Hypoxia-inducible Factor-1α Levels Research Articles

Effect of the Combined Treatment of Taoren Chengqi Decoction with Laser Photocoagulation on Diabetic Retinopathy

This study investigates the therapeutic effect of the combined treatment of Taoren Chengqi decoction with laser photocoagulation on DR. In animal experiments, A total of 18 rats were used and randomly divided into blank groups, model groups, and treatment groups. The rats in the model group and treatment group were intraperitoneally injected with 60 mg/kg streptozotocin. Moreover, the TCT group was gavaged with Tao-ren Cheng-qi decoction, while the other groups received an equivalent volume of normal saline once daily for 8 weeks. Fasting blood glucose (FBG) and serum interleukin (IL)-6 and IL-1β were determined. In the clinical trial, a total of 80 patients with DR were divided into a laser group (treated with laser photocoagulation) and a combination group (treated with laser photocoagulation+Taoren Chengqi decoction). The central macular thickness (CMT), best-corrected visual acuity (BCVA), and serum levels of high-density lipoprotein cholesterol, low-density-lipoprotein cholesterol, total cholesterol, triglycerides, glycated hemoglobin, and hypoxia-inducible factor 1α were measured in the patients. In animal experiments, we observe that Tao-ren Cheng-qi decoction can effectively reduce the levels of FBG, IL-6, and IL-1β in rats models with DR. Clinical studies revealed that the combination of Taoren Chengqi decoction with laser photocoagulation reduced the levels of blood glucose and lipid-related indices, and improved the inflammatory state, BCVA, and CMT. Therefore, this study suggests that the combined treatment of laser surgery and Taoren Chengqi decoction can be considered an optimal therapeutic approach for patients with DR.

GIP attenuates neuronal oxidative stress by regulating glucose uptake in spinal cord injury of rat.

Glucose-dependent insulinotropic polypeptide (GIP) is a ligand of glucose-dependent insulinotropic polypeptide receptor (GIPR) that plays an important role in the digestive system. In recent years, GIP has been regarded as a hormone-like peptide to regulate the local metabolic environment. In this study, we investigated the antioxidant role of GIP on the neuron and explored the possible mechanism. Cell counting Kit-8 (CCK-8) was used to measure cell survival. TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect apoptosis invitro and invivo. Reactive oxygen species (ROS) levels were probed with2', 7'-Dichloro dihydrofluorescein diacetate (DCFH-DA), and glucose intake was detected with 2-NBDG. Immunofluorescence staining and western blot were used to evaluate the protein level in cells and tissues. Hematoxylin-eosin (HE) staining, immunofluorescence staining and tract-tracing were used to observe the morphology of the injured spinal cord. Basso-Beattie-Bresnahan (BBB) assay was used to evaluate functional recovery after spinal cord injury. GIP reduced the ROS level and protected cells from apoptosis in cultured neurons and injured spinal cord. GIP facilitated wound healing and functional recovery of the injured spinal cord. GIP significantly improved the glucose uptake of cultured neurons. Meanwhile, inhibition of glucose uptake significantly attenuated the antioxidant effect of GIP. GIP increased glucose transporter 3 (GLUT3) expression via up-regulating the level of hypoxia-inducible factor 1α (HIF-1α) in an Akt-dependent manner. GIP increases GLUT3 expression and promotes glucose intake in neurons, which exerts an antioxidant effect and protects neuronal cells from oxidative stress both invitro and invivo.

Association of Polymorphism with Periodontitis and Salivary Levels of Hypoxia-Inducible Factor-1α.

This investigation aims to investigate the association between HIF-1α genetic polymorphism and periodontitis and examine and contrast the levels of HIF-1α present in the saliva of subjects afflicted with periodontitis and in the control group. Additionally, this study aims to establish diagnostic proficiency of this biomarker in distinguishing between periodontal health and disease. This study entailed the collection of venous blood samples and unstimulated saliva samples from a total of 160 participants, encompassing 80 individuals diagnosed with periodontitis and 80 periodontitis-free individuals. The periodontal parameters were evaluated, involving the measurement of clinical attachment loss, the probing pocket depth, and the bleeding on probing percentage. Subsequently, genetic analysis of HIF-1α using polymerase chain reaction (PCR) technique, DNA sequencing, and enzyme-linked immunosorbent assays was conducted. The genetic analysis of 352 bp of the HIF-1α gene revealed the presence of 66 single-nucleotide polymorphisms (SNPs) in control samples, whereas 78 SNPs were found in periodontitis sample. The nucleotide A was replaced with a C nucleotide at position 207 of the amplified PCR fragments. The homozygous AA pattern was predominant in the control group, with significant differences between the two groups. In contrast, the homozygous CC pattern was more dominant in the periodontitis group, with significant differences between the two groups. The analysis of Hardy-Weinberg equilibrium for the comparison between the observed and the expected genotypes showed significant differences between the observed and the expected values in the control and periodontitis groups, as well as the total sample. The highest mean values of the measured periodontal parameters were found in the periodontitis group (clinical attachment loss = 4.759, probing pocket depth = 4.050, and bleeding on probing = 30.950) with statistically significant differences between the groups. The periodontitis group showed significantly higher salivary HIF-1α levels compared to control group (p < 0.001). Besides, HIF-1α is a good biomarker in distinguishing between periodontal health and periodontitis. rs1951795 SNP of HIF-1α has no significant impact on the progression of periodontitis and the salivary level HIF-1α. Periodontitis results in a notable elevation in HIF-1α salivary levels, with an outstanding diagnostic ability to distinguish between periodontitis and periodontal health.

