Qing Zao Fang (QZF) Alleviates the Inflammatory Microenvironment of the Submandibular Gland in Sjögren's Syndrome Based on the PI3K/Akt/HIF-1α/VEGF Signaling Pathway.

Sjögren's syndrome (SS) which could lead to a disorder of our immune system is a chronic autoimmune disease characterized by invading exocrine glands such as salivary glands and lacrimal glands and other exocrine glands. Its common symptom is dry mouth and dry eyes, often accompanied by a large number of lymphocyte infiltrations and can involve other organs to cause complex clinical manifestations. In this study, we aimed at investigating the effect of QZF in SS, identifying the molecular mechanism in modulating autoimmune response, and determining the important roles of these factors' function as a modulator in the pathogenesis of SS. The NOD mice were utilized to establish the rats' model of Sjögren's syndrome. After 10 weeks' hydroxychloroquine and QZF in different dose interference, submandibular gland tissue was collected. The therapeutic effect of QZF on SS rats was identified, and the results suggest the comparable potential to hydroxychloroquine. In submandibular gland tissue, interleukin- (IL-) 17 was significantly lower in high-dose QZF than that in SS rats and the focal lymphocytes were highly attenuated. Moreover, we found that PI3K/Akt signals were activated and the downstream HIF-1α/VEGF signals were enhanced in SS rats whose protein expression could be inhibited by QZF treatment. In addition, QZF could modulate autophagy in submandibular gland tissue and then inhibit the inflammation response and therefore facilitate the tissue repair.

Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/ß-catenin transcriptional cofactor BCL9.

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/ß-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/ß-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and ß-catenin expression and BCL9-ß-catenin co-localization. In addition, BCL9 formed a complex with ß-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/ß-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/ß-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

HIF­1α in cerebral ischemia (Review).

Cerebral ischemic injury may lead to a series of serious brain diseases, death or different degrees of disability. Hypoxia­inducible factor­1α (HIF­1α) is an oxygen­sensitive transcription factor, which mediates the adaptive metabolic response to hypoxia and serves a key role in cerebral ischemia. HIF­1α is the main molecule that responds to hypoxia. HIF­1α serves an important role in the development of cerebral ischemia by participating in numerous processes, including metabolism, proliferation and angiogenesis. The present review focuses on the endogenous protective mechanism of cerebral ischemia and elaborates on the role of HIF­1α in cerebral ischemia. In addition, it focuses on cerebral ischemia interventions that act on the HIF­1α target, including biological factors, non­coding RNA, hypoxic­ischemic preconditioning and drugs, and expands upon the measures to strengthen the endogenous compensatory response to support HIF­1α as a therapeutic target, thus providing novel suggestions for the treatment of cerebral ischemia.

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

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

The impact of hypoxia on immune state in cancer.

Hypoxia is a known feature of solid tumors and a critical promoter of tumor hallmarks. Hypoxia influences tumor immunity in a way favoring immune evasion and resistance. Extreme hypoxia and aberrant hypoxia-inducible factor-1 (HIF-1) activity in tumor microenvironment (TME) is a drawback for effective immunotherapy. Infiltration and activity of CD8+ T cells is reduced in such condition, whereas regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) show high activities. Highly hypoxic TME also impairs maturation and activity of dendritic cell (DCs) and natural killer (NK) cells. In addition, the hypoxic TME positively is linked positively with metabolic changes in cells of immune system. These alterations are indicative of a need for hypoxia modulation as a complementary targeting strategy to go with immune checkpoint inhibitor (ICI) therapy.

Apigenin increases radiosensitivity of glioma stem cells by attenuating HIF-1α-mediated glycolysis.

