Effects of three weeks base training at moderate simulated altitude with or without hypoxic residence on exercise capacity and physiological adaptations in well-trained male runners.

Objectives: To test the hypothesis that 'live high-base train high-interval train low' (HiHiLo) altitude training, compared to 'live low-train high' (LoHi), yields greater benefits on performance and physiological adaptations. Methods: Sixteen young male middle-distance runners (age, 17.0 ± 1.5 y; body mass, 58.8 ± 4.9 kg; body height, 176.3 ± 4.3 cm; training years, 3-5 y; training distance per week, 30-60 km.wk-1) with a peak oxygen uptake averaging ~65 ml.min-1.kg-1 trained in a normobaric hypoxia chamber (simulated altitude of ~2,500 m, monitored by heart rate ~170 bpm; thrice weekly) for 3 weeks. During this period, the HiHiLo group (n = 8) stayed in normobaric hypoxia (at ~2,800 m; 10 h.day-1), while the LoHi group (n = 8) resided near sea level. Before and immediately after the intervention, peak oxygen uptake and exercise-induced arterial hypoxemia responses (incremental cycle test) as well as running performance and time-domain heart rate variability (5-km time trial) were assessed. Hematological variables were monitored at baseline and on days 1, 7, 14 and 21 during the intervention. Results: Peak oxygen uptake and running performance did not differ before and after the intervention in either group (all P > 0.05). Exercise-induced arterial hypoxemia responses, measured both at submaximal (240 W) and maximal loads during the incremental test, and log-transformed root mean square of successive R-R intervals during the 4-min post-run recovery period, did not change (all P > 0.05). Hematocrit, mean reticulocyte absolute count and reticulocyte percentage increased above baseline levels on day 21 of the intervention (all P < 0.001), irrespective of group. Conclusions: Well-trained runners undertaking base training at moderate simulated altitude for 3 weeks, with or without hypoxic residence, showed no performance improvement, also with unchanged time-domain heart rate variability and exercise-induced arterial hypoxemia responses.

TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

Expression of HIF­α and their association with clinicopathological parameters in clinical renal cell carcinoma.

Objectives: This study aimed to assess the cellular localization and expression levels of hypoxia-inducible factor (HIF) -α proteins (specifically HIF-1α, HIF-2α, and HIF-3α) that play a role in the hypoxia pathway and to determine their correlation with clinicopathological parameters and patient survival in renal cell carcinoma (RCC). Materials and methods: Tissue microarray (TMA) with cores from 150 clear cell RCCs and 31 non-ccRCC samples. HIF-1α, HIF-2α, and HIF-3α antibodies were used for immunohistochemistry (IHC) of TMA to evaluate the cellular localization and expression levels of HIF-α proteins, specifically in relation to the hypoxia pathway. Results: The expression levels of the HIF-α proteins were higher in the nucleus than in the cytoplasm. Furthermore, the nuclear expression levels of all HIF-α proteins were significantly higher in clear cell RCC (ccRCC) than in non-ccRCC. Cytoplasmic HIF-3α expression was also higher in ccRCC than in non-ccRCC, whereas cytoplasmic HIF-1α and HIF-2α expression levels were similar between the different RCC types. In ccRCC, nuclear HIF-1α expression levels correlated with both nuclear HIF-2α and HIF-3α levels, whereas cytoplasmic HIF-3α expression levels were associated with HIF-1α only.In non-ccRCC, there was a positive correlation observed between nuclear HIF-1α and HIF-3α expression, but no correlation was found with HIF-2α. In patients with ccRCC, the nuclear expressions of HIF-1α and HIF-3α was significantly associated with cancer-specific survival (CSS) in univariate analysis. This association was no longer evident in multivariate analysis. Notably, there was no correlation observed between nuclear HIF-2α expression and CSS in these patients. In contrast, cytoplasmic expression levels showed no association with CSS. Conclusion: The expression levels of the three primary HIF-α proteins were found to be higher in the nucleus than in the cytoplasm. Furthermore, the results indicated that HIF-3α and HIF-1α expression levels were significant univariate factors associated with CSS in patients with clear cell RCC. These results highlight the critical role that HIF-3α and HIF-1α play in the hypoxia pathway.

Hypobaric hypoxia aggravates osteoarthritis via the alteration of the oxygen environment and bone remodeling in the subchondral zone.