Elevated aerobic glycolysis driven by p62-mTOR axis promotes arsenic-induced oncogenic phenotypes in human mammary epithelial cells.

Chronic arsenic exposure is considered to increase the risk of breast cancer. p62 is a multifunctional adaptor protein that controls myriad cellular processes and is overexpressed in breast cancer tissues. Although previous studies have indicated the involvement of p62 accumulation in arsenic tumorigenesis, the underlying mechanism remains obscure. Here, we found that 0.1 µM or 0.5 µM arsenite exposure for 24 weeks induced oncogenic phenotypes in human mammary epithelial cells. Elevated aerobic glycolysis, cell proliferation capacity, and activation of p62-mTOR pathway, as indicated by increased protein levels of p62, phosphorylated-mTOR (p-mTOR) and hypoxia-inducible factor 1α (HIF1α), were observed in chronically arsenite-exposed cells, and of note in advance of the onset of oncogenic phenotypes. Moreover, p62 silencing inhibited acquisition of oncogenic phenotypes in arsenite-exposed cells. The protein levels of p-mTOR and HIF1α, as well as aerobic glycolysis and cell proliferation, were suppressed by p62 knockdown. In addition, re-activation of p‑mTOR reversed the inhibitory effects of p62 knockdown. Collectively, our data suggest that p62 exerts an oncogenic role via mTORC1 activation and acts as a key player in glucose metabolism during arsenite-induced malignant transformation, which provides a new mechanistic clue for the arsenite carcinogenesis.

To observe the clinical effect of lipoic acid combined with continuous positive airway pressure ventilation in treating obstructive sleep apnea-hypopnea syndrome and its effect on peripheral blood γ-aminobutyric acid and melatonin levels.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common respiratory disease with potential lethality. At present, the commonly used treatment method is continuous positive airway pressure ventilation, but with the prolongation of the course of the disease, the effect of single ventilation on the improvement of oxidative stress levels is not good. Lipoic acid is a commonly used antioxidant in clinics. In this paper, lipoic acid combined with continuous positive airway pressure ventilation is used to explore whether it has a better therapeutic effect on patients. To probe into the clinical efficacy of lipoic acid combined with continuous positive airway pressure ventilation in the therapy of OSAHS. 82 patients with OSAHS who were cured in our hospital from March 2021 to September 2022 were prospectively collected as subjects. Based on different treatment methods, patients were grouped into a control group (43 cases) and an observation group (39 cases). The control group was treated with continuous positive airway pressure (CPAP), and the observation group was treated with lipoic acid based on control group. The therapeutic effects were measuredby apnea hypopnea index (AHI), oxygen saturation (SpO2), mean oxygen saturation (MSpO2), serum malondialdehyde (MDA), superoxide dismutase (SOD), hypoxia inducible factor-1α (HIF-1α) levels, peripheral blood γ-aminobutyric acid, melatonin levels. The clinical effectiveness of the observation group was better (P < 0.05). After treatment, AHI, the levels of MDA and HIF-1α in the observation group were lower and SpO2, MSpO2 and the level of SOD, γ- aminobutyric acid, and melatonin were higher than those in the control group (P < 0.05). The levels of γ- aminobutyric acid and melatonin were negatively correlated with the severity of symptoms, ESS, and AIS scores (P < 0.05). The clinical effect of lipoic acid combined with CPAP in the treatment of OSAHS is better, and it has a positive effect on the levels of γ-aminobutyric acid and melatonin in peripheral blood. Lipoic acid was added to the original method for treatment, and the therapeutic effect was greatly improved.