Apigenin, a natural flavonoid compound present in a variety of edible plants and health foods, has an anti-tumor effect and inhibits hypoxia inducible factor-lα (HIF-1α) expression in hypertrophic cardiac tissues. However, whether or not apigenin has a radiosensitization effect on glioma stem cells (GSCs) is unknown. Our present study aimed to investigate the effect of apigenin and its possible mechanisms. The human GSCs SU3 and its radioresistance line SU3-5R were treated with apigenin, radiation, or their combination, and the cell proliferation, migration, colony formation, and intracellular lactic acid and glycolytic related protein expressions were determined. Additionally, a cell model with hypoxia-induced HIF-1α expression was used and treated with apigenin. The current results displayed that the combination of apigenin and radiation could synergically reduce the viability, colony formation, and migration of the both GSCs. Moreover, this combination could also decrease the radiation-induced increments of glycolytic production lactic acid in the both GSCs and related protein expressions, including HIF-1α, glucose transporter (GLUT)-1/3, nuclear factor kappa B (NF-κB) p65, and pyruvate kinase isozyme type M2 (PKM2). Further study confirmed that after treatment of hypoxia-cultured SU3 or SU3-5R cells with apigenin, the expression levels of HIF-1α, GLUT-1/3, NF-κB p65, and PKM2 proteins were reduced. These results demonstrated that apigenin could increase the radiosensitivity of GSCs and its radiosensitization mechanisms were attributable to the attenuation of glycolysis, which might result from the inhibition of HIF-1α expression and subsequent reductions of GLUT-1/3, NF-κB, and PKM2 expressions.

Regulation of the Hypoxia-Inducible Factor (HIF) by Pro-Inflammatory Cytokines.

Hypoxia and inflammation are frequently co-incidental features of the tissue microenvironment in a wide range of inflammatory diseases. While the impact of hypoxia on inflammatory pathways in immune cells has been well characterized, less is known about how inflammatory stimuli such as cytokines impact upon the canonical hypoxia-inducible factor (HIF) pathway, the master regulator of the cellular response to hypoxia. In this review, we discuss what is known about the impact of two major pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), on the regulation of HIF-dependent signaling at sites of inflammation. We report extensive evidence for these cytokines directly impacting upon HIF signaling through the regulation of HIF at transcriptional and post-translational levels. We conclude that multi-level crosstalk between inflammatory and hypoxic signaling pathways plays an important role in shaping the nature and degree of inflammation occurring at hypoxic sites.

Astragalus polysaccharide ameliorates steroid-induced osteonecrosis of femoral head through miR-206/HIF-1α/BNIP3 axis.

Declining autophagy and rising apoptosis are the main factors driving the development of steroid-induced osteonecrosis of the femoral head (SONFH). Here, we showed that astragalus polysaccharide (APS) improved femoral head necrosis via regulation of cell autophagy and apoptosis through microRNA (miR)-206/hypoxia inducible factor-1 (HIF-1α)/BCL2 interacting protein 3 (BNIP3) axis. The expression of miR-206, HIF-1α, and BNIP3 in SONFH specimens and cell model were measured using qPCR. SONFH cell model was treated with APS. Cell autophagy was evaluated using LC3-immunofluorescence assays. Flow cytometry was conducted to assess cell apoptosis. Apoptosis-related proteins and autophagy-related proteins were determined using western blot. Besides, dual-luciferase reporter assay was employed to investigate the relationship between miR-206 and HIF-1α. Here we showed that miR-206 expression was upregulated in SONFH tissues and cell model. APS promoted autophagy and inhibited apoptosis in SONFH cell model via downregulating miR-206. What is more, HIF-1α was the target of miR-206. Knockdown of HIF-1α reversed the recovery effect of miR-206 inhibitor on SONFH cell model. Furthermore, BNIP3 was the target of HIF-1α. HIF-1α overexpression promoted autophagy and inhibited apoptosis, and knockdown of BNIP3 abolished the recovery effect of HIF-1α overexpression in SONFH cell model. These results provided evidence that APS reduced miR-206 expression, and the downregulated miR-206 increased BNIP3 expression by targeting HIF-1α to promote autophagy and inhibit bone cell apoptosis. Our research proved that APS effectively improved SONFH by regulating cell autophagy and apoptosis.

Macrophage Motility in Wound Healing Is Regulated by HIF-1α via S1P Signaling.

Accumulating evidence indicates that the molecular pathways mediating wound healing induce cell migration and localization of cytokines to sites of injury. Macrophages are immune cells that sense and actively respond to disturbances in tissue homeostasis by initiating, and subsequently resolving, inflammation. Hypoxic conditions generated at a wound site also strongly recruit macrophages and affect their function. Hypoxia inducible factor (HIF)-1α is a transcription factor that contributes to both glycolysis and the induction of inflammatory genes, while also being critical for macrophage activation. For the latter, HIF-1α regulates sphingosine 1-phosphate (S1P) to affect the migration, activation, differentiation, and polarization of macrophages. Recently, S1P and HIF-1α have received much attention, and various studies have been performed to investigate their roles in initiating and resolving inflammation via macrophages. It is hypothesized that the HIF-1α/S1P/S1P receptor axis is an important determinant of macrophage function under inflammatory conditions and during disease pathogenesis. Therefore, in this review, biological regulation of monocytes/macrophages in response to circulating HIF-1α is summarized, including signaling by S1P/S1P receptors, which have essential roles in wound healing.