A high prevalence of osteoarthritis (OA) has been observed among individuals living at high altitudes, and hypobaric hypoxia (HH) can cause bone mass and strength deterioration. However, the effect of HH on OA remains unclear. In this study, we aimed to explore the impact of HH on OA and its potential mechanisms. A rat knee OA model was established by surgery, and the rats were bred in an HH chamber simulating a high-altitude environment. Micro-computed tomography (Micro-CT), histological analysis, and RNA sequencing were performed to evaluate the effects of HH on OA in vivo. A hypoxic co-culture model of osteoclasts and osteoblasts was also established to determine their effects on chondrogenesis in vitro. Cartilage degeneration significantly worsened in the HH-OA group compared to that in the normoxia-OA (N-OA) group, 4 weeks after surgery. Micro-CT analysis revealed more deteriorated bone mass in the HH-OA group than in the N-OA group. Decreased hypoxia levels in the cartilage and enhanced hypoxia levels in the subchondral bone were observed in the HH-OA group. Furthermore, chondrocytes cultured in a conditioned medium from the hypoxic co-culture model showed decreased anabolism and extracellular matrix compared to those in the normoxic model. RNA sequencing analysis of the subchondral bone indicated that the glycolytic signaling pathway was highly activated in the HH-OA group. HH-related OA progression was associated with alterations in the oxygen environment and bone remodeling in the subchondral zone, which provided new insights into the pathogenesis of OA.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation.

BACKGROUND: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation. METHODS: In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1. RESULTS: Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment. CONCLUSIONS: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

Assessment of hypoxemia among young adults with sickle cell anaemia in steady state in southwestern Nigeria: a crosssectional study.

OBJECTIVES: Hypoxia is a known feature of sickle cell anaemia (SCA) which results from chronic anaemia and recurrent vaso-occlusive crisis (VOC) which can cause tissue ischaemia that leads to an end organ damage. The hallmark of SCA is chronic anaemia and recurrent vaso-occlusive crisis. The aim of this study is to compare the oxygen saturation of sickle cell anaemic individuals with the normal haemoglobin type (Hb AA) control and also to determine the prevalence of hypoxemia among SCA. RESULTS: Two-hundred and twenty-two (136 Hb SS and 86 Hb AA) participated in the study. The mean ± SD of age (years), oxygen saturation (%) and pulse rate (bpm) of participants with sickle cell anaemia and Hb AA control were 21.85 ± 3.04 and 22.14 ± 3.18 (t = 0.701, p = 0.436), 95.21 ± 3.02 and 98.07 ± 0.81 (t=-8.598, p < 0.0001) and 77.10 ± 9.28 and 73.16 ± 8.52 (t = 3.173, p = 0.002) respectively. The prevalence of hypoxemia among SCA participants was 47.1%. Prevalence of hypoxemia in males with SCA was 60.9% while 39.1% of the females had hypoxemia.

Mitophagy mediated by HIF-1α/FUNDC1 signaling in tubular cells protects against renal ischemia/reperfusion injury.

Acute kidney injury (AKI) is associated with a high mortality rate. Pathologically, renal ischemia/reperfusion injury (RIRI) is one of the primary causes of AKI, and hypoxia-inducible factor (HIF)-1α may play a defensive role in RIRI. This study assessed the role of hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy in protection against RIRI in vitro and in vivo. The human tubular cell line HK-2 was used to assess hypoxia/reoxygenation (H/R)-induced mitophagy through different in vitro assays, including western blotting, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reactive oxygen species (ROS) measurement. Additionally, a rat RIRI model was established for evaluation by renal histopathology, renal Doppler ultrasound, and transmission electron microscopy to confirm the in vitro data. The selective HIF-1α inhibitor LW6 reduced H/R-induced mitophagy but increased H/R-induced apoptosis and ROS production. Moreover, H/R treatment enhanced expression of the FUN14 domain-containing 1 (FUNDC1) protein. Additionally, FUNDC1 overexpression reversed the effects of LW6 on the altered expression of light chain 3 (LC3) BII and voltage-dependent anion channels as well as blocked the effects of HIF-1α inhibition in cells. Pretreatment of the rat RIRI model with roxadustat, a novel oral HIF-1α inhibitor, led to decreased renal injury and apoptosis in vivo. In conclusion, the HIF-1α/FUNDC1 signaling pathway mediates H/R-promoted renal tubular cell mitophagy, whereas inhibition of this signaling pathway protects cells from mitophagy, thus aggravating apoptosis, and ROS production. Accordingly, roxadustat may protect against RIRI-related AKI.

[Best evidence summary for awake prone positioning in treating hypoxemic due to COVID-19 infection].