Morusin suppresses the stemness characteristics of gastric cancer cells induced by hypoxic microenvironment through inhibition of HIF-1α accumulation

Gastric cancer (GC) is a common, life-threatening malignancy that contributes to the global burden of cancer-related mortality, as conventional therapeutic modalities show limited effects on GC. Hence, it is critical to develop novel agents for GC therapy. Morusin, a typical prenylated flavonoid, possesses antitumor effects against various cancers. The present study aimed to demonstrate the inhibitory effect and mechanism of morusin on the stemness characteristics of human GC in vitro under hypoxia and to explore the potential molecular mechanisms. The effects of morusin on cell proliferation and cancer stem cell-like properties of the human GC cell lines SNU-1 and AGS were assessed by MTT assay, colony formation test, qRT-PCR, flow cytometry analysis, and sphere formation test under hypoxia or normoxia condition through in vitro assays. The potential molecular mechanisms underlying the effects of morusin on the stem-cell-like properties of human GC cells in vitro were investigated by qRT-PCR, western blotting assay, and immunofluorescence assay by evaluating the nuclear translocation and expression level of hypoxia-inducible factor-1α (HIF-1α). The results showed that morusin exerted growth inhibitory effects on SNU-1 and AGS cells under hypoxia in vitro. Moreover, the proportions of CD44+/CD24– cells and the sphere formation ability of SNU-1 and AGS reduced in a dose-dependent manner following morusin treatment. The expression levels of stem cell-related genes, namely Nanog, OCT4, SOX2, and HIF-1α, gradually decreased, and the nuclear translocation of the HIF-1α protein was apparently attenuated. HIF-1α overexpression partially reversed the abovementioned effects of morusin. Taken together, morusin could restrain stemness characteristics of GC cells by inhibiting HIF-1α accumulation and nuclear translocation and could serve as a promising compound for GC treatment.

Pioglitazone, a peroxisome proliferator‑activated receptor γ agonist, induces cell death and inhibits the proliferation of hypoxic HepG2 cells by promoting excessive production of reactive oxygen species.

Hypoxia is a hallmark of solid tumors. Hypoxic cancer cells adjust their metabolic characteristics to regulate the production of cellular reactive oxygen species (ROS) and facilitate ROS-mediated metastasis. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that regulates the transcription of fatty acid metabolism-related genes that have a key role in the survival and proliferation function of hypoxic cancer cells. In the present study, mRNA expression in HepG2 cells under chemically induced hypoxia was assessed. The protein expression levels of hypoxia-inducible factor 1α (HIF-1α) were measured using western blotting. Following treatment with the PPARγ agonist pioglitazone, cell viability was assessed using a Cell Counting Kit-8 assay, whilst cell proliferation and death were determined using 5-ethynyl-2'-deoxyuridine incorporation staining, and calcein-acetoxymethyl ester and propidium iodide staining, respectively. Cellular ROS production was assessed using dihydroethidium staining. Cobalt chloride was used to induce hypoxia in HepG2 cells, which was evaluated using HIF-1α expression. The results revealed that the mRNA expression of PPARγ, CD36, acetyl-co-enzyme A dehydrogenase (ACAD) medium chain (ACADM) and ACAD short-chain (ACADS) was downregulated in hypoxic HepG2 cells. The PPARγ agonist pioglitazone decreased the cell viability of hypoxic HepG2 cells by inhibiting cell proliferation and inducing cell death. Following treatment with the PPARγ agonist pioglitazone, hypoxic HepG2 cells produced excessive ROS. ROS-mediated cell death induced by the PPARγ agonist pioglitazone was rescued with the antioxidant N-acetyl-L-cysteine. The downregulated mRNA expression of PPARγ, CD36, ACADM and ACADS was not reverted by a PPARγ agonist in hypoxic HepG2 cells. By contrast, the PPARγ agonist suppressed the mRNA expression of BCL2, which was upregulated in hypoxic HepG2 cells. In summary, the PPARγ agonist stimulated excessive ROS production to inhibit cell proliferation and increase the death of hypoxic HepG2 cells by decreasing BCL2 mRNA expression, suggesting a negative association between PPARγ and BCL2 in the regulation of ROS production in hypoxic HepG2 cells.

The measurement of hypoxia-inducible factor-1α and interleukin-6 levels in pregnancies with threatened preterm labor

The measurement of hypoxia-inducible factor-1α and interleukin-6 levels in pregnancies with threatened preterm labor

Effect of high-fat diet on cerebral pathological changes of cerebral small vessel disease in SHR/SP rats.