Elevated glucose increases genomic instability by inhibiting nucleotide excision repair.

We investigated potential mechanisms by which elevated glucose may promote genomic instability. Gene expression studies, protein measurements, mass spectroscopic analyses, and functional assays revealed that elevated glucose inhibited the nucleotide excision repair (NER) pathway, promoted DNA strand breaks, and increased levels of the DNA glycation adduct N 2 -(1-carboxyethyl)-2'-deoxyguanosine (CEdG). Glycation stress in NER-competent cells yielded single-strand breaks accompanied by ATR activation, γH2AX induction, and enhanced non-homologous end-joining and homology-directed repair. In NER-deficient cells, glycation stress activated ATM/ATR/H2AX, consistent with double-strand break formation. Elevated glucose inhibited DNA repair by attenuating hypoxia-inducible factor-1α-mediated transcription of NER genes via enhanced 2-ketoglutarate-dependent prolyl hydroxylase (PHD) activity. PHD inhibition enhanced transcription of NER genes and facilitated CEdG repair. These results are consistent with a role for hyperglycemia in promoting genomic instability as a potential mechanism for increasing cancer risk in metabolic disease. Because of the pleiotropic functions of many NER genes beyond DNA repair, these results may have broader implications for cellular pathophysiology.

Transcriptional and Microenvironmental Landscape of Macrophage Transition in Cancer: A Boolean Analysis.

The balance between pro- and anti-inflammatory immune system responses is crucial to face and counteract complex diseases such as cancer. Macrophages are an essential population that contributes to this balance in collusion with the local tumor microenvironment. Cancer cells evade the attack of macrophages by liberating cytokines and enhancing the transition to the M2 phenotype with pro-tumoral functions. Despite this pernicious effect on immune systems, the M1 phenotype still exists in the environment and can eliminate tumor cells by liberating cytokines that recruit and activate the cytotoxic actions of TH1 effector cells. Here, we used a Boolean modeling approach to understand how the tumor microenvironment shapes macrophage behavior to enhance pro-tumoral functions. Our network reconstruction integrates experimental data and public information that let us study the polarization from monocytes to M1, M2a, M2b, M2c, and M2d subphenotypes. To analyze the dynamics of our model, we modeled macrophage polarization in different conditions and perturbations. Notably, our study identified new hybrid cell populations, undescribed before. Based on the in vivo macrophage behavior, we explained the hybrid macrophages' role in the tumor microenvironment. The in silico model allowed us to postulate transcriptional factors that maintain the balance between macrophages with anti- and pro-tumoral functions. In our pursuit to maintain the balance of macrophage phenotypes to eliminate malignant tumor cells, we emulated a theoretical genetically modified macrophage by modifying the activation of NFκB and a loss of function in HIF1-α and discussed their phenotype implications. Overall, our theoretical approach is as a guide to design new experiments for unraveling the principles of the dual host-protective or -harmful antagonistic roles of transitional macrophages in tumor immunoediting and cancer cell fate decisions.

Up-down regulation of HIF-1α in cancer progression.

Hypoxia induicible factor-1 alpha (HIF-1α) is a key transcription factor in cancer progression and target therapy in cancer. HIF-1α acts differently depending on presence or absence of Oxygen. In an oxygen-immersed environment, HIF-1α completely deactivated and destroyed by the ubiquitin proteasome pathway (UPP). In contrast, in the oxygen-free environment, it escapes destruction and enters to the nucleus of cells then upregulates many genes involved in cancer progression. Overexpressed HIF-1α and downstream genes support cancer progression through various mechanisms including angiogenesis, proliferation and survival of cells, metabolism reprogramming, invasion and metastasis, cancer stem cell maintenance, induction of genetic instability, and treatment resistance. HIF-1α can be provoked by signaling pathways unrelated to hypoxia during cancer progression. Therefore, cancer development and progression can be modulated by targeting HIF-1α and its downstream signaling molecules. In this regard, HIF-1α inhibitors which are categorized into the agents that regulate HIF-1α in gene, mRNA and protein levels used as an efficient way in cancer treatment. Also, HIF-1α expression can be negatively affected by the agents suppressing the activation of mTOR, PI3k/Akt and MAPK pathways.