Objective: To retrieve, evaluate, and summarize the best evidence for the treatment of hypoxemia in patients with COVID-19 infection using the awake prone positioning, with the aim of guiding healthcare professionals in the standardized implementation of this therapy. Methods: A systematic search was conducted in databases including UpToDate, BMJ Best Practice, JBI Evidence-Based Healthcare Center, American Association of Critical-Care Nurses, Intensive Care Society, European Respiratory Society, World Health Organization website, Cochrane Library, PubMed, China National Knowledge Infrastructure (CNKI), and Wanfang. The retrieved literature was subjected to quality assessment and evidence extraction. Results: A total of ten publications were included, consisting of one thematic evidence summary, one guideline, two systematic reviews, three randomized controlled trials, and three expert consensus statements. This summary synthesizes thirty key pieces of evidence in five categories: organizational management and training, risk assessment, preparatory operations, implementation key points, and risk control. Conclusions: Awake prone positioning is beneficial for improving hypoxemia in patients with COVID-19 and is easy to implement. Medical institutions should develop nursing management systems, operational standards, and best practices for awake prone positioning based on evidence-based evidence in order to improve the quality of care management for such patients.

[Techniques for quantifying endotypes of obstructive sleep apnea].

Obstructive sleep apnea (OSA) is the frequent occurrence of apnea and/or hypopnea during sleep, leading to intermittent hypoxia, hypercapnia, and disruption of sleep architecture, further resulting in multisystem damage. The pathophysiological mechanisms include abnormal anatomical structure, low arousal threshold, high loop gain, and poor muscle reactivity, etc. As there are individual differences in the underlying mechanisms of OSA (i.e. endotypes), the effectiveness of treatment and prognosis may also vary according to these characteristics. Understanding the endotype of OSA is critical to understanding which patients are most likely to benefit from non-invasive ventilation therapy. Quantification of endotypes is central to the precision treatment of OSA and may provide the basis for accurate clinical treatment of OSA based on endotypes.

[Theory of centralized aerobic-anaerobic energy balance compensation and non-pharmaceutical treatment methods of arterial hypertension]. / Teoriya tsentralizovannoi kompensatsii aerobno-anaerobnogo energeticheskogo balansa i nelekarstvennye metody lecheniya arterial'noi gipertenzii.

Several chronic non-communicable diseases are associated with arterial hypertension and are closely related to increased blood pressure. The theory of centralized aerobic-anaerobic energy balance compensation (TCAAEBC) was formulated in connection with the above-mentioned processes. This theory, including the hypothesis of the «egoistic brain¼, is a broader concept. The key point of TCAAEBC is hypoxic anaerobic metabolism, which affects reflex vascular zones, including the neurons of the respiratory and cardiovascular centers of the rhomboid fossa of the medulla oblongata. Hypoxia correction using manual techniques, physical exercises, and other non-pharmaceutical methods under certain conditions can stabilize the level of blood pressure and has a curative effect in the case of arterial hypertension syndrome.

Xinnaotongluo liquid protects H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3.

Xinnaotongluo liquid has been used to improve the clinical symptoms of patients with myocardial infarction. However, the molecular mechanism of Xinnaotongluo liquid is not completely understood. H9c2 cells exposed to hypoxia/reoxygenation (H/R) was used to simulate damage to cardiomyocytes in myocardial infarction in vitro. The biological indicators of H9c2 cells were measured by cell counting kit-8, enzyme linked immunoabsorbent assay, and western blot assay. In H/R-induced H9c2 cells, a markedly reduced murine double minute 2 (MDM2) was observed. However, the addition of Xinnaotongluo liquid increased MDM2 expression in H/R-induced H9c2 cells. And MDM2 overexpression strengthened the beneficial effects of Xinnaotongluo liquid on H9c2 cells from the perspective of alleviating oxidative damage, cellular inflammation, apoptosis and ferroptosis of H/R-induced H9c2 cells. Moreover, MDM2 overexpression reduced the protein expression of p53 and Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3). Whereas, STEAP3 overexpression hindered the function of MDM2-overexpression in H/R-induced H9c2 cells. Our results insinuated that Xinnaotongluo liquid could protect H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3, which provide a potential theoretical basis for further explaining the working mechanism of Xinnaotongluo liquid.

Management of six episodes of intraoperative cardiac arrests in an infant with traumatic bronchial rupture: A case report.