Cerebral small vessel diseases (CSVD) are neurological disorders associated with microvessels, manifested pathologically as white matter (WM) changes and cortical microbleeds, with hypertension as a risk factor. Additionally, a high-fat diet (HFD) can affect peripheral vessel health. Our study explored how HFD affects cerebral small vessels in normotensive WKY, hypertensive SHR, and SHR/SP rats. The MRI results revealed that HFD specifically increased WM hyperintensity in SHR/SP rats. Pathologically, it increased WM pallor and vacuolation in SHR and SHR/SP rats. Levels of blood-brain barrier (BBB) protein claudin 5 were decreased in SHR and SHR/SP compared to WKY, with HFD having minimal impact on these levels. Conversely, collagen IV levels remained consistent among the rat strains, which were increased by HFD. Consequently, HFD caused vessel leakage in all rat strains, particularly within the corpus callosum of SHR/SP rats. To understand the underlying mechanisms, we assessed the levels of hypoxia-inducible factor-1α (HIF-1α), Gp91-phox, and neuroinflammatory markers astrocytes, and microglia were increased in SHR and SHR/SP compared to WKY and were further elevated by HFD in all rat strains. Gp91-phox was also increased in SHR and SHR/SP compared to WKY, with HFD causing an increase in WKY but little effect in SHR and SHR/SP. In conclusion, our study demonstrates that HFD, in combined with hypertension, intensifies cerebral pathological alterations in CSVD rats. This exacerbation involves increased oxidative stress and HIF-1α in cerebral vessels, triggering neuroinflammation, vascular basement membrane remodeling, IgG leakage, and ultimately WM damage.

Evaluation of nocturnal apnea and airflow limitation as indicators for cognitive dysfunction in patients with chronic obstructive pulmonary disease/obstructive sleep apnea hypopnea syndrome overlap syndrome.

The aim of this study is to investigate how much intermittent hypoxemia and airflow limitation contribute to cognitive impairment in overlap syndrome (OS), which is the coexistence of two common diseases, obstructive sleep apnea hypopnea syndrome (OSAHS) and chronic obstructive pulmonary disease (COPD). We conducted a cross-sectional study of patients with OSAHS, COPD or OS, compared with normal controls, to determine the association between sleep apnea/pulmonary function-related indicators and cognitive dysfunction in individuals with OSAHS, COPD or OS. A total of 157 participants were recruited. Both OSAHS and OS presented lower adjusted Montreal cognitive assessment (MoCA) scores compared with COPD group. In addition, the MoCA score was significantly lower in COPD group compared with control group. The incidence of cognitive impairment was 57.4% in OSAHS group, and 78% in OS group, which were significantly higher than COPD group (29%) and control group (8.8%). Furthermore, a broader range of cognitive domains were affected in OS group compared with OSAHS group. Elevated levels of oxygen desaturation index (ODI) and/or apnea hypopnea index (AHI) were positively correlated with increased Epworth sleeping scale (ESS) in OSAHS and OS. Forced vital capacity (FVC), forced expiratory volume in 1s (FEV1) and peak expiratory flow (PEF) were positively correlated with cognitive scores in OSAHS but not in OS. Serum level of hypoxia-inducible factor-1α (HIF-1α) was significantly higher in OS. Logistic regression identified ODI as an independent risk factor for cognitive impairment in OS, while severity of snoring and PEF were independent risk factors in OSAHS. This study revealed significant cognitive impairment in OS, OSAHS and COPD. Sleep-related indicators are warranted in OS patients for detection, differentiation and grading of cognitive impairment, whereas pulmonary functions are warranted in OSAHS patients for detection and early intervention of cognitive impairment.

A preventative role of nitrate for hypoxia-induced intestinal injury

BackgroundStudying effective interventions for hypoxia-induced injury is crucial, particularly in high-altitude areas. Symptoms stemming from intestinal injuries have a significant impact on the health of individuals transitioning from plains to plateau regions. This research explores the effects and mechanisms of nitrate supplementation in preventing hypoxia-induced intestinal injury. MethodsA hypoxia survival mouse model was established using 7% O2 conditions. The intervention with 4 mM sodium nitrate (NaNO3) in drinking water commenced 7 days prior to hypoxia exposure. Weight monitoring, hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and intestinal permeability assays were employed for physiological, histological, and functional analyses. Quantitative PCR (qPCR), Western blot, and immunofluorescence were utilized to analyze the levels of tight junction (TJ) proteins and hypoxia-inducible factor 1α (Hif 1α). RNA sequencing (RNA-seq) identified nitrate's target, and chromatin immunoprecipitation (ChIP) verified the transcriptional impact of Hif 1α on TJ proteins. Villin-cre mice infected with AAV9-FLEX-EGFP-Hif 1α were used for mechanism validation. ResultsThe results demonstrated that nitrate supplementation significantly alleviated small intestinal epithelial cell necrosis, intestinal permeability, disruption of TJs, and weight loss under hypoxia. Moreover, the nitrate-triggered enhancement of TJs is mediated by Hif 1α nuclear translocation and its subsequent transcriptional function. The effect of nitrate supplementation on TJs was largely attributed to the stimulation of the EGFR/PI3K/AKT/mTOR/Hif 1α signaling pathways. ConclusionNitrate serves as a novel approach in preventing hypoxia-induced intestinal injury, acting through Hif 1α activation to promote the transcription of TJ proteins. Furthermore, our study provides new and compelling evidence for the protective effects of nitrate in hypoxic conditions, especially at high altitudes.

The HIF-1α/EGF/EGFR Signaling Pathway Facilitates the Proliferation of Yak Alveolar Type II Epithelial Cells in Hypoxic Conditions.