HIF-1 α may play a role in late pregnancy hypoxia-induced autism-like behaviors in offspring rats.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that can be caused by various factors. The present study aimed to determine whether prenatal hypoxia can lead to ASD and the role of hypoxia-inducible factor-1α (HIF-1α) in this process. We constructed a prenatal hypoxia model of pregnant rats by piping nitrogen and oxygen mixed gas, with an oxygen concentration of 10 ± 0.5 %, into the self-made hypoxia chamber. Rats were subjected to different extents of hypoxia treatments at different points during pregnancy. The results showed that hypoxia for 6 h on the 17th gestation day is most likely to lead to autistic behavior in offspring rats, including social deficits, repetitive behaviors, and impaired learning and memory. The mRNA expression level of TNF-α also increased in hypoxia-induced autism group and valproic acid (VPA) group. Western blotting analysis showed increased levels of hypoxia inducible factor 1 alpha (HIF-1α) and decreased levels of phosphatase and tensin homolog (PTEN) in the hypoxic-induced autism group. Meanwhile, N-methyl d-aspartate receptor subtype 2 (NR2A) and glutamate ionotropic receptor AMPA type subunit 2 (GluR2) were upregulated in the hypoxic-induced autism group. HIF-1α might play a role in hypoxia-caused autism-like behavior and its regulatory effect is likely to be achieved by regulating synaptic plasticity.

The Role of HIF1α-PFKFB3 Pathway in Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness for adults in developed countries. Both microvasculopathy and neurodegeneration are implicated in mechanisms of DR development, with neuronal impairment preceding microvascular abnormalities, which is often underappreciated in the clinic. Most current therapeutic strategies, including anti-vascular endothelial growth factor (anti-VEGF)-antibodies, aim at treating the advanced stages (diabetic macular edema and proliferative diabetic retinopathy) and fail to target the neuronal deterioration. Hence, new therapeutic approach(es) intended to address both vascular and neuronal impairment are urgently needed. The hypoxia-inducible factor 1α (HIF1α)-6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) pathway is critically implicated in the islet pathology of diabetes. Recent evidence highlighted the pathway relevance for pathologic angiogenesis and neurodegeneration, two key aspects in DR. PFKFB3 is key to the sprouting angiogenesis, along with VEGF, by determining the endothelial tip-cell competition. Also, PFKFB3-driven glycolysis compromises the antioxidative capacity of neurons leading to neuronal loss and reactive gliosis. Therefore, the HIF1α-PFKFB3 signaling pathway is unique as being a pervasive pathological component across multiple cell types in the retina in the early as well as late stages of DR. A metabolic point-of-intervention based on HIF1α-PFKFB3 targeting thus deserves further consideration in DR.

Adenylate Kinase 4 Promotes Inflammatory Gene Expression via Hif1α and AMPK in Macrophages.

Macrophages comprise the front line of defense against various pathogens. Classically activated macrophages (M1), induced by IFN-γ and LPS, highly express inflammatory cytokines and contribute to inflammatory processes. By contrast, alternatively activated macrophages (M2) are induced by IL-4 and IL-13, produce IL-10, and display anti-inflammatory activity. Adenylate kinase 4 (Ak4), an enzyme that transfers phosphate group among ATP/GTP, AMP, and ADP, is a key modulator of ATP and maintains the homeostasis of cellular nucleotides which is essential for cell functions. However, its role in regulating the function of macrophages is not fully understood. Here we report that Ak4 expression is induced in M1 but not M2 macrophages. Suppressing the expression of Ak4 in M1 macrophages with shRNA or siRNA enhances ATP production and decreases ROS production, bactericidal ability and glycolysis in M1 cells. Moreover, Ak4 regulates the expression of inflammation genes, including Il1b, Il6, Tnfa, Nos2, Nox2, and Hif1a, in M1 macrophages. We further demonstrate that Ak4 inhibits the activation of AMPK and forms a positive feedback loop with Hif1α to promote the expression of inflammation-related genes in M1 cells. Furthermore, RNA-seq analysis demonstrates that Ak4 also regulates other biological processes in addition to the expression of inflammation-related genes in M1 cells. Interestingly, Ak4 does not regulate M1/M2 polarization. Taken together, our study uncovers a potential mechanism linking energy consumption and inflammation in macrophages.

mTOR inhibition as a possible pharmacological target in the management of systemic inflammatory response and associated neuroinflammation by lipopolysaccharide challenge in rats.

