HIF-1α Protein Expression Research Articles

Hypoxia and collagen deposition in the kidneys infected with Acanthamoeba sp.

Acanthamoeba spp. are facultative, opportunistic pathogens that are found in diverse environments. In the hosts, they lead to multi-organ disease. Recent studies reported that they may induce changes in the kidneys of hosts. The aim of the study was to determine the influence of Acanthamoeba sp. on hypoxia and collagen deposition in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba sp. The results strongly suggest that Acanthamoeba sp. induces hypoxia in mice with normal and reduced immune response by increasing gene and/or protein expression of HIF1α as well as HIF2α. Additionally, the activation of these factors is probably induced via NOX2/ROS. Hypoxia promotes vessel formation, and we found that angiogenesis occurs in the kidneys of mice infected with the parasite regardless of their immunological status. The proangiogenic factors released in hypoxic conditions cause modulation and inflammation in the kidney cells, which in turn leads to collagen deposition via TGF-β. This work reveals mechanisms occurring in the hosts infected with Acanthamoeba sp., highlights as well as supports the relevance of pathophysiology in the kidneys in hosts with systematic acanthamoebiasis.

Eleutheroside B alleviates oxidative stress and neuroinflammation by inhibiting the JAK2/STAT3 signaling pathway in a rat high altitude cerebral edema model

BackgroundHigh altitude cerebral edema (HACE) is a condition where the central nervous system experiences severe impairment as a result of sudden oxygen deprivation at high elevations. At present, effective measures for preventing and treating this condition are still lacking. Eleutheroside B (EB), the primary natural active compound found in the Eleutheroside senticosus, has demonstrated various biological functions. It has also shown significant potential in addressing acute mountain sickness and various neurological disorders. However, additional investigation is required to explore the potential protective effects and its underlying mechanisms of EB on HACE.MethodsThe male rats received pre-treatment with either vehicle, EB 100 mg/kg or 50 mg/kg, Dexamethasone 4 mg/kg, or coumermycin A1 100 μg/kg. To simulate the hypobaric hypoxia environment at a plateau of 6,000 m, a hypobaric hypoxia chamber was utilized. The therapeutic effects of EB were assessed through measurements of brain water content, histopathological observation, and evaluation of oxidative stress and inflammatory factors using immunofluorescence and ELISA. Furthermore, molecular docking, molecular dynamics simulation and Western blot were employed to clarify its molecular mechanism. Through these analyses, the underlying mechanism by which EB on HACE was identified.ResultsPre-treatment with EB demonstrated a significant protective effect against HACE by effectively reducing brain water content, down-regulating HIF-1α and AQP4 protein expression induced by hypoxia and reversing pathological changes in brain tissue and neuron damage. Compared to the group treated with HACE alone, the group pre-treated with EB showed a significant reduction in levels of ROS and MDA, as well as an increase in GSH. In addition, pre-treatment with EB led to a significant decrease in the levels of IL-1β, IL-6, and TNF-α. Molecular docking and dynamics simulations indicated that EB has a strong binding affinity to the JAK2/STAT3 signaling pathway. Western blot further confirmed that EB significantly downregulated the expression of JAK2/STAT3 related proteins in the brain tissue of HACE rats. Additionally, coumermycin A1, an agonist of the JAK2, reversed the anti-oxidative stress and neuroinflammation against HACE of EB.ConclusionEB exerts its antioxidant stress and anti-neuroinflammatory effects by inhibiting the JAK2/STAT3 signaling pathway in a rat HACE model.

The impact of X-rays on cardiac hydrometabolism and the regulatory role of AS-IV

BackgroundRadiation-induced cardiac injury has emerged as a significant pathological entity, with many studies focusing on the fibrotic changes in myocardial tissue. However, these do not offer solutions for the clinical prevention and treatment of radiation-induced heart disease. Regulating hydrometabolism presents a potential therapeutic target for the management of cardiovascular diseases. This research seeks to explore the impacts of irradiation on cardiac hydrometabolism and its regulatory mechanisms. MethodsThe impact of X-ray radiation on cardiac and cardiomyocyte hydrometabolism was studied through in vivo and in vitro experiments, examining the pharmacological effects and mechanisms of PX-478 and AS-IV interventions in cardiomyocytes. Results28 days after direct chest irradiation with 20 Gy X-rays, C57BL/6 mice exhibited an increased heart wet-to-dry weight ratio, significant enlargement of cardiomyocyte cross-sectional area, and elevated protein expression of HIF-1α, AQP1, AQP4, Cx43, Caspase3, and Bax, with decreased expression of Bcl-2. Irradiation with 6 Gy X-rays induced edema and damage in AC16 and HL-1 cardiomyocytes at 24, 48, and 72 h, with increased expression of HIF-1α, AQP1, AQP4, and Cx43 proteins post-radiation. Inhibition of HIF-1α ameliorated edema and apoptosis in AC16 and HL-1 cardiomyocytes, reducing the expression of HIF-1α, AQP1, AQP4, and Cx43 proteins. AS-IV demonstrated strong binding affinity with HIF-1α, and successfully attenuated the expression levels of HIF-1α, AQP1, AQP4, and Cx43 proteins, alleviating edema, mitochondrial swelling, and apoptosis in AC16 and HL-1 cardiomyocytes. Furthermore, AS-IV improved cardiomyocyte edema by restoring the activity of Na/K-ATPase. ConclusionAberrant activation of the HIF-1α/AQPs/Cx43 axis is a key mechanism in X-ray-induced cardiomyocyte edema and damage. AS-IV can ameliorate X-ray induced cardiac damage by regulating hydrometabolism.