RATIONALE: Traumatic bronchial rupture in infants usually necessitates surgical intervention, with few reports documenting instances of multiple cardiac arrests occurring during surgery under conditions of severe hypoxemia. PATIENT CONCERNS: A 3-year-old boy after trauma presented with severe hypoxemia for 2 days and was urgently transferred to the operating room for surgery, 6 episodes of cardiac arrest happend during surgery. DIAGNOSES: The baby was diagnosed with bronchial rupture based on the history of trauma, clinica manifestations, and intraoperative findings. INTERVENTIONS: Intrathoracic cardiac compression and intravenous adrenaline were administrated. OUTCOMES: The normal sinus rhythm of the heart was successfully restored within 1 minute on each occasion, facilitating the smooth completion of the surgical procedure. By the end of surgery, SpO2 levels had rebounded to 95% and remained stable. LESSONS: Inadequate management of bronchial ruptures in infants frequently coincides with severe hypoxemia, necessitating immediate surgical intervention. Prompt identification and management of cardiac arrest by anesthetists during surgery is imperative to reduce mortality.

Oxygenation saturation index in neonatal hypoxemic respiratory failure.

BACKGROUND: This study aimed to assess the validity of the oxygenation saturation index (OSI) and the ratio of oxygen saturation to the fraction of inspired oxygen (FIO2) (S/F ratio) with percutaneous oxygen saturation (OSISpO2 and the Sp/F ratio) and to evaluate the correlation between these values and the oxygen index (OI). It also determined their cut-off values for predicting OI in accordance with neonatal hypoxic respiratory failure severity. METHODS: We reviewed the data of 77 neonates (gestational age 31.7 ± 6.1 weeks; birthweight, 1768 ± 983 g) requiring invasive mechanical ventilation between 2013 and 2020, 1233 arterial blood gas samples in total. We calculated the OI, OSISpO2, OSI with arterial oxygen saturation (SaO2) (OSISaO2), Sp/F ratio, and the ratio of SaO2 to FIO2 (Sa/F ratio). RESULTS: The regression and Bland-Altman analysis showed good agreement between OSISpO2 or the Sp/F ratio and OSISaO2 or the Sa/F ratio. Although a significant positive correlation was found between OSISpO2 and OI, OSISpO2 was overestimated in SpO2 > 98% with a higher slope of the fitted regression line than that below 98% of SpO2. Furthermore, receiver-operating characteristic curve analysis using only SpO2 ≤ 98% samples showed that the optimal cut-off points of OSISpO2 and the Sp/F ratio for predicting OI were: OI 5, 3.0 and 332; OI 10, 5.3 and 231; OI 15, 7.7 and 108; OI 20, 11.0 and 149; and OI 25, 17.1 and 103, respectively. CONCLUSION: The cut-off OSISpO2 and Sp/F ratio values could allow continuous monitoring for oxygenation changes in neonates with the potential for wider clinical applications.

[Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells].

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

[Astragali Radix-Curcumae Rhizoma inhibits colon cancer progression and enhances 5-FU efficacy by regulating hypoxia-inducible factors and tumor stem cells].

The animal and cell models were used in this study to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in inhibiting colon cancer progression and enhancing the efficacy of 5-fluorouracil(5-FU) by regulating hypoxia-inducible factors and tumor stem cells. The animal model was established by subcutaneous transplantation of colon cancer HCT116 cells in nude mice, and 24 successfully modeled mice were randomized into model, 5-FU, HQEZ, and 5-FU+HQEZ groups. The tumor volume was measured every two days. Western blot was employed to measure the protein levels of epidermal growth factor receptor(EGFR), dihydropyrimidine dehydrogenase(DPYD), and thymidylate synthase(TYMS), the key targets of the hypoxic core region, as well as the hypoxia-inducible factors HIF-1α and HIF-2α and the cancer stem cell surface marker CD133 and SRY-box transcription factor 2(SOX2). The results of animal experiments showed that HQEZ slowed down the tumor growth and significantly increased the tumor inhibition rate of 5-FU. Compared with the model group, HQEZ significantly down-regulated the protein levels of EGFR and DPYD, and 5-FU+HQEZ significantly down-regulated the protein levels of EGFR and TYMS in tumors. Compared with the model group, HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, SOX2, and CD133 in the hypoxic core region. Compared with the 5-FU group, 5-FU+HQEZ lowered the protein levels of HIF-1α, HIF-2α, and SOX2. The cell experiments showed that the protein le-vels of HIF-1α and HIF-2α in HCT116 cells elevated significantly after low oxygen treatment. Compared with 5-FU(1.38 µmol·L~(-1)) alone, HQEZ(40 mg·mL~(-1)) and 5-FU+HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, and TYMS. In conclusion, HQEZ can inhibit the expression of hypoxia-responsive molecules in colon cancer cells and reduce the properties of cancer stem cells, thereby enhancing the therapeutic effect of 5-FU on colon cancer.

[Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats].

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P<0.05 or P<0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P<0.05 or P<0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P<0.05 or P<0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P<0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P<0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

BACKGROUND: Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. METHOD: The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. RESULTS: HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. CONCLUSION: HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.