Neuroinflammation plays a critical role during sepsis triggered by microglial activation. Mammalian target of rapamycin (mTOR) has gained attraction in neuroinflammation, however, the mechanism remains unclear. Our goal was to assess the effects of mTOR inhibition by rapamycin on inflammation, microglial activation, oxidative stress, and apoptosis associated with the changes in the inhibitor-κB (IκB)-α/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α) pathway activity following a systemic challenge with lipopolysaccharide (LPS). Rats received saline (10 mL/kg), LPS (10 mg/kg), and (or) rapamycin (1 mg/kg) intraperitoneally. Inhibition of mTOR by rapamycin blocked phosphorylated form of ribosomal protein S6, NF-κB p65 activity by increasing degradation of IκB-α in parallel with HIF-1α expression increased by LPS in the kidney, heart, lung, and brain tissues. Rapamycin attenuated the increment in the expression of tumor necrosis factor-α and interleukin-1ß, the inducible nitric oxide synthase, gp91phox, and p47phox in addition to nitrite levels elicited by LPS in tissues or sera. Concomitantly, rapamycin treatment reduced microglial activation, brain expression of caspase-3, and Bcl-2-associated X protein while it increased expression of B cell lymphoma 2 induced by LPS. Overall, this study supports the hypothesis that mTOR contributes to the detrimental effect of LPS-induced systemic inflammatory response associated with neuroinflammation via IκB-α/NF-κB/HIF-1α signaling pathway.

Molecular biological investigation of temozolomide and KC7F2 combination in U87MG glioma cell line.

Glioblastom Multiforme (GBM) is the most invasive and malignant member of the IV grade of the subclass Astrocytoma according to the last assessment of the 2016 WHO report. Due to the resistance to treatment and weak response, as well as the topographical structure of the blood brain barrier, the treatment is also difficult due to the severe clinical manifestation, and new treatment methods and new therapeutic agents are needed. Temozolomide (TMZ) is widely used in the treatment of glioblastoma and is considered as the primary treatment modality. TMZ, a member of the class of cognitive agents, is currently considered the most effective drug because it can easily pass through the blood brain barrier. Glucose metabolism is a complex energy producing machine that, a glucose molecule produces 38 molecules of ATP after full glycolytic catabolism. According to Otto Warburg's numerous studies cancer cells perform the first glycolytic step without entering the mitochondrial step. These cells produce lactic acid and make the micro-media more acidic even in aerobic conditions. This phenomenon is attributed to the Warburg hypothesis and either as aerobic glycolysis. Although glycolysis enzymes are the primary actors of this phenotypic expression, some genetic and epigenetic factors are no exception. We experimentally used KC7F2 active ingredient to target cancer metabolism. In our study, we evaluated cancer metabolism in combination with the effect of TMZ chemotherapeutic agent, examining the effect of two different agents separately and in combination to observe the effects of cancer cell proliferation, survival, apoptosis and expression of metabolism genes on expression. We observed that the combined effect of reduced the effective dose of the TMZ alkylating agent and that the effect was increased and the effect of the combined teraphy is assessed from a metabolic point of view and that it suppresses aerobic glycolysis.

Naringin Targets NFKB1 to Alleviate Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury in PC12 Cells Via Modulating HIF-1α/AKT/mTOR-Signaling Pathway.