Neuroprotective effect of chelidonic acid through oxidative stress reduction in paclitaxel-induced peripheral neuropathy in rats.

The present study evaluated the effect of chelidonic acid on paclitaxel-induced neuropathy in male Wistar rats. Neuropathy was induced by administering paclitaxel (2mg/kg, i.p.) on 0, 2, 4, and 6days of the protocol. Chelidonic acid treatment (at doses of 10, 20, and 40mg/kg orally, for 21days) was initiated simultaneously with paclitaxel administration. After completion of the protocol, mechanical allodynia, hyperalgesia, and thermal hyperalgesia were measured. Additionally, nerve conduction velocity was assessed. The study investigated inflammatory cytokines, oxidative stress, and histological changes in the sciatic nerve. Furthermore, the Nrf2 was measured, and the protein expression of pAMPK and HIF-1α was assessed using western blotting. The results showed that chelidonic acid significantly normalized mechanical allodynia, hyperalgesia, and thermal hyperalgesia. It also led to a significant improvement in motor and sensory nerve conduction velocity and reduced oxidative stress compared with paclitaxel alone. Inflammatory markers such as TNF-alpha, IL-6, and IL-1β concentrations, as well as nitric oxide and C-reactive protein, were significantly decreased in the chelidonic acid-treated group. Histopathological examination revealed reduced neuronal damage, demyelination, and leukocyte infiltration with chelidonic acid treatment. Chelidonic acid treatment also resulted in a considerable rise in Nrf2 levels (p < 0.001) and increased protein expression of pAMPK in the sciatic nerve. Conversely, HIF-1α expression was significantly declined in the chelidonic acid treatment. Overall, the findings suggest that chelidonic acid treatment attenuates paclitaxel-induced neuropathic pain.

Design, Synthesis, and Hypoxia-Inducible Factor-1α Inhibitory Activity Evaluation of Panaxadiol Derivatives Containing a Thiazole Moiety.

The hypoxia-inducible factor-1α (HIF-1α) pathway has been implicated in tumor angiogenesis, growth, and metastasis. Therefore, the inhibition of this pathway is an important therapeutic target for cancer. Thiazole derivatives have been reported to have diverse biological activities, especially in terms of anti-tumor. Consequently, we hypothesized that the introduction of a thiazole functional group in PD was likely to improve the biological potency. Here, three series of PD derivatives containing a thiazole moiety were synthesized, including (a) sulfonyl-containing thiazole derivatives (5 a-l), (b) urea-containing thiazole derivatives (7 a-i), and (c) thiourea-containing thiazole derivatives (9 a-i), and evaluated for HIF-1α inhibitory activity using a Hep3B cell-based luciferase reporter assay. The results showed that about 1/3 of the target compounds showed moderate or strong HIF-1α inhibitory activity, among which compounds 5 d and 7 b showed the strongest inhibitory activity with IC50 values of 17.37 and 6.42 μM, respectively, and did not show any significant cytotoxicity. Western blot assay results indicated that these two compounds exhibited more potent inhibition, compared with panaxadiol, of the expression of HIF-1α protein in Hep3B cells at a concentration of 50 μM. Molecular docking experiments were also performed to investigate the structure-activity relationship. Compounds 5 d and 7 b can be used as leads for further study and development of novel antitumor drugs.