The yak is a unique creature that thrives in low-oxygen environments, showcasing its adaptability to high-altitude settings with limited oxygen availability due to its unique respiratory system. However, the impact of hypoxia on alveolar type II (AT2) epithelial cell proliferation in yaks remains unexplored. In this study, we investigated the effects of different altitudes on 6-month-old yaks and found an increase in alveolar septa thickness and AT2 cell count in a high-altitude environment characterized by hypoxia. This was accompanied by elevated levels of hypoxia-inducible factor-1α (HIF-1α) and epidermal growth factor receptor (EGFR) expression. Additionally, we observed a significant rise in Ki67-positive cells and apoptotic lung epithelial cells among yaks inhabiting higher altitudes. Our in vitro experiments demonstrated that exposure to hypoxia activated HIF-1α, EGF, and EGFR expression leading to increased proliferation rates among yak AT2 cells. Under normal oxygen conditions, activation of HIF-1α enhanced EGF/EGFR expressions which subsequently stimulated AT2 cell proliferation. Furthermore, activation of EGFR expression under normoxic conditions further promoted AT2 cell proliferation while simultaneously suppressing apoptosis. Conversely, inhibition of EGFR expression under hypoxic conditions had contrasting effects. In summary, hypoxia triggers the proliferation of yak AT2 cells via activation facilitated by the HIF-1α/EGF/EGFR signaling cascade.

The protease Lon prolongs insect lifespan by responding to reactive oxygen species and degrading mitochondrial transcription factor A

Diapause is a widespread adaptation of insects that enables them to survive during unfavorable seasons and is characterized by suppressed metabolism and increased lifespan. Previous works have demonstrated that high levels of reactive oxygen species (ROS) and hypoxia-inducible factor-1α (HIF-1α) in the pupal brain of the moth Helicoverpa armigera induce diapause and extend lifespan by downregulating mitochondrial transcription factor A (TFAM). However, the molecular mechanisms of ROS-HIF-1α regulating metabolic activity to extend lifespan are still poorly understood. Here, we show that the mitochondrial abundance in diapause-type pupal brains is markedly lower than that in their nondiapause-type pupae, suggesting that ROS-HIF-1α signaling negatively regulates the number of mitochondria. The protease Lon, a major mitochondrial matrix protease, can respond to ROS signals. It is activated by transcription factor HIF-1α, which specifically binds the LON promoter to promote its expression. A high level of LON mediates the degradation of TFAM, which is a crucial factor in regulating mitochondrial abundance and metabolic activity. We believe this is the first report that a previously unrecognized regulatory pathway, ROS-HIF-1α-LON-TFAM, reduces mitochondrial activity to induce diapause, extending insect lifespan.

Pt-Se-Bonded Nanoprobe for High-Fidelity Detection of Non-small Cell Lung Cancer and Enhancement of NIR II Photothermal Therapy.

Non-small cell lung cancer (NSCLC) accounts for a high proportion of lung cancer cases globally, but early detection remains challenging, and insufficient oxygen supply at tumor sites leads to suboptimal treatment outcomes. Therefore, the development of core-shell Au@Pt-Se nanoprobes (Au@Pt-Se NPs) with peptide chains linked through Pt-Se bonds was designed and synthesized for NSCLC biomarker protein calcium-activated neutral protease 2 (CAPN2) and photothermal therapy (PTT) enhancement. The NP can be specifically cleaved by CAPN2, resulting in fluorescence recovery to realize the detection. The Pt-Se bonds exhibit excellent resistance to biologically abundant thiols such as glutathione, thus avoiding "false-positive" results and enabling precise detection of NSCLC. Additionally, the platinum (Pt) shell possesses catalase-like properties that catalyze the generation of oxygen from endogenous hydrogen peroxide within the tumor, thereby reducing hypoxia-inducible factor-1α (HIF-1α) levels and alleviating the hypoxic environment at the tumor site. The Au@Pt-Se NPs exhibit strong absorption bands, enabling the possibility of PTT in the near-infrared II region (NIR II). This study presents an effective approach for the early detection of NSCLC while also serving as an oxygen supplier to alleviate the hypoxic environment and enhance NIR II PTT.

