissue also likely contribute to systemic metabolic processes. Increased production of local and systemic adipokines and cytokines, polarization of macrophages, T helper subtype changes could contribute to pathologies linking obesity to diabetes, both by decreasing insulin sensitivity, by compromising β-cell function and disturbing adipose tissue metabolism and distribution. Tissue oxygen (O2) levels, hypoxia inducible factor (s) (HIFs) secretion differences regulate the plasticity of macrophages and the polarization of macrophages controls functionally divergent processes in cells. A hypoxic and inflammatory phenotype has been reported in adipose tissue during obesity. Therefore, the present review focuses HIFs-mediated effects of hypoxia in adipocyte inflammation and macrophage polarization associated with obesity pathogenesis.

The effect of hypoxia inducible factor on articular chondrocytes / 医学研究生学报

Because articular chondrocytes have a long-term exposure to different degrees of hypoxic environment , hypoxia induc-ible factor is considered to be one of the most important regulation factors to adapt to low oxygen levels .It plays a significant role in the metabolism, repair, and degeneration of cartilage chondrocyts .This article reviews the comprehensive effects of hypoxia inducible fac-tor in cartilage chondrocyts .

Pathogenesis of anemia in chronic aristolochic acid nephropathy rats / 中华肾脏病杂志

Objective To study the pathogenesis of anemia in chronic aristolochic acid nephmpathy(CAAN) rats. Methods The hemoglobin(Hb)values of sixty-two male SD rats were assayed to determine its normal range.Among them,24 rats with normal Hb value were randomly divided into 2 groups:model group (MG)in which rats received the extract of Aristololochia manshuriensis Kom (AmK) by gavage,and control group (CG) received tap water only by gavage.Body weisht(BW),Hb,24 h urinary protein excretion(UP)and creatinine clearance (Ccr)of 6 rats in each group were measured before administration and at the end of the 8th week, respeetively.then these rats were sacrificed.The relative area of renal interstitial fibrosis was measured by microscopy.The mRNA expression of erythropoietin (EPO)in kidney tissue Was determined by real-time RT-PCR;protein expression of type I collagen(Coll),aminopeptidase P (APP),hypoxia indHeible factor let and 2α(HIF-1α and HIF-2α)in kidney tissue Was examined by immunohistochemistry staining. Results Hb values of normal rats presented normal distribution. The normal Hb was (155.9±16.5) g/L. Rat anemia was diagnosed when Hb was below 123.6 g/L. There was no difference in all the examination results between CG and MG before administration (P>0.05). Compared with CG, the Hb and Cer in MG were significantly decreased [(121.66±15.68) g/L vs (169.00±12.89) g/L, (0.63±0.13) ml/min vs (1.27±0.18) ml/min, P< 0.01], and the UP in MG was significantly increased at the end of the 8th week [(27.04±9.40) mg/d vs (6.11±0.84) mg/d, P<0.01]; the relative areas of fibrosis and Col l in renal interstitium of MG were significantly enlarged [(12.89±2.33)% vs (0.55±0.10)%, (13.92±2.92)% vs (1.32±0.84)%, P<0.01]; the protein expression of APP and the mRNA expression of EPO in the kidney tissue of MG were significantly down-regulated [(0.55±0.23)% vs (3.77±1.06)%, 0.005±0.001 vs 0.032±0.013, P<0.01]; the protein expression of HIF-lα and HIF-2α in the kidney tissue of MG was significantly up-regulated (2.55±0.16 vs 1.12±0.46, 2.33±0.33 vs 1.15±0.27, P<0.01), at the end of the 8th week. Conclusions The pathogenesis of anemia in CAAN may be due to the decreased production of EPO caused by the destruction of peritubular capillary. The compensatory up-regulation of HIF-lα and HIF-2α expression can not prevent the anemia development.

Effects of curcumin on expression of HIF-1? in human hepatocellular carcinoma BEL-7402 cells / 中国药理学通报

Aim To investigate the effects of curcumin on expression of HIF-1? in human hepatocellular carcinoma BEL-7402 cells.Methods Different concentrations of 0,2.5,5,10,15 and 20 ?mol?L~(-1) curcumin-treated human hepatocellular carcinoma BEL-7402 cells were cultured under hypoxia for 6 hours.The cell proliferation and cell viability were assayed by WST-8 and the trypan blue dye exclusion method.The expression level of HIF-1? in human hepatocellular carcinoma BEL-7402 cells was checked by RT-PCR and Western Blot;0,10 ?mol?L~(-1) curcumin,10 ?mol?L~(-1) MG-132,and 10 ?mol?L~(-1) curcumin +10 ?mol? L~(-1) MG-132-treated human hepatocellular carcinoma BEL-7402 cells were cultured under hypoxia for 6 hours.The protein expression level of HIF-1? protein in human hepatocellular carcinoma BEL-7402 cells was checked by Western Blot.Results ① No significant difference was found in the cell proliferation rate and cell viability between control and different concentrations of curcumin.② Curcumin can down-regulate the expression of HIF-1? protein with the increasing concentrations.③ the effect of curcumin on expression of HIF-1?mRNA had no significant difference between control and different concentrations.④ MG-132 can reverse the curcumin-mediated decrease of HIF-1? protein.Conclusion Inhibitory effect of curcumin on expression of HIF-1? in human hepatocellular carcinoma BEL-7402 cells was acted through posttranscriptional mechanisms and the proteasome pathway.

Hypoxia-inducible factors and oxygen sensors / 第二军医大学学报

The erythropoietin(EPO) system was used to study the molecular mechanisms associated with the induction of hypoxia responsive genes and from these investigations the hypoxia-inducible factors (HIFs) was identified as a key transcriptional hypoxic regulator of EPO. Subsequent research has now found that a large number of other hypoxia-inducible genes are also induced by HIF under hypoxic conditions. The HIF transcriptional complex is a heterodimer consisting of an alpha subunit and a beta subunit. Recently, two oxygen sensor, oxygen-dependent prolyl and asparaginyl hydroxylation were found,and it is the first time that the oxygen sensor had been described for higher organisms. This review focused on the structure and functions of HIF and the regulation of HIF proteins by hypoxia and oxygen sensors.