This study was designed to investigate the effect of naringin in oxygen-glucose deprivation/reoxygenation (OGD/R) model and its mechanism. The target gene of naringin and the enriched pathways of the gene were searched and identified using bioinformatics analysis. Then OGD/R model was built using PC12 cells, after which the cells were treated with different concentrations of naringin. Subsequently, cell proliferation and apoptosis were evaluated by cell counting kit-8 (CCK-8) and flow cytometry assays, respectively. Meanwhile, the expression of NFKB1 in PC12 cells underwent OGD/R-induced injury was detected by qRT-PCR, while apoptosis-related and pathway-related proteins were checked by Western blot. DCF-DA kit was utilized to measure the level of ROS. Our results revealed that NFKB1, which was upregulated in MACO rats and OGD/R-treated PC12 cells, was a target gene of naringin. Naringin could alleviate OGD/R-induced injury via promoting the proliferation, and repressing the apoptosis of PC12 cells through regulating the expression of NFKB1 and apoptosis-associated proteins and ROS level. Besides, the depletion of NFKB1 was positive to cell proliferation but negative to cell apoptosis. Moreover, the depletion of NFKB1 enhanced the influences of naringin on cell proliferation and apoptosis as well as the expression of apoptosis-related proteins and ROS level. Western blotting indicated that both naringin treatment and depletion of NFKB1 could increase the expression of HIF-1α, p-AKT, and p-mTOR compared with OGD/R group. What's more, treatment by naringin and si-NFKB1 together could significantly increase these effects. Nevertheless, the expression of AKT and mTOR among each group was almost not changed. In conclusion, naringin could prevent the OGD/R-induced injury in PC12 cells in vitro by targeting NFKB1 and regulating HIF-1α/AKT/mTOR-signaling pathway, which might provide novel ideas for the therapy of cerebral ischemia-reperfusion (I/R) injury.

Hepatocyte growth factor regulates HIF-1α-induced nucleus pulposus cell proliferation through MAPK-, PI3K/Akt-, and STAT3-mediated signaling.

Intervertebral discs are important for maintaining mobility and offer support to the body trunk. If these discs lose their biomechanical features, lower back pain can occur. We previously reported that hepatocyte growth factor (HGF) promotes cell proliferation and suppresses apoptosis, inflammation, and matrix degradation in nucleus pulposus (NP) cells. In the present study, we investigated the molecular mechanisms of how HGF promotes the proliferation of NP cells in hypoxic conditions. Hypoxic stimulation promoted modest cell proliferation, which was further upregulated by HGF. Expression of hypoxia-inducible factor (HIF-1α) protein, which contributes to the maintenance of homeostasis in NP cells, was also upregulated in hypoxia-treated cell groups; HGF further increased HIF-1α expression in NP cells. Additionally, knockdown of HIF-1α expression significantly reduced the proliferation of NP cells. An MAPK inhibitor inhibited the expression of HIF-1α and pERK, as well as cell proliferation in a dose-dependent manner. Similarly, inhibiting the PI3K/Akt and STAT3 pathways also decreased the expression of HIF-1α and cell proliferation. These results show that under hypoxic conditions, HGF promotes NP cell proliferation via HIF-1α-, MAPK-, PI3K/Akt-, and STAT3-mediated signaling which is involved in this pathway. The control of these signaling pathways may be a target for potential therapeutic strategies for the treatment of disc degeneration in hypoxic conditions.

HIF-1α inhibits mitochondria-mediated apoptosis and improves the survival of human adipose-derived stem cells in ischemic microenvironments.

BACKGROUND: Human adipose mesenchymal stem cells (hADSCs) show poor survival after transplantation, limiting their clinical application. Tissue regeneration resulting from stem cell treatment may be caused by attenuation of hypoxia-inducible factor-1α (HIF-1α). In this study, we constructed hADSCs stably expressing HIF-1α and investigated the potential effects of HIF-1α expression in the ischemic microenvironment on mitochondrial apoptosis and survival of hADSCs, and studied the mechanisms involved. METHOD: Apoptosis was induced by an ischemic microenvironment in vitro. ADSCs with stable HIF-1α expression were established. Cell survival and apoptosis were observed by CCK-8 assay, western blotting, flow cytometry, and fluorescence staining. ADSCs were subcutaneously transplanted into nude mice in the location where a hypoxia ischemic microenvironment was simulated in vivo. After 1, 3, and 7 d, mitochondrial apoptotic proteins were evaluated by immunohistochemistry and immunofluorescence staining. RESULTS: Exogenous HIF-1α downregulated mitochondrial reactive oxygen species, cytochrome c, caspase-9, and caspase-3, but inhibited mitochondrial membrane potential depolarization and increased the Bcl-2/bax ratio. HIF-1α prevented apoptosis and promoted vascular endothelial growth factor (VEGF) secretion as demonstrated by enzyme-linked immunosorbent assay (ELISA), terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and flow cytometry analysis. HIF-1α enhanced the survival of transplanted ADSCs in nude mice. CONCLUSION: We have shown that through inhibition of the mitochondria-mediated apoptotic pathway and promotion of VEGF secretion in hADSCs in an ischemic microenvironment, HIF-1α may potentially be applied in clinical therapy and as an alternative strategy for improving hADSC therapy.