Shenfu Injection alleviates sepsis-associated lung injury by regulating HIF-1α

Shenfu Injection(SFI) is praised for the high efficacy in the treatment of septic shock. However, the precise role of SFI in the treatment of sepsis-associated lung injury is not fully understood. This study investigated the protective effect of SFI on sepsis-associated lung injury by a clinical trial and an animal experiment focusing on the hypoxia-inducing factor-1α(HIF-1α)-mediated mitochondrial autophagy. For the clinical trial, 70 patients with sepsis-associated lung injury treated in the emergency intensive care unit of the First Affiliated Hospital of Zhengzhou University were included. The levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-α were measured on days 1 and 5 for every patient. Real-time quantitative polymerase chain reaction(RT-qPCR) was performed to determine the mRNA level of hypoxia inducible factor-1α(HIF-1α) in the peripheral blood mononuclear cells(PBMCs). For the animal experiment, 32 SPF-grade male C57BL/6J mice(5-6 weeks old) were randomized into 4 groups: sham group(n=6), SFI+sham group(n=10), SFI+cecal ligation and puncture(CLP) group(n=10), and CLP group(n=6). The body weight, body temperature, wet/dry weight(W/D) ratio of the lung tissue, and the pathological injury score of the lung tissue were recorded for each mouse. RT-qPCR and Western blot were conducted to determine the expression of HIF-1α, mitochondrial DNA(mt-DNA), and autophagy-related proteins in the lung tissue. The results of the clinical trial revealed that the SFI group had lowered levels of inflammatory markers in the blood and alveolar lavage fluid and elevated level of HIF-1α in the PBMCs. The mice in the SFI group showed recovered body temperature and body weight. lowered TNF-α level in the serum, and decreased W/D ratio of the lung tissue. SFI reduced the inflammatory exudation and improved the alveolar integrity in the lung tissue. Moreover, SFI down-regulated the mtDNA expression and up-regulated the protein levels of mitochondrial transcription factor A(mt-TFA), cytochrome c oxidase Ⅳ(COXⅣ), HIF-1α, and autophagy-related proteins in the lung tissue of the model mice. The findings confirmed that SFI could promote mitophagy to improve mitochondrial function by regulating the expression of HIF-1α.

Correlation of serum levels of HIF-1α, SMAD3, and HDAC3 with the disease severity in stroke patients.

Explore serum levels of hypoxia-inducible factor-1α (HIF-1α), signal transduction molecule 3 (SMAD3), and histone deacetylase (HDAC) and their correlation with the severity of the condition of stroke patients. Clinical records of 93 stroke patients and 93 healthy individuals were retrospectively analyzed. Serum levels of HIF-1α, SMAD3, and HDAC3 in patients with different disease degrees and lesion areas were compared between the two groups. Correlation between serum levels of HIF-1α, SMAD3, and HDAC3 and the severity and lesion area of the observation group were analyzed. Serum levels of HIF-1α, SMAD3, and HDAC3 in the observation group were higher than those in the control group (p<0.05). Serum levels of HIF-1α, SMAD3, and HDAC3 in patients with moderate and severe disease were significantly higher than those in patients with mild disease and were the highest in patients with severe disease (p<0.05). Serum levels of HIF-1α, SMAD3, and HDAC3 in patients with moderate and large areas of cerebral infarction were significantly higher than those in patients with small areas of cerebral infarction and the highest in patients with large areas of cerebral infarction (p<0.05). Spearman correlation analysis showed that serum levels of HIF-1α, SMAD3, and HDAC3 significantly positively correlated with the severity of stroke and lesion area (p<0.05). Serum levels of HIF-1α, SMAD3, and HDAC3 in stroke patients are highly expressed, and the increase positively correlates with the severity of the disease and the area of the lesion.

HIF-1α-Dependent Autophagy Deficiency in Bone Marrow Endothelial Progenitor Cells Contributes to Acute Graft-Versus-Host Disease after Allotransplant