Hypoxia drives estrogen receptor β-mediated cell growth via transcription activation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with a poor prognosis. Hypoxia conditions affect multiple cellular processes promoting the adaptation and progression of cancer cells via the activation of hypoxia-inducible factors (HIF) and subsequent transcription activation of their target genes. Preliminary studies have suggested that estrogen receptor β (ERβ) might play a promoting role in the progression of NSCLC. However, the precise mechanisms, particularly its connection to HIF-1α-mediated modulation under hypoxia, remain unclear. Our findings demonstrated that the overexpression of ERβ, not ERα, increased cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. Tissue microarray staining revealed a strong correlation between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in CoCl2-induced hypoxia, 1% O2 incubation, or HIF-1α overexpressing cells. ChIP identified HIF-1α binding to a hypoxia response element in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the tumor growth, thus emphasizing the promising prospects of targeting HIF-1α and ERβ as a therapeutic approach for the treatment of NSCLC. KEY MESSAGES: ERβ, not ERα, increases cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. A strong correlation exists between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in hypoxic cells via binding to HRE in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the NSCLC tumor growth.

ALKBH4 functions as a hypoxia-responsive tumor suppressor and inhibits metastasis and tumorigenesis.

The human AlkB homolog (ALKBH) dioxygenase superfamily plays a crucial role in gene regulation and is implicated in cancer progression. Under hypoxic conditions, hypoxia-inducible factors (HIFs) dynamically regulate methylation by controlling various dioxygenases, thereby modulating gene expression. However, the role of hypoxia-responsive AlkB dioxygenase remains unclear. The molecular events were examined using real-time PCR and Western blot analysis. Tumor cell aggressiveness was evaluated through migration, invasion, MTT, trypan blue exclusion, and colony formation assays. In vivo metastatic models and xenograft experiments were conducted to evaluate tumor progression. Here, we examined the expression of the ALKBH superfamily under hypoxic conditions and found that ALKBH4 expression was negatively regulated by hypoxia. Knockdown of ALKBH4 enhanced the epithelial-mesenchymal transition (EMT), cell migration, invasion, and growth in vitro. The silencing of ALKBH4 enhanced metastatic ability and tumor growth in vivo. Conversely, overexpression of ALLKBH4 reversed these observations. Furthermore, overexpression of ALKBH4 significantly reversed hypoxia/HIF-1α-induced EMT, cell migration, invasion, tumor metastasis, and tumorigenicity. Notably, high expression of ALKBH4 was associated with better outcomes in head and neck cancer and breast cancer patients. Enrichment analysis also revealed that ALKBH4 was negatively enriched in hypoxia-related pathways. Clinically, a negative correlation between ALKBH4 and HIF-1α protein expression has been observed in tissues from both head and neck cancers and breast cancers. These findings collectively suggest that ALKBH4 acts as a tumor suppressor and holds therapeutic potential for hypoxic tumors.

β2-integrins control HIF1α activation in human neutrophils.

During inflammation, human neutrophils engage β2-integrins to migrate from the blood circulation to inflammatory sites with high cytokine but low oxygen concentrations. We tested the hypothesis that the inhibition of prolyl hydroxylase domain-containing enzymes (PHDs), cytokines, and β2-integrins cooperates in HIF pathway activation in neutrophils. Using either the PHD inhibitor roxadustat (ROX) (pseudohypoxia) or normobaric hypoxia to stabilize HIF, we observed HIF1α protein accumulation in adherent neutrophils. Several inflammatory mediators did not induce HIF1α protein but provided additive or even synergistic signals (e.g., GM-CSF) under pseudohypoxic and hypoxic conditions. Importantly, and in contrast to adherent neutrophils, HIF1α protein expression was not detected in strictly suspended neutrophils despite PHD enzyme inhibition and the presence of inflammatory mediators. Blocking β2-integrins in adherent and activating β2-integrins in suspension neutrophils established the indispensability of β2-integrins for increasing HIF1α protein. Using GM-CSF as an example, increased HIF1α mRNA transcription via JAK2-STAT3 was necessary but not sufficient for HIF1α protein upregulation. Importantly, we found that β2-integrins led to HIF1α mRNA translation through the phosphorylation of the essential translation initiation factors eIF4E and 4EBP1. Finally, pseudohypoxic and hypoxic conditions inducing HIF1α consistently delayed apoptosis in adherent neutrophils on fibronectin under low serum concentrations. Pharmacological HIF1α inhibition reversed delayed apoptosis, supporting the importance of this pathway for neutrophil survival under conditions mimicking extravascular sites. We describe a novel β2-integrin-controlled mechanism of HIF1α stabilization in human neutrophils. Conceivably, this mechanism restricts HIF1α activation in response to hypoxia and pharmacological PHD enzyme inhibitors to neutrophils migrating toward inflammatory sites.

Intracellular Methylglyoxal Accumulation in Classically Activated Mouse Macrophages is Mediated by HIF-1α.

Approximately one million cases of sepsis in the U.S.A. occur annually. The early phase of sepsis features dramatic changes in host metabolism and inflammation. While examining the effects of metabolic pathways on inflammation, we discovered that the highly reactive glycolytic metabolite, methylglyoxal (MG), accumulates intracellularly during classical activation of macrophages. Herein, we explored the role of glycolysis and the master regulator of glycolysis, Hypoxia-Inducing Factor-1α (HIF-1α), in inflammation and MG accumulation in mouse and human macrophages. To determine how HIF-1α regulates the inflammatory response of macrophages, we correlated HIF-1α stabilization with proinflammatory gene expression and MG-adduct accumulation in WT vs HIF1a-deficient macrophages treated with LPS or LPS+IFN-γ. A nearly complete loss of HIF-1α protein expression in response to the hypoxia mimetic, cobalt chloride, confirmed the phenotype of the HIF1a-deficient macrophages. Moreover, absence of HIF-1α was also associated with decreased MG accumulation. Increasing the glucose concentration in cultured macrophages was sufficient to cause accumulation of endogenous MG-adducts which correlated with increased Tnf and Il1b expression during classical activation. Use of the MG antagonist, aminoguanidine, led to a significant decrease in Tnf and Il1b expression in both mouse macrophages and in the THP-1 human macrophage cell line. Although off-target effects cannot be ruled out, these results are consistent with the possibility that MG regulates cytokine expression in classically activated macrophages. Collectively, this work suggests that HIF-1α stabilization is upstream of MG accumulation and that targeting the activity of HIF-1α in macrophages may be therapeutic during sepsis by limiting endogenous MG accumulation.

HSPA12A stimulates "Smurf1-Hif1α-aerobic glycolysis" axis to promote proliferation of renal tubular epithelial cells after hypoxia/reoxygenation injury.

Proliferation of renal tubular epithelial cells (TECs) is critical for the recovery after kidney ischemia/reperfusion (KI/R). However, there is still a lack of ideal therapies for promoting TEC proliferation. Heat shock protein A12A (HSPA12A) shows abundant expression in kidney in our previous studies. To investigate the role of HSPA12A in TEC proliferation after KI/R, an in vitro KI/R model was simulated by hypoxia (12h) and reoxygenation (12h) in human kidney tubular epithelial HK-2 cells. We found that, when hypoxia/reoxygenation (H/R) triggered HK-2 cell injury, HSPA12A expression was downregulated, and extracellular lactate, the readout of glycolysis, was also decreased. Loss and gain of functional studies showed that HSPA12A did not change cell viability after hypoxia but increased cell proliferation as well as glycolytic flux of HK-2 cells after H/R. When blocking glycolysis by 2-deoxy-D-glucose or oxamate, the HSPA12A promoted HK-2 cell proliferation was also abolished. Further analysis revealed that HSPA12A overexpression increased hypoxia-inducible factor 1α (Hif1α) protein expression and nuclear localization in HK-2 cells in response to H/R, whereas HSPA12A knockdown showed the opposite effects. Notably, pharmacologicalinhibition of Hif1α with YC-1 reversed the HSPA12A-induced increases of both glycolytic flux and proliferation of H/R HK-2 cells. Moreover, the HSPA12A increased Hif1α protein expression was not via upregulating its transcription but through increasing its protein stability in a Smurf1-dependent manner. The findings indicate that HSPA12A might serve as a promising target for TEC proliferation to help recovery after KI/R.