Background: Acute Graft-Versus-Host Disease (aGVHD) constitutes a prevalent component after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Among the components of the Bone Marrow（BM)microenvironment, Endothelial Progenitor Cells (EPCs) hold significant importance in hematopoietic-supporting and immune reconstitution in post-transplantation patients. Consequently, a discernible correlation emerges between the impairment of EPCs and the incidence and severity of GVHD. Our recent study has shown impaired EPC function and heightened reactive oxygen species (ROS) levels in aGVHD patients (2018, BJH). Additionally, reduced levels of hypoxia-inducible factor-1α (HIF-1α) and autophagy were observed in BM EPCs from aGVHD patients in comparison to those without GVHD（non-GVHD）. HIF-1α is closely linked to ROS generation. Therefore， exploring the role of HIF-1α and autophagy in the development of aGVHD after allo-HSCT is significant. Aims： To conduct a comparative analysis of the functions of EPCs and assess the variations in the expression levels of HIF-1α and autophagy-related proteins between aGVHD and non-GVHD patients. Additionally, the study aims to assess whether the low HIF-1α level is responsible for the autophagy deficiency and mitochondrial dysfunction observed in EPCs of aGVHD patients, and to delineate the specific pathways through which this downregulation influences their capacity of hematopoiesis-supporting. Methods: The current prospective clinical case-matched study included 20 patients with aGVHD and 40 patients without GVHD. BM mononuclear cells（BMMNCs） were cultured as former publications reported. EPCs（CD34+, CD133+, CD309+) were flow-sorted for evaluating apoptosis, ROS, and autophagy levels. SiRNA transfection knocked down HIF-1α in human umbilical vein endothelial cells (HUVECs). Endothelial cells(ECs） gene expression levels were determined using Western blot, RT-qPCR. ECs function was assessed by angiogenesis, Transwell cell migration, CCK-8 cell proliferation, and JC-1 mitochondrial staining. ECs were co-cultured with hematopoietic stem cells （HSCs） for seven days, and HSCs were subjected to colony-forming unit assays and other experiments to evaluate hematopoietic stem cell function. ECs NAD+ and NADH levels were assessed by the kit. Results: Downregulation of autophagy signaling pathway in HUVECs following knockdown of HIF-1α, leading to inhibited cell proliferation, migration, and angiogenesis. Similarly, patients affected by aGVHD exhibited reduced HIF-1α expression, impaired autophagy, and cellular function in EPCs compared to non-GVHD patients. HSCs co-culturing with HUVECs with HIF-1α knockdown and EPCs from aGVHD patients resulted in increased apoptosis, elevated levels of ROS, and impaired colony formation ability. However, intervention with HIF-1α activators Roxadustat, which served to upregulate HIF-1α levels in ECs, activated autophagy, and improved cellular function, also enhanced the hematopoiesis-supporting ability. Furthermore, the knockdown of HIF-1α in HUVECs and EPCs of aGVHD patients led to the abnormal activation of SIRT1 and PARP1 and other NAD+-consuming proteins, which subsequently caused a decrease in NAD+ levels and resulted in abnormal mitochondrial membrane potential. The supplementation of NAD+ precursors, specifically β-nicotinamide mononucleotide, effectively increased NAD+ levels in ECs and restored cellular function. Conclusion: The results revealed that the autophagy deficiency was mediated by reduced expression of HIF-1α in EPCs of patients with aGVHD. The deficiency of autophagy also led to abnormal activation of SIRT1 and PARP1, which in turn affected the levels of NAD+ and mitochondrial membrane potential, ultimately impairing cellular function and hematopoiesis-supporting ability. These molecular changes may be identified as potential contributors to the pathogenesis of aGVHD after allo-HSCT. On the other hand, Roxadustat upregulates HIF-1α expression in ECs, which subsequently enhanced the autophagy level and improved mitochondrial function. As a result, cellular function and hematopoiesis-supporting ability were restored. This promising therapeutic approach highlights a novel strategy for preventing and treating of patients with aGVHD after allo-HSCT.

Sirtuin4 alleviates severe acute pancreatitis by regulating HIF-1α/HO-1 mediated ferroptosis.

Acute pancreatitis (AP) is a common emergency of the digestive system and serious cases can develop into severe acute pancreatitis (SAP), which ortality rates up to 30%. Sirtuin4 (SIRT4) is a member of the sirtuin family, and plays a key role in inflammation and oxidative stress. However, the potential role of SIRT4 in SAP has yet to be elucidated. In the present study, we found that the expression level of SIRT4 in human AP was downregulated by screening a public database, suggesting that SIRT4 may play a role in AP. Subsequently, we used L-arginine (L-Arg) to induce SAP in SIRT4 knockout (SIRT4_KO) and SIRT4 overexpression (AAV_SIRT4) mice. The results showed that the pancreatic tissue injury and related lung and kidney injury were serious in SIRT4_KO mice after SAP induction, but were significantly reduced in AAV_SIRT4 mice. More importantly, we found that the levels of antioxidant factors GSH and SOD were decreased in SIRT4_KO mice, and the production of oxidative products and lipid peroxidation markers was increased, suggesting that SIRT4 was involved in inflammation and oxidative stress during SAP. Further studies showed that the absence or overexpression of SIRT4 affected the expression level of Hypoxia-inducible factor-1α (HIF-1α) after SAP induction, and regulated the expression of ferroptosis related proteins by mediating HIF-1α/HO-1 pathway. Collectively, our study revealed that SIRT4 plays a protective role in SAP by regulating the HIF-1α/HO-1 pathway to inhibit ferroptosis.