Novel mechanisms of intestinal flora regulation in high-altitude hypoxia

BackgroundThis study investigates the molecular mechanisms behind firmicutes-mediated macrophage (Mψ) polarization and glycolytic metabolic reprogramming through HIF-1α in response to intrinsic mucosal barrier injury induced by high-altitude hypoxia. MethodsEstablishing a hypoxia mouse model of high altitude, we utilized single-cell transcriptome sequencing to identify key cell types involved in regulating intestinal mucosal barrier damage caused by high-altitude hypoxia. Through proteomic analysis of colonic tissue Mψ and metabolomic analysis of Mψ metabolites, we determined crucial proteins and metabolic pathways influencing intestinal mucosal barrier damage induced by high-altitude hypoxia. Mechanistic validation was conducted using RAW264.7 Mψ in vitro by assessing cell viability with CCK-8 assay following treatment with different metabolites. The hypoxia mouse model was further validated in vivo by transplanting gut microbiota of Firmicutes. Histological examinations through H&E staining assessed colonic cell morphology and structure, while the FITC-dextran assay evaluated intestinal tissue permeability. Hypoxia probe signal intensity in mouse colonic tissue was assessed via metronidazole staining. Various experimental techniques, including flow cytometry, immunofluorescence, ELISA, Western blot, and RT-qPCR, were employed to study the impact of HIF-1α/glycolysis pathway and different gut microbiota metabolites on Mψ polarization. ResultsBioinformatics analysis revealed that single-cell transcriptomics identified Mψ as a key cell type, with their polarization pattern playing a crucial role in the intestinal mucosal barrier damage induced by high-altitude hypoxia. Proteomics combined with metabolomics analysis indicated that HIF-1α and the glycolytic pathway are pivotal proteins and signaling pathways in the intestinal mucosal barrier damage caused by high-altitude hypoxia. In vitro cell experiments demonstrated that activation of the glycolytic pathway by HIF-1α led to a significant upregulation of mRNA levels of IL-1β, IL-6, and TNFα while downregulating mRNA levels of IL-10 and TGFβ, thereby promoting M1 Mψ activation and inhibiting M2 Mψ polarization. Further mechanistic validation experiments revealed that the metabolite butyric acid from Firmicutes bacteria significantly downregulated the protein expression of HIF-1α, GCK, PFK, PKM, and LDH, thus inhibiting the HIF-1α/glycolytic pathway that suppresses M1 Mψ and activates M2 Mψ, consequently alleviating the hypoxic symptoms in RAW264.7 cells. Subsequent animal experiments confirmed that Firmicutes bacteria inhibited the HIF-1α/glycolytic pathway to modulate Mψ polarization, thereby mitigating intestinal mucosal barrier damage in high-altitude hypoxic mice. ConclusionThe study reveals that firmicutes, through the inhibition of the HIF-1α/glycolysis pathway, mitigate Mψ polarization, thereby alleviating intrinsic mucosal barrier injury in high-altitude hypoxia.

Homoharringtonine promotes non-small-cell lung cancer cell death via modulating HIF-1α/ERβ/E2F1 feedforward loop.

Hypoxia conditions promote the adaptation and progression of non-small-cell lung cancer (NSCLC) via hypoxia-inducible factors (HIF). HIF-1α may regulate estrogen receptor β (ERβ) and promote the progression of NSCLC. The phytochemical homoharringtonine (HHT) exerts strong inhibitory potency on NSCLC, with molecular mechanism under hypoxia being elusive. The effects of HHT on NSCLC growth were determined by cell viability assay, colony formation, flow cytometry, and H460 xenograft models. Western blotting, molecular docking program, site-directed mutagenesis assay, immunohistochemical assay, and immunofluorescence assay were performed to explore the underlying mechanisms of HHT-induced growth inhibition in NSCLC. HIF-1α/ERβ signaling-related E2F1 is highly expressed and contributes to unfavorable survival and tumor growth. The findings in hypoxic cells, HIF-1α overexpressing cells, as well as ERβ- or E2F1-overexpressed and knockdown cells suggest that the HIF-1α/ERβ/E2F1 feedforward loop promotes NSCLC cell growth. HHT suppresses HIF-1α/ERβ/E2F1 signaling via the ubiquitin-proteasome pathway, which is dependent on the inhibition of the protein expression of HIF-1α and ERβ. Molecular docking and site-directed mutagenesis revealed that HHT binds to the GLU305 site of ERβ. HHT inhibits cell proliferation and colony formation and promotes apoptosis in both NSCLC cells and xenograft models. The formation of the HIF-1α/ERβ/E2F1 feedforward loop promotes NSCLC growth and reveals a novel molecular mechanism by which HHT induces cell death in NSCLC.

Tujia medicine Toddalia asiatica improves synovial pannus in rats with collagen-induced arthritis through the PI3K/Akt signaling pathway

To investigate the therapeutic mechanism of Tujia medicine Toddalia asiatica alcohol extract (TAAE) for synovial pannus formation in rats with college-induced arthritis (CIA). Sixty male SD rats were randomized into normal control group, CIA model group, TGT group, 3 TAAE treatment groups at low, medium and high doses (n=10). Except for those in the normal control group, all the rats were subjected to CIA modeling using a secondary immunization method and treatment with saline, TGT or TAAE by gavage once daily for 35 days. The severity of arthritis was assessed using arthritis index (AI) score, and knee joint synovium pathologies were examined with HE staining. Serum levels of TNF-α, IL-6, and IL-1β were detected with ELISA; the protein expressions of PI3K, Akt, p-PI3K, p-Akt, VEGF, endostatin, HIF-1α, MMP1, MMP3, and MMP9 in knee joint synovial tissues were determined using Western blotting, and the mRNA expressions of TNF‑α, IL-6, IL-1β, VEGF, HIF-1α, PI3K, and Akt were detected with RT-PCR. Treatment of CIA rat models with TAAE and TGT significantly alleviated paw swelling, lowered AI scores, and reduced knee joint pathology, neoangiogenesis, and serum levels of inflammatory factors. TAAE treatment obviously increased endostatin protein expression, downregulated p-PI3K, p-Akt, MMP1, MMP3, MMP9, VEGF, and HIF-1α proteins, and reduced TNF‑α, IL-6, IL-1β, PI3K, Akt, VEGF, and HIF-1α mRNA levels in the synovial tissues, and these changes were comparable between high-dose TAAE group and TGT group. TAAE can improve joint symptoms and inhibit synovial pannus formation in CIA rats by regulating the expressions of HIF-1α, VEGF, endostatin, MMP1, MMP3, and MMP9 via the PI3K/Akt signalling pathway.

Ruthenium Complex Suppresses Proliferation of Residual Hepatocellular Carcinoma after Incomplete Radiofrequency Ablation Therapy.

Radiofrequency ablation (RFA) is an effective therapy for hepatocellular carcinoma (HCC). However, incomplete radiofrequency ablation (IRFA) can promote the progression of residual cancer cells, which is a serious problem in the clinical application of RFA. Therefore, it is of great significance to explore the mechanism and countermeasures of the progression of residual tumors after IRFA. Our previous study confirmed that IRFA can activate the hypoxia/ autophagy pathway of residual tumors in mice and then induce the proliferation of residual tumor cells. Additionally, we found a metal ruthenium complex [Ru(bpy)2(ipad)](ClO4)2 (Ru, where bpy = 2,2'-bipyridine and ipad = 2-(anthracene-9,10-dione-2-yl)imidazo[4,5-f][1,10]phenanthroline) can effectively inhibit hypoxia-inducible factor (HIF-1α) and has good anti-tumor effect in a hypoxic environment; however, whether Ru could suppress the proliferation of residual tumor cells after IRFA is unknown. This study intends to evaluate the effect of Ru in suppressing the proliferation of residual hepatocellular carcinoma after IRFA in a mice model. The Hepa1-6 xenograft mouse model was established in C57BL/6 mice to simulate clinical IRFA. H&E staining was used to evaluate the biosafety of major organs in the treated mice. TUNEL assay was employed to assess the antitumor effect. Immunohistochemically and immunofluorescence staining was performed to detect the expression of HIF-1α and autophagy-related proteins. The ELISA assay was used to examine the cytokines of interferon-gamma (IFN-γ) and interleukin 10 (IL-10). Our findings revealed that the residual tumor relapsed via the HIF-1α/LC3B/P62 autophagy- related pathway after IRFA, while Ru could suppress this process. In addition, it was demonstrated that Ru could effectively activate the immune system of the mice and reverse the tumor immune suppression microenvironment after IRFA. The ruthenium complex Ru could suppress the proliferation of residual hepatocellular carcinoma cells after IRFA in the mice model. This study introduces a novel approach that combines the use of ruthenium complexes with IRFA, offering a potential solution to address the reoccurrence of residual liver cancer following IRFA in clinical settings.

Human umbilical cord mesenchymal stem cells derived-exosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity.

To observe the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) secretions on the relevant factors in mouse retinal astrocytes, and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A (VEGF-A) and to observe the therapeutic effect on the mouse model of retinopathy of prematurity (ROP). Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group. MTT assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect related indicators. Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored. At last, the efficacy of exosomes of UCMSCs in a mouse ROP model was explored. Graphpad6 was used to comprehensively process data information. The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation. Reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased, and the ROP cell model was established after 6h of hypoxia. The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α, the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent. Compared with the ROP cell model group, the expression of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signal pathway related factors in the hUCMSCs exocrine group is significantly decreased. The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1α in ROP model tissues. HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion. In a hypoxia induced mouse retinal astrocyte model, hUCMSCs exosomes are found to effectively reduce the expression of HIF-1α and VEGF-A, which are positively correlated with the concentration of hUCMSCs exosomes. HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1α and VEGF-A proteins in ROP mice, and are positively correlated with drug dosage. Besides, they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Protective effects of 2-methoxyestradiol against hypoxic pulmonary hypertension in neonatal rats

To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 μg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.

Effects and mechanisms of Gegen Qinlian Decoction in inhibiting M1 polarization of macrophages under inflammatory hypoxia microenvironment

To explore the effect and mechanism of Gegen Qinlian Decoction(GQD) in inhibiting M1 polarization of macrophages under inflammatory hypoxia by simulating intestinal hypoxia microenvironment in vitro. A tri-gas incubator was used to simulate normal physiological hypoxia of the colon and inflammatory hypoxia microenvironments of ulcerative colitis(UC). RAW264.7 macrophages were divided into 18.5% O_(2 )(normoxia group), 4% O_2(physiological hypoxia group), and 1% O_2(inflammatory hypoxia group), and they were induced by lipopolysaccharide(LPS) for 24 h. M1 polarization was detected by flow cytometry. Under the condition of 1% inflammatory hypoxia, they were divided into blank group, model group, and GQD-containing serum low, medium, and high dose groups. Flow cytometry was used to detect M1 polarization marker CD86, and ELISA was used to detect the expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell supernatant. The mRNA expression of hypoxia-inducible factor-1α(HIF-1α), TNF-α, and IL-1β was detected by qRT-PCR. Western blot was used to detect the expression of HIF-1α/nuclear transcription factor-κB(NF-κB) signaling pathway-related proteins. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The results showed that the positive rate of CD86 in the 1% O_2 group was the highest. Under the condition of 1% inflammatory hypoxia, compared with the blank group, the expression of CD86, TNF-α, IL-1β, and HIF-1α in the model group increased. Compared with the model group, each group of GQD could reduce the expression of CD86, TNF-α, IL-1β, and HIF-1α. Compared with the blank group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/β, and p-IκBα in the model group increased. Compared with the model group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/β, and p-IκBα in GQD groups was significantly decreased. Compared with the blank group, NF-κB p65 in the model group entered the nucleus significantly. Compared with the model group, the nuclear expression of NF-κB p65 was decreased in each GQD group. Studies have shown that GQD may protect the intestine by down-regulating the HIF-1α/NF-κB signaling pathway to inhibit M1 polarization of macrophages and secretion of related inflammatory factors under 1% inflammatory hypoxia.

Mechanism of Liangfang Wenjing Decoction in treatment of hypoxia on endometriosis with cold coagulation and blood stasis by regulating CHCHD4 expression

To explore the mechanism of Liangfang Wenjing Decoction regulating coiled-coil-helix coiled-coil-helix domain containing 4(CHCHD4) in the treatment of hypoxia on endometriosis(EMs) with cold coagulation and blood stasis. The rat model of cold coagulation and blood stasis syndrome was prepared by the ice-water bath method, and then the EMs model was established by autologous intimal transplantation. The rats were randomly divided into model group, low, medium, and high(4.7, 9.4, and 18.8 g·kg~(-1)) dose groups of Liangfang Wenjing Decoction, Shaofu Zhuyu Decoction group, and sham group, with 10 rats in each group. The rats were given intragastric administration for four weeks. During the modeling, the general condition and vaginal smear of rats were observed, and the blood flow of ears and uterus were detected by laser speckle contrast imaging(LSCI) to judge the syndrome of cold coagulation and blood stasis. After the administration, the general condition of the rats was observed, and the area of ectopic lesions was measured by caliper. The localization and expression of CHCHD4 and hypoxia inducible factors-1α(HIF-1α) were detected by immunohistochemistry, and the mRNA and protein expressions of CHCHD4 and HIF-1α were detected by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot. The primary culture of ectopic endometrial stromal cells(ESCs) from EMs patients was performed, and the CHCHD4 overexpression plasmid was constructed and transfected to establish the ESCs model of CHCHD4 overexpression. The cells were divided into the control group, CHCHD4 overexpression group, CHCHD4 overexpression+control serum group, and CHCHD4 overexpression+Liangfang Wenjing Decoction serum group. The protein expression of CHCHD4 and HIF-1α was detected by Western blot, and the glucose consumption and lactic acid level were detected. The cell proliferation was detected by MTT assay. The experiment found that compared with normal rats, the modeling rats showed symptoms of cold coagulation and blood stasis, such as mental malaise, reduced diet and drinking water, disordered estrous cycle, and blocked blood circulation in ears and uterine microvessels. Compared with the sham group, the ectopic lesions in the model group were uplifted, and the mRNA and protein expressions of CHCHD4 and HIF-1α were significantly increased(P&lt;0.05). Compared with the model group, the symptoms of cold coagulation and blood stasis in each treatment group were improved, and the area of ectopic lesions was significantly reduced(P&lt;0.05 or P&lt;0.01). The mRNA and protein expression levels of CHCHD4 and HIF-1α were significantly decreased(P&lt;0.05 or P&lt;0.01). In the cell model, compared with the control group, the expression of CHCHD4, HIF-1α protein, glucose consumption, lactic acid level, and cell proliferation activity in the CHCHD4 overexpression group were significantly increased(P&lt;0.01). Compared with the CHCHD4 overexpression group, there was no significant change in each index in the control serum group, while the protein expression of CHCHD4 and HIF-1α in the Liangfang Wenjing Decoction serum group was decreased significantly(P&lt;0.05 or P&lt;0.01). The glucose consumption, lactic acid level, and cell proliferation activity decreased significantly(P&lt;0.01). It can be seen from the above that the therapeutic effect of Liangfang Wenjing Decoction on EMs with cold coagulation and blood stasis might be related to reducing the expression of CHCHD4 and then improving the hypoxia of ectopic lesions and ectopic ESCs.