#5449 CALYCOSIN ATTENUATES RENAL ISCHEMIA/REPERFUSION INJURY BY SUPPRESSING INFLAMMATION VIA PPARγ/EGR1 PATHWAY

Abstract Background and Aims Renal ischemia reperfusion injury (IRI) is a leading and common cause of AKI, and delayed recovery of IRI contributes to chronic kidney disease and even end-stage kidney disease. However, there is no effective prevention and treatment for IRI-AKI. Calycosin (CAL), an isoflavonoid phytoestrogen isolated from Radix astragali, has various pharmacological activities. However, whether CAL has a protective effect on renal IRI and its mechanism remains elusive. In this study, we aim to explore the effect and mechanism of CAL on IRI-AKI through bioinformatics and experiments in vitro and in vivo models, so as to provide potential candidate drugs for early intervention treatment of IRI-AKI. Method (1) In the first part, mice were intragastrically given CAL daily for 7 d, and subjected to bilateral renal artery occlusion for 35 min followed by reperfusion 24 h to establish the renal IRI model. HE staining was used to evaluate the renal pathological injury, and the renal function including serum creatinine (SCr) and blood urea nitrogen (BUN), was also examined. Meanwhile, inflammatory factors were detected. In cell experiments, HK-2 cells pretreated with 8,16 and 32 μM CAL were subjected with hypoxia/reoxygenation (H/R), and the effect of CAL on H/R induced cell injury were investigated by qRT-PCR and ELISA. (2) In the second part, the differentially expressed gene (DEGs) of IRI-AKI were obtained by bioinformatics analysis of GSE52004 dataset. Subsequently, the hub gene with the highest score was obtained by protein-protein interaction (PPI) analysis, and its upstream transcription factors were predicted by JASPAR database. In IRI-AKI mice, the predicted results of bioinformatics were verified by qRT-PCR, Western Blot and immunohistochemistry. In HK-2 cells, the regulatory relationship between the hub gene and its upstream transcription factor and the role of them in inflammatory process were verified by double luciferase reporter gene and siRNA. Furthermore, molecular docking was used to predict the interaction between CAL and the transcription factor, and the role of the target protein in the protection of H/R HK-2 cells by calycosin was verified by siRNA. Results (1) In the mice with renal IRI, CAL dose dependently alleviated SCr and BUN levels and renal tubular injury score. Meanwhile, the mRNA levels and concentrations of inflammatory cytokines were markedly inhibited by CAL pretreatment. (2) In the pre-experiment in vitro, the toxicity of CAL was negligible at concentrations of 32 μM or less for 24 h incubation. In H/R induced HK-2 cells, CAL incubation reduced the levels of hypoxia-inducible factor 1α (HIF-1α) and the inflammatory cytokines. (3) 58 DEGs were identified in the kidney of IRI mice. PPI analysis showed early growth response 1 (EGR1) is the hub gene the highest score. JASPAR database predicted that peroxisome proliferator-activated receptor γ (PPARγ) is the upstream transcription factor of EGR1. (4) In kidney after IRI, EGR1 was up-regulated and PPARγ was down-regulated, while the pretreatment of CAL decreased EGR1 and increased PPARγ in a dose-dependent manner. (5) In H/R induced HK-2 cells, after inhibited the expression of EGR1 by siRNA, the level of inflammatory factors decreased significantly, suggesting that EGR1 participated in and promoted the inflammatory process of tubular cells. In addition, dual luciferase reporter gene assay confirmed that PPARγ interact with EGR1 promoter, as EGR1 increased after EGR1 promoter plasmid transfection but decreased after PPARγ overexpression. (6) In mice after renal IRI, CAL promote PPARγ expression markedly. According to molecular docking, CAL, as a ligand, could be embedded into the orthosteric pocket of PPARγ with a binding energy of -6.46 kcal/mol. In CAL incubated cells, PPARγ increased and EGR1 decreased considerably. However, the protective effect of CAL on H/R induced inflammation was absent when PPARγ knockdown, indicated that CAL ameliorate renal IRI and inhibit inflammation via PPARγ/EGR1. Conclusion CAL could significantly modulate inflammation by targeting the PPARγ/EGR1 pathway, contributing to the alleviation of renal IRI.

Prognostic Value of HIF-1α-Induced Genes in Sepsis/Septic Shock.

Hypoxia is characterized as one of the main consequences of sepsis, which is recognized as the leading cause of death in intensive care unit (ICU) patients. In this study, we aimed to examine whether the expression levels of genes regulated under hypoxia could be utilized as novel biomarkers for sepsis prognosis in ICU patients. Whole blood expression levels of hypoxia-inducible factor-1α (HIF1A), interferon-stimulated gene 15 (ISG15), hexokinase 2 (HK2), lactate dehydrogenase (LDHA), heme oxygenase-1 (HMOX1), erythropoietin (EPO), and the vascular endothelial growth factor A (VEGFA) were measured on ICU admission in 46 critically ill, initially non-septic patients. The patients were subsequently divided into two groups, based on the development of sepsis and septic shock (n = 25) or lack thereof (n = 21). HMOX1 mRNA expression was increased in patients who developed sepsis/septic shock compared to the non-septic group (p < 0.0001). The ROC curve, multivariate logistic regression, and Kaplan-Meier analysis demonstrated that HMOX1 expression could be utilized for sepsis and septic shock development probability. Overall, our results indicate that HMOX1 mRNA levels have the potential to be a valuable predictive factor for the prognosis of sepsis and septic shock in ICU patients.