Effect of 2-phenylethyl-beta-glucopyranoside isolated from Huaizhong No. 1 Rehmannia glutinosa on hypoxic pulmonary hypertension by regulating PI3K/Akt/m TOR/HIF-1α pathway

The study investigated the protective effect and mechanism of 2-phenylethyl-beta-glucopyranoside(Phe) from Huaizhong No.1 Rehmannia glutinosa on hypoxic pulmonary hypertension(PH), aiming to provide a theoretical basis for clinical treatment of PAH. Male C57BL/6N mice were randomly divided into normal group, model group, positive drug(bosentan, 100 mg·kg~(-1)) group, and low-and high-dose Phe groups(20 and 40 mg·kg~(-1)). Except for the normal group, all other groups were continuously subjected to model induction in a 10% hypoxic environment for 5 weeks, with oral administration for 14 days starting from the 3rd week. The cardiopulmonary function, right ventricular pressure, cough and asthma index, lung injury, cell apoptosis, oxidative stress-related indicators, immune cells, and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/hypoxic inducible factor 1α(HIF-1α) pathway-related proteins or mRNA levels were examined. Furthermore, hypoxia-induced pulmonary arterial smooth muscle cell(PASMC) were used to further explore the mechanism of Phe intervention in PH combined with PI3K ago-nist(740Y-P). The results showed that Phe significantly improved the cardiopulmonary function of mice with PH, decreased right ventricular pressure, cough and asthma index, and lung injury, reduced cell apoptosis, oxidative stress-related indicators, and nuclear levels of phosphorylated Akt(p-Akt) and phosphorylated mTOR(p-mTOR), inhibited the expression levels of HIF-1α and PI3K mRNA and proteins, and maintained the immune cell homeostasis in mice. Further mechanistic studies revealed that Phe significantly reduced the viability and migration ability of hypoxia-induced PASMC, decreased the expression of HIF-1α and PI3K proteins and nuc-lear levels of p-Akt and p-mTOR, and this effect was blocked by 740Y-P. Therefore, it is inferred that Phe may exert anti-PH effects by alleviating the imbalance of oxidative stress and apoptosis in lung tissues and regulating immune levels, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR/HIF-1α pathway. This study is expected to provide drug references and research ideas for the treatment of PH.

Exploring the Mechanism of Zhishi-Xiebai-Guizhi Decoction for the Treatment of Hypoxic Pulmonary Hypertension Based on Network Pharmacology and Experimental Analyses.

Hypoxic Pulmonary Hypertension (HPH), a prevalent disease in highland areas, is a crucial factor in various complex highland diseases with high mortality rates. Zhishi-Xiebai-Guizhi Decoction (ZXGD), traditional Chinese medicine with a long history of use in treating heart and lung diseases, lacks a clear understanding of its pharmacological mechanism. This study aimed to investigate the pharmacological effects and mechanisms of ZXGD on HPH. We conducted a network pharmacological prediction analysis and molecular docking to predict the effects, which were verified through in vivo experiments. Network pharmacological analysis revealed 51 active compounds of ZXGD and 701 corresponding target genes. Additionally, there are 2,116 target genes for HPH, 311 drug-disease co-target genes, and 17 core target genes. GO functional annotation analysis revealed that the core target genes primarily participate in biological processes such as apoptosis and cellular response to hypoxia. Furthermore, KEGG pathway enrichment analysis demonstrated that the core targets are involved in several pathways, including the phosphatidylinositol- 3 kinase/protein kinase B (PI3K/Akt) signaling pathway and Hypoxia Inducible Factor 1 (HIF1) signaling pathway. In vivo experiments, the continuous administration of ZXGD demonstrated a significant improvement in pulmonary artery pressure, right heart function, pulmonary vascular remodeling, and pulmonary vascular fibrosis in HPH rats. Furthermore, ZXGD was found to inhibit the expression of PI3K, Akt, and HIF1α proteins in rat lung tissue. In summary, this study confirmed the beneficial effects and mechanism of ZXGD on HPH through a combination of network pharmacology and in vivo experiments. These findings provided a new insight for further research on HPH in the field of traditional Chinese